# FROM STRUCTURE TO SIGNALOSOMES: NEW PERSPECTIVES ABOUT MEMBRANE RECEPTORS AND CHANNELS

EDITED BY : Yi Ma, Kai He and Gerald A. Berkowitz PUBLISHED IN : Frontiers in Plant Science

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## FROM STRUCTURE TO SIGNALOSOMES: NEW PERSPECTIVES ABOUT MEMBRANE RECEPTORS AND CHANNELS

Topic Editors: Yi Ma, University of Connecticut, United States Kai He, Lanzhou University, China Gerald A. Berkowitz, University of Connecticut, United States

Citation: Ma, Y., He, K., Berkowitz, G. A., eds. (2019). From Structure to Signalosomes: New Perspectives About Membrane Receptors and Channels. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-973-5

# Table of Contents


Helen R. Irving, David M. Cahill and Chris Gehring

*16 Magnesium Transporter MGT6 Plays an Essential Role in Maintaining Magnesium Homeostasis and Regulating High Magnesium Tolerance in*  Arabidopsis

Yu-Wei Yan, Dan-Dan Mao, Lei Yang, Jin-Liang Qi, Xin-Xin Zhang, Qing-Lin Tang, Yang-Ping Li, Ren-Jie Tang and Sheng Luan

*29* SlCNGC1 *and* SlCNGC14 *Suppress* Xanthomonas oryzae *pv.* oryzicola*-Induced Hypersensitive Response and Non-host Resistance in Tomato*

Xuan-Rui Zhang, You-Ping Xu and Xin-Zhong Cai

*38 Mapping of* HKT1;5 *Gene in Barley Using GWAS Approach and Its Implication in Salt Tolerance Mechanism*

Khaled M. Hazzouri, Basel Khraiwesh, Khaled M. A. Amiri, Duke Pauli, Tom Blake, Mohammad Shahid, Sangeeta K. Mullath, David Nelson, Alain L. Mansour, Kourosh Salehi-Ashtiani, Michael Purugganan and Khaled Masmoudi

*51 Auxin and Gibberellins are Required for the Receptor-Like Kinase ERECTA Regulated Hypocotyl Elongation in Shade Avoidance in Arabidopsis*

Junbo Du, Hengke Jiang, Xin Sun, Yan Li, Yi Liu, Mengyuan Sun, Zhou Fan, Qiulin Cao, Lingyang Feng, Jing Shang, Kai Shu, Jiang Liu, Feng Yang, Weiguo Liu, Taiwen Yong, Xiaochun Wang, Shu Yuan, Liang Yu, Chunyan Liu and Wenyu Yang

*63 Activation of the LRR Receptor-Like Kinase PSY1R Requires Transphosphorylation of Residues in the Activation Loop*

Christian B. Oehlenschlæger, Lotte B. A. Gersby, Nagib Ahsan, Jesper T. Pedersen, Astrid Kristensen, Tsvetelina V. Solakova, Jay J. Thelen and Anja T. Fuglsang

*75 Excessive Cellular* S*-nitrosothiol Impairs Endocytosis of Auxin Efflux Transporter PIN2*

Min Ni, Lei Zhang, Ya-Fei Shi, Chao Wang, Yiran Lu, Jianwei Pan and Jian-Zhong Liu


Yue Niu, Dong Qian, Baiyun Liu, Jianchao Ma, Dongshi Wan, Xinyu Wang, Wenliang He and Yun Xiang

*140 Transcriptome and Differential Expression Profiling Analysis of the Mechanism of Ca2+ Regulation in Peanut (*Arachis hypogaea*) Pod Development*

Sha Yang, Lin Li, Jialei Zhang, Yun Geng, Feng Guo, Jianguo Wang, Jingjing Meng, Na Sui, Shubo Wan and Xinguo Li

*150* ERECTA *Regulates Cell Elongation by Activating Auxin Biosynthesis in*  Arabidopsis thaliana

Xiaoya Qu, Zhong Zhao and Zhaoxia Tian


Yang Gao, Yujun Wu, Junbo Du, Yanyan Zhan, Doudou Sun, Jianxin Zhao, Shasha Zhang, Jia Li and Kai He

# Editorial: From Structure to Signalsomes: New Perspectives About Membrane Receptors and Channels

Yi Ma<sup>1</sup> \*, Kai He<sup>2</sup> and Gerald A. Berkowitz <sup>1</sup> \*

*<sup>1</sup> Plant Science, University of Connecticut, Mansfield, CT, United States, <sup>2</sup> School of Life Sciences, Lanzhou University, Lanzhou, China*

Keywords: ion channels, signalosome, stress response and growth, signal transduction, membrane receptors

**Editorial on the Research Topic**

#### Edited by:

*Kendal Hirschi, Baylor College of Medicine, United States*

#### Reviewed by:

*Roberto Adrian Gaxiola, Arizona State University, United States June M. Kwak, Daegu Gyeongbuk Institute of Science and Technology (DGIST), South Korea Wayne Versaw, Texas A&M University, United States*

#### \*Correspondence:

*Yi Ma yi.ma@uconn.edu Gerald A. Berkowitz gerald.berkowitz@uconn.edu*

#### Specialty section:

*This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science*

Received: *09 April 2019* Accepted: *06 May 2019* Published: *22 May 2019*

#### Citation:

*Ma Y, He K and Berkowitz GA (2019) Editorial: From Structure to Signalsomes: New Perspectives About Membrane Receptors and Channels. Front. Plant Sci. 10:682. doi: 10.3389/fpls.2019.00682* **From Structure to Signalsomes: New Perspectives About Membrane Receptors and Channels**

The plasma membrane (PM) forms a selective barrier between the interior of the cell and the external environment. The PM structure and constituents can act as a sentry to facilitate perception of changes in the environment to trigger intricate signaling cascades, leading to fine-tuned adaptive responses. The PM contains a plethora of proteins that are channels, transporters, receptors or enzymes. These proteins are involved in a number of cell activities such as the following: (a) transport of molecules or other substances across the plasma membrane, (b) perception of external environmental cues that cause perturbations in plant homeostasis (for example, temperature stress and salinity), and (c) patterning the cellular response to internal (plant) signal molecules such as hormones or peptides. In these cases of PM function, the transduction of signal perception orchestrates downstream events in the cytosol that modify cell function to respond to the signal (Palme, 2012). The proteins could function together as a signalosome in a discreet functional unit (a plasma membrane "nanodomain") to facilitate the signal transduction processes that are responsible for plant growth and development or defense responses (Tapken and Murphy, 2015).

This Frontiers Research Topic have compiled a collection of cutting-edge research articles and up to date reviews on the study of plasma membrane proteins that are involved in abiotic or biotic stress, cell growth and plant development. The article collection that composes this Research Topic includes 13 original research articles, one review and one perspective. Contributions were made by 115 authors from Australia, China, Denmark, Italy, Tunisia, United Arab Emirates, and the United States.

Regulation of plasma membrane transporters is crucial for ion homeostasis in plants under a variety of abiotic stress conditions. Hazzouri et al. identified the HKT1;5 gene and its associated QTL in barley. This work provides new insights on the breeding of salt tolerant barley. Among the small grains, this crop is often grown in soil with saline stress. Another HKT member in rice, OsHKT2;4, was shown to be a low-affinity Mg2<sup>+</sup> transporter that mediates Mg2<sup>+</sup> homeostasis under high Mg2<sup>+</sup> conditions (Zhang et al.). In addition, Yan et al. demonstrated that Mg2<sup>+</sup> transporters MGT6 and MGT7 has additive dual functions in plant adaptation to a low Mg2<sup>+</sup> environment and detoxification of excess Mg2<sup>+</sup> under high Mg2<sup>+</sup> conditions. Niu et al. showed that the lipid flippase activity of the P4-type ATPase ALA6 plays a critical role for membrane stability under heat stress. Yu et al. demonstrated that overexpression of poplar ABA receptors PtPYRL1 or PtPYRL5 significantly enhanced cold, drought, and osmotic tolerance in poplar trees.

Cyclic nucleotide-gated ion channels (CNGCs) and Ca2<sup>+</sup> have been shown to play vital roles in plant defense responses to various pathogens (Ma and Berkowitz, 2011; Moeder et al., 2011). Zhang et al. showed that silencing of tomato CNGC1 and CNGC14 enhanced Xanthomonas oryzae pv. oryzicola (Xoc) induced hypersensitive responses and non-host resistance to Xoc, suggesting a negative regulatory role of these CNGCs in non-host resistance and basal immune responses (also referred to as pathogen associated molecular pattern (PAMP)-triggered immunity). Studies have revealed contradictory roles of CNGCs in pathogen resistance; clearly this ion channel-mediated signal transduction system responding to pathogen perception should be the focus of further studies in the future to better understand how Ca2<sup>+</sup> and CNGCs regulate plant immune defense responses to microbial pathogens.

Plasma membrane localized transporters and receptors regulate hormone mediated cell elongation. Among the transporters facilitating this signaling are the PIN-FORMED (PIN) family of auxin transporters. Ni et al. showed that Snitrosoglutathione-regulated PIN2 endocytosis is important for root elongation. Some members of the large family of leucinerich repeat receptor-like kinases (LRR-RLKs) act as receptors for hormones and peptides; in some cases these receptors can transduce developmental signals into altered cell growth. Among these LRR-RLKs involved in facilitating peptide signals and modulating growth is ERECTA. Qu et al. demonstrated that ERECTA regulates hypocotyl elongation by activating auxin biosynthesis. In another study of ERECTA, in this case focusing on shade avoidance, Du et al. showed that under shade, ERECTA-regulated hypocotyl elongation is controlled by both auxin and gibberellins.

Yang et al. and Li et al. employed transcriptomic approaches to reveal genes involved in Ca2<sup>+</sup> regulated peanut pod development, which could also be regulated by hormonal signaling. Ca2<sup>+</sup> signaling is involved as a secondary cytosolic messenger responding to ligand perception by cell membrane receptors in myriad signal transduction pathways, including those responding to pathogen perception during defense response, and in response to hormone/peptide cues patterning growth and development. One such signaling pathway that impacts cell expansion involves the tyrosine-sulfated pentapeptide phytosulfokine (PSK) acting as a peptide hormone signal that activates cell responses upon binding to its cognate LRR-RLK receptor PSKR1. Irving et al. focused on the signaling pathway activated by PSKR1 perception of the PSK ligand in a review of a subset of LRR-RLK cell membrane receptors. Irving et al. noted that, in addition to having cytosol kinase domains that facilitate downstream signaling, PSKR1 (and similar LRR-RLKs) also facilitate downstream signaling involving cGMP generation and CNGC activation which then leads to cytosolic Ca2<sup>+</sup> elevation. Irving et al. provide some insightful context for signaling in presenting the PSK/PSKR1 interaction as impacting other membrane proteins beyond CNGCs. They point out that PSKR1 is thought to function as a complex with other proteins including the coreceptor BAK1, a CNGC, and the cell membrane proton pumps AHA1 and AHA2. These proteins might act as a unit, or signalosome in a nanodomain at the cell surface.

A peptide hormone/LRR-RLK cell membrane receptor signaling pathway similar to PSK/PSKR1 involves another tyrosine-sulfated peptide, PSY1, and its cognate receptor PSY1R. Oehlenschlæger et al. studied PSY1-dependent transphosphorylation of residues in the activation loop of PSY1R, which are critical for the activation of the receptor and downstream signaling. They demonstrated that PSY1R activation involves interaction (transphosphorylation) with the BAK1 coreceptor (and other members of the BAK1 membrane protein family) in a fashion similar to PSKR1. Intriguingly, signaling by the PSKR1 and PSY1R receptors also involves activation of a cell membrane proton pump [in this case, only AHA1; (Fuglsang et al., 2014)], and both receptors are involved in cell expansion activated during developmental programs in plants. The two pathways downstream from these receptors are also involved in signaling that leads to plant defense against pathogen invasion (Mosher and Kemmerling, 2013). Gao et al. studied another aspect of the coreceptor BAK1 involvement in immune signaling. In their study, they found that some signal associated with light interacts with programmed cell death; one possible downstream outcome from immune signaling. The immune responses involving BAK1 as an upstream component were found to involve proteins of the salicylic acid signaling pathway.

Gehring and Turek summarized the current progress of work on cyclic nucleotide monophosphates (cAMP and cGMP), which activate CNGCs. And, as mentioned above, Irving et al. provided a review and perspectives on the research of cyclic nucleotides using cGMP and PSKR1 as an example. Although the presence of plant nucleotide cyclases that synthesize cAMP and cGMP in plant cells is still controversial, more and more evidence supports their roles in numerous signal transduction pathways in plant cells; the focus of some of the contributions to this Research Topic.

More than 80% of the global population live in poverty; most of this problem can be attributed to adverse agricultural conditions such as salinity, drought, lack of nutrition, temperature stress or pathogen infection. The studies included in this Research Topic could provide new insights on how to solve or ameliorate such problems through the modifications of traits controlled by certain genes.

### AUTHOR CONTRIBUTIONS

YM wrote the editorial article. GB edited and modified the article. KH proofread the article.

### REFERENCES


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Ma, He and Berkowitz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Moonlighting Proteins and Their Role in the Control of Signaling Microenvironments, as Exemplified by cGMP and Phytosulfokine Receptor 1 (PSKR1)

Helen R. Irving1,2 \*, David M. Cahill<sup>3</sup> and Chris Gehring<sup>4</sup>

<sup>1</sup> Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, Australia, <sup>2</sup> La Trobe Institute for Molecular Science, La Trobe University, Bendigo, VIC, Australia, <sup>3</sup> Faculty of Science Engineering and Built Environment, Deakin University, Geelong, VIC, Australia, <sup>4</sup> Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy

Signal generating and processing complexes and changes in concentrations of messenger molecules such as calcium ions and cyclic nucleotides develop gradients that have critical roles in relaying messages within cells. Cytoplasmic contents are densely packed, and in plant cells this is compounded by the restricted cytoplasmic space. To function in such crowded spaces, scaffold proteins have evolved to keep key enzymes in the correct place to ensure ordered spatial and temporal and stimulusspecific message generation. Hence, throughout the cytoplasm there are gradients of messenger molecules that influence signaling processes. However, it is only recently becoming apparent that specific complexes involving receptor molecules can generate multiple signal gradients and enriched microenvironments around the cytoplasmic domains of the receptor that regulate downstream signaling. Such gradients or signal circuits can involve moonlighting proteins, so called because they can enable finetune signal cascades via cryptic additional functions that are just being defined. This perspective focuses on how enigmatic activity of moonlighting proteins potentially contributes to regional intracellular microenvironments. For instance, the proteins associated with moonlighting proteins that generate cyclic nucleotides may be regulated by cyclic nucleotide binding directly or indirectly. In this perspective, we discuss how generation of cyclic nucleotide-enriched microenvironments can promote and regulate signaling events. As an example, we use the phytosulfokine receptor (PSKR1), discuss the function of its domains and their mutual interactions and argue that this complex architecture and function enhances tuning of signals in microenvironments.

Keywords: calcium, cyclic nucleotides, cyclic GMP (cGMP), kinases, intracellular signals, microenvironment, molecular crowding, phytosulfokine receptor (PSKR1)

## INTRODUCTION

Often, despite the beautiful illustrations from Goodsell (1993, 2016), it is forgotten how crowded it is within the cytoplasmic space (Fulton, 1982). The term molecular crowding is used to indicate that 25 to 40% of space in the cytoplasm is occupied by many different, large biomolecules (**Figure 1A**). The number of individual biomolecules is estimated to range from 10 to 1000 molecules per cell

### Edited by:

Yi Ma, University of Connecticut, United States

### Reviewed by:

Ian Max Møller, Aarhus University, Denmark John Hancock, University of the West of England, United Kingdom

> \*Correspondence: Helen R. Irving h.irving@latrobe.edu.au

### Specialty section:

This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science

Received: 21 August 2017 Accepted: 14 March 2018 Published: 28 March 2018

### Citation:

Irving HR, Cahill DM and Gehring C (2018) Moonlighting Proteins and Their Role in the Control of Signaling Microenvironments, as Exemplified by cGMP and Phytosulfokine Receptor 1 (PSKR1). Front. Plant Sci. 9:415. doi: 10.3389/fpls.2018.00415

**8**

(Srere, 1967; Luby-Phelps, 2000; Ellis, 2001). Water is closely associated with the surface of large biomolecules while the flow apart from this encasing layer reflects diffusion (Srere, 1980, 1981, 2000; Saenger, 1987; Luby-Phelps, 2000, 2013; Ellis, 2001; Saxton, 2012; Phillip and Schreiber, 2013).

In plant cells, the cytoplasm is found in a relatively narrow layer between the plasma membrane and large organelles such as the vacuole, compounding the problem of molecular crowding. Underlying the plasma membrane is a network of cytoskeletal proteins that support and interact with other proteins, and are involved in organelle movement (Williamson, 1993; Shimmen, 2007; Goldstein and van de Meert, 2015). Cytoplasmic streaming involves various biophysical pathways resulting in movement that can be along cellular edges or alternatively create turbulence throughout the cytoplasm (Goldstein and van de Meert, 2015). Over short distances diffusion of small molecules is faster than cytoplasmic streaming (Vestergaard et al., 2012). However, if the small molecules have affinity to large biomolecules, they may have restricted capacity to diffuse (Geremia et al., 2006).

Molecular crowding occurs despite the limited copies of individual proteins present (Luby-Phelps, 2000, 2013; Ellis, 2001), so correct spatial arrangements of individual enzymes is necessary for signals to be relayed through signal networks to elicit cellular responses. Several metabolic pathways or metabolons employ molecular channeling to efficiently deliver the product from the first enzyme to form the substrate for the next enzyme (Srere, 1985, 2000; Miles et al., 1999; Winkel, 2004; Moller, 2010; Sweetlove and Fernie, 2013; Zhang et al., 2017). Correct positioning of individual enzymes and scaffold proteins enhances signal cascades via molecular channeling (Rohwer et al., 1998; Wheeldon et al., 2016). Positioning in this way creates subcellular microenvironments containing microcues of concentrated signaling molecules that in turn activate downstream points of signal cascades, thereby emphasizing the importance of spatial and temporal regulation of protein expression.

The crowded intracellular space combined with cellular compartmentalization and intracellular molecular gradients, have led biological systems toward microenvironments (or localized signaling circuits). We propose that a component of these signaling circuits are moonlighting proteins. In a general sense, moonlighting proteins are proteins that can perform more than one function and/or act in more than one spot in the cell (Jeffery, 2003, 2009, 2014). Examples of the latter type of moonlighting proteins include various mitochondrial proteins that also act in the nucleus (Monaghan and Whitmarsh, 2015) where they contribute to cellular signaling pathways. It is thought that during evolution, ancestral proteins have acquired additional functions including transcriptional regulation and signal transduction. For instance, it has been suggested that many of the newly emerging RNA-binding proteins are moonlighting as they have enzymatic activities, for example in metabolism, as well as functioning as regulators of transcription and RNA turnover (Hentze and Preiss, 2010; Marondedze et al., 2016b). In this perspective, we will concentrate on moonlighting proteins with roles in signal transduction. Many of these moonlighting proteins are receptor kinases that contain a cytosolic main function, a kinase, and an additional cryptic function, a cyclase (Wong and Gehring, 2013; Wong et al., 2015). The spatial arrangement of these two domains is somewhat unexpected and since both enzymatic activities are affected by second messengers such as calcium ions and the catalytic product of each domain, these moonlighting proteins are likely to serve as molecular tuners. For these reasons, we propose that such moonlighting signaling proteins are well-suited to operate in or generate subcellular microenvironments containing ions or small molecules that provide points of control in signal cascades (**Figure 1B**). Concepts of metabolons and molecular crowding are well-established while the concept of proteins generating their own small molecule microenvironmental regulatory milieu is less established. This perspective focuses on recent advances in cGMP signaling in plants, and how enigmatic activity of moonlighting proteins can contribute to regional intracellular microenvironments. First, we discuss the importance of small incremental changes in cGMP microenvironments and then we use the phytosulfokine receptor (PSKR1) as an example of a moonlighting protein that generates phospho- and cGMPmicroenvinvironments.

### SIGNAL STRENGTH AND SPECIFICITY

A considerable body of literature exists on the biological functions and mechanisms of action of cyclic nucleotide signaling in lower and higher eukaryotes (Lemtiri-Chlieh et al., 2011). In fact, cGMP and cAMP are accepted as key signaling molecules in developmental and environmental stress response cascades. However, acceptance that cyclic nucleotides have such a role in plant signaling was slow. One reason was that, in plants, cellular cyclic nucleotide levels seem generally lower than in

FIGURE 1 | The molecularly crowded cytoplasm and the presence of small signal-enriched microenvironments within the cytoplasm. (A) A small portion of the cytoplasm depicting molecular crowding as illustrated by David S. Goodsell, the Scripps Research Institute. Cytoskeletal components are shown along with ribosomes (large blue molecules). The scale bar = 30 nm and is based on the measured width of ribosomes (Haga et al., 1970). (B) Diagram showing how specific signal-enriched areas can occur in the cytoplasm. Three areas with different micro-enriched signals (gray, pale green, and pale yellow) are depicted that affect separate groups of proteins and represent areas with diameters of 100–200 nm. The microenvironments may be created by proteins within the group or generated from external sources such as ion fluxes from internal organelles or the extracellular environment.

animals or lower eukaryotes (Newton et al., 1999; Gehring, 2010; Marondedze et al., 2017). An additional reason for the initially reluctant acceptance of these signaling molecules in higher plants was that molecular evidence for mononucleotide cyclases in higher plants only came after the first plant draft genome was published in 2000 (Ludidi and Gehring, 2003). Since the publication of the first mononucleotide cyclase, the number of identified and experimentally tested mononucleotide cyclases has increased steadily and there are indications that there are >50 candidate cyclases in the Arabidopsis thaliana proteome and that they come in many different domain organizations (Meier et al., 2007; Wong and Gehring, 2013). The multitude of candidates and domain partners points to a diverse spectrum of biological functions for mononucleotide cyclases and their catalytic products.

Invariably the question of how a single messenger, like cAMP or cGMP, is capable of triggering highly specific responses to different developmental and/or environmental stimuli arises. It seems obvious that saturating the cell with either cAMP or cGMP cannot be the answer. To illustrate the point, such an approach would be like attempting to regulate traffic flow in a city with only one "gigantic traffic light" that is either red or green. Since the "gigantic traffic light" is unlikely to work, two solutions come to mind. One solution relies on strict compartmentalization of the messenger(s) and the other on the combination and integration of several messengers, e.g., cAMP/cGMP with cytoplasmic calcium ions and/or pH. A recent review has highlighted the interplay of calcium ion signatures with cGMP in plant–microbe interactions (Yuan et al., 2017). Specific response signatures and cooperation between messengers arises through spatial clustering of stimulus-dependent cyclases and their downstream signaling components and/or through the specific binding of the cyclic nucleotides to effector molecules such as kinases (Kwezi et al., 2011; Isner and Maathuis, 2016; Wheeler et al., 2017) or channel subunits (Hoshi, 1995; Zelman et al., 2012). A recent study using a constitutively expressed mammalian guanylate cyclase in Arabidopsis that produced intracellular cGMP levels >50 fold above normal resulted in mis-signaling and down-regulation of many proteins in systemic acquired resistance (Hussain et al., 2016). This study and others where calcium ion influxes flood intracellular compartments (Sanders et al., 2002; Yuan et al., 2017) highlight the need for transient and controlled levels of signaling molecules to generate appropriate responses to environmental and developmental stimuli within defined cytoplasmic areas or cellular compartments.

An affinity pull-down approach has been applied to obtain a cGMP-dependent interactome (Donaldson and Meier, 2013; Donaldson et al., 2016) where several of the cGMP-binding candidates have critical functions in the Calvin–Benson– Bassham cycle and the photorespiration pathway and they also contain cyclic nucleotide-binding domains. It is conceivable that the enzyme activity of these molecules may be directly or indirectly modified by cGMP. Since the Calvin–Benson– Bassham cycle is confined to the stroma of the chloroplast, we might imagine cGMP is generated specifically in the stroma to modulate these enzymes without affecting, for example, cGMP-dependent channels found in the plasma membrane of guard cells. Incidentally, it has also been demonstrated that the activity of the cGMP-binding photorespiration enzyme glycolate oxidase (GOX1) is dampened by cGMP and NO treatment. Since GOX1 produces H2O<sup>2</sup> in response to Pseudomonas (Pst DC3000 AvrRpm1), it implicates cGMP-mediated processes in the crosstalk between NO and H2O<sup>2</sup> signaling during defense responses (Donaldson et al., 2016).

If we agree that the "gigantic traffic light" does not work, we may find it easier to accept that small transients in cellular cAMP and cGMP are not a problem, but rather the solution to highly differentiated stimulus-specific cellular signaling in plants. The "gigantic traffic light" has additional implications; predominantly in relation to systems-based investigations of cAMP- and cGMP-dependent processes where the experimental set-up includes cell-permeant cyclic mononucleotides at high concentrations. Such investigations, particularly at the system level, can give insights into cyclic mononucleotide-dependent phosphoproteome (Marondedze et al., 2016a), but merely identify target rather than resolve stimulus-specific signaling cascades. In addition to the generators of the cyclic nucleotide signal, we must also consider the signal-off state. The role and, to this date, lack of genetic evidence for suitable phosphodiesterases that degrade cyclic nucleotides to mononucleotide phosphates has been excellently reviewed (Grosse and Durner, 2016). To generate greater insights into the formation of subcellular microenvironments, specific signaling pathways need to be examined in detail as complex interactions are likely between proteins and the immediate microenvironment (**Figure 1B**). Below we describe the evidence supporting the formation of a subcellular microenvironment surrounding the moonlighting phytosulfokine receptor (PSKR1) as an example of how plants may utilize enigmatic enzymatic centers in homeostatic function.

### PSKR1 AND THE FORMATION OF MICROENVIRONMENTS

Phytosulfokine (PSK) was first discovered as a secreted sulfated pentapeptide promoting growth in cell cultures and the receptor via ligand-based affinity chromatography (Matsubayashi and Sakagami, 1996; Matsubayashi et al., 2002). Characterization of PSK:PSKR1 ligand-receptor interactions has shown that they have extensive roles in plant growth and development (Wheeler and Irving, 2010; Matsubayashi, 2014; Sauter, 2015). PSKR1 is a member of the leucine-rich repeat receptor like kinase family that typically contain a large extracellular ligandbinding domain composed of leucine-rich repeats, a single transmembrane spanning domain and an intracellular catalytic kinase domain (Matsubayashi et al., 2002). There are five genes encoding PSK that are expressed in different tissues of the plant (Matsubayashi et al., 2006). Active PSK needs to be sulfated and this is achieved by tyrosylprotein sulfotransferase (TPST) found in the Golgi apparatus. Genetic approaches have been a powerful tool used to study PSK:PSKR interactions in plants and much of this work has been carried out in Arabidopsis. All sulfated residues are removed in tpst mutants as TPST is the single enzyme catalyzing sulfation of tyrosine residues in Arabidopsis,

while triple knockouts of pskr1, pskr2 and pysr1 are used to create the null pskr receptor background. These plants show defects in growth and development. Specifically, the pskr null background has reduced root and shoot growth and revealed that the PSK:PSKR receptor system is involved in promoting root and shoot growth in addition to roles in development of xylem vessels and pollen tubes (Sauter, 2015). Analysis of plant pathogen interactions has revealed that PSKR also has roles in protecting plants. While plant growth is promoted by PSK, pattern-triggered immune responses such as those activated by the biotrophic bacteria Pseudomonas syringae are attenuated by PSK (Igarashi et al., 2012). Interestingly, although the pskr or tpst null mutants are more resistant to biotrophic pathogens such as P. syringae, the oomycete Hyaloperonospora arabidopsidis and the nematode Meloidogyne incognita, they are more susceptible to necrotrophic pathogens such as the fungus Alternaria brassicicola (Mosher et al., 2013; Rodiuc et al., 2016). One of the features biotrophic pathogens have in common is that on penetrations of host cells, they stimulate the formation of specialized cell structures (e.g., haustoria) and it appears that these pathogens have co-opted the PSK signaling system to promote cell differentiation (Rodiuc et al., 2016). Moreover, PSK is expressed in nodules in Lotus japonicus and application of exogenous PSK increases nodule numbers (Wang C. et al., 2015) where rhizobia may be co-opting PSK in the formation of the specialized nodule.

Binding of PSK to PSKR1 stimulates allosteric changes throughout the receptor resulting in heterodimerization interactions with other integral membrane proteins such as the somatic embryogenesis receptor-like kinase (SERKs) including BRI1-associated receptor kinase (BAK1)/SERK3 (Wang J. et al., 2015). Leucine-rich repeat receptor like kinase homo- and heterodimerization is well-established following the characterization of the brassinosteroid receptor brassinosteroid insensitive 1 (BRI1) (Clouse, 2011). Similarly, the damage ligand, AtPep1, binds to its leucine-rich repeat receptor like kinase, PEPR1, and causes heterodimerization with BAK1 (Tang et al., 2015). BAK1 is a promiscuous molecule that was first discovered associated with BRI1 but also interacts with many other LRR RLKs (Chinchilla et al., 2009). Life time fluorescence imaging revealed that PSKR1 interacted with H+-ATPases AHA1 and AHA2 and also BAK1 to form a receptor complex (Ladwig et al., 2015). This complex involving PSKR1, BAK1, AHA1, and AHA2 also associates with the cyclic nucleotide-gated cation channel 17 (CNGC17) although PSKR1 does not directly bind to it (Ladwig et al., 2015). Since CNGC17 is regulated by calmodulin and cGMP (Zelman et al., 2012; Fischer et al., 2013, 2017), there is a possibility that a localized receptor complex microenvironment involving calcium ions and possibly cyclic nucleotides such as cGMP is generated (**Figure 2**).

In vitro studies provided the first clues that PSKR1 may form subcellular microenvironments containing cGMP. Within the kinase domain of PSKR1 is a sequence motif predictive of a guanylate cyclase center (Kwezi et al., 2007). Studies using recombinant cytoplasmic domains of PSKR1 revealed that the protein could indeed produce cGMP albeit at low levels that were enhanced with additional calcium ions (Kwezi et al., 2011; Muleya et al., 2014, 2016). Both BRI1 and PEPR1 also have a similar guanylate cyclase center that can generate cGMP (Kwezi et al., 2007; Qi et al., 2010; Wheeler et al., 2017). Initial studies used a small recombinant fragment of the BRI1 kinase domain containing the guanylate cyclase center that showed cGMP generation (Kwezi et al., 2007) although a later study using most of the kinase domain failed to show cGMP production (Bojar et al., 2014). However, a more sensitive detection method has since demonstrated cGMP production by the full length BRI1 kinase domain (Wheeler et al., 2017). Notably, this recombinant protein also contained the N and C terminal regions necessary for homodimerization (Bojar et al., 2014) that has been predicted to be necessary for the catalytic conversion of GTP to cGMP (Freihat et al., 2014; Muleya et al., 2016). Arabidopsis mesophyll protoplasts treated with exogenous application of PSK generated increased cGMP levels compared to the controls treated with the non-sulfated backbone PSK pentapeptide (Kwezi et al., 2011). Furthermore, transfection of protoplasts with full-length PSKR1 considerably raised basal levels of cGMP (Kwezi et al., 2011). In addition, the ligand for PEPR1, Pep1, stimulates intracellular increases in cGMP measured using an in vivo cGMP reporter in root cells (Ma et al., 2012). Within the catalytic guanylate cyclase center, G924 in PSKR1 is predicted to have roles in determining substrate specificity for GTP (Sunahara et al., 1998; Tucker et al., 1998; Wong and Gehring, 2013; Wong et al., 2015). When glycine is mutated to lysine (G924K), cGMP production is reduced in vitro (Kwezi et al., 2011; Muleya et al., 2014). Complementation studies with this full length mutant in the pskr null background showed that it could not restore root growth (Ladwig et al., 2015). However, although the G924K mutant did not significantly impair phosphorylation of the SOX substrate (Muleya et al., 2014), it was unable to phosphorylate myelin basic protein (Kaufmann et al., 2017), so questions arise about its ability to properly phosphorylate PSKR1 downstream substrates in vivo.

Phosphorylation has long been recognized as a means of regulation of proteins as the number and specific residues phosphorylated create ionic enriched micro-environments. Like BRI1, PSKR1 is a dual kinase with the ability to phosphorylate tyrosine as well as threonine and serine residues (Oh et al., 2009; Muleya et al., 2016) and has a complex autophosphorylation profile involving dimerization (Hartmann et al., 2015; Muleya et al., 2016). Complementation studies with the PSKR1 kinase inactive mutant K762E in the pskr null background demonstrated that kinase activity is essential for root and shoot growth (Hartmann et al., 2014). The phosphorylation status of PSKR1 is important in regulating the guanylate cyclase activity as well as its kinase activity. For instance, phosphorylated mimetic mutations of phosphorylated serine residues located at the juxtamembrane region enhance kinase activity in vitro and also have differential effects on growth, promoting growth in the root but not the shoot (Muleya et al., 2016; Kaufmann et al., 2017). Interestingly, the mimetic non-phosphorylated mutation decreases kinase activity and both mutations are associated with a lack of guanylate cyclase activity, whereas other mutations that modify kinase activity (Y888E or Y888F) have little effect on guanylate cyclase activity (Muleya et al., 2016). These differences may be related to the ability of the phospho-mimetic mutants to form homodimers,

indicated by a dashed arrow. For further details see the text. AHA, H+-ATPases; BAK1, BRI1 associated kinase; CNGC, cyclic nucleotide-gated channel; PSK,

which are important at least in the tyrosine kinase activity and potentially in the guanylate cyclase activity (Muleya et al., 2016).

phytosulfokine; PSKR1, phytosulfokine receptor.

There appears to be considerable intramolecular crosstalk occurring as not only is kinase activity associated with specific residues being available for phosphorylation, but it is decreased by the guanylate cyclase product cGMP. Thus PSKR1 can generate an enriched environment of cGMP that in turn suppresses its predominant kinase function. The PSKR1 receptor complex also includes CNGC17 (Ladwig et al., 2015) which can be regulated by cGMP. Together these findings suggest that cGMP acts as a regional traffic signal within the PSKR1 receptor complex (**Figure 2**). BRI1 also displays a complex autophosphorylation status that impacts on its effects on growth (Clouse, 2011). Like PSKR1, cGMP inhibits kinase activity in BRI1 and certain kinase inactive mutants no longer generate any cGMP (Kwezi et al., 2011; Wheeler et al., 2017). To date, it is not known how cGMP inhibits kinase activity of PSKR1 or BRI1 but it is potentially by binding at intracellular allosteric sites on the receptors. Following activation of PSKR1 and PePR1 and association with BAK1, they are then internalized by a clathrindependent pathway that is important in sustaining immune responses (Mbengue et al., 2016; Rodiuc et al., 2016).

Intracellular calcium ion concentration is tightly controlled to ensure that cells can rapidly respond to specific patterns of spatial and temporal changes in calcium ion levels (Ehrhardt et al., 1996; Sanders et al., 2002; Kudla et al., 2010; Steinhorst and Kudla, 2013; Chen et al., 2015; Yuan et al., 2017). Changes in calcium ion concentration begin at localized points in the cell via influx from external sources that in turn can amplify release via both internal and further external sources generating calcium ion waves (Tuteja and Mahajan, 2007). Calcium ion concentration is returned to basal levels via various internalization mechanisms. In vitro studies have shown that PSKR1 can directly respond to physiological calcium ion concentrations of 0.1–10 µM via a reversible inhibition of kinase activity (Muleya et al., 2014). Notably at these same concentrations, guanylate cyclase activity is enhanced and this appears to be a reciprocal effect as lower calcium ion concentrations are associated with high kinase activity (Muleya et al., 2014). It is possible that even higher levels of calcium ions override this effect since 100 µM did not inhibit kinase activity using myelin basic protein as a substrate (Kaufmann et al., 2017). PSKR1 also contains a predicted calmodulin binding site within its kinase domain that interacts with calmodulins (Hartmann et al., 2014). Although complementation studies using PSKR1 W831S mutants in the pskr null background suggest that calmodulin binding is necessary for growth responses (Hartmann et al., 2014) it has since been identified that this mutation removes kinase activity (Kaufmann et al., 2017). There is a need to investigate if specific PSK signaling modulates changes in calcium ion concentrations and how these may affect the receptor and immediate surrounding microenvironment.

### CONCLUSION AND FUTURE QUESTIONS

We argue that small amplitude signals have a critical part to play in plant homeostasis and that these begin with the development of micro-signaling environments within the cytoplasm that set up the potential for specific signal cascades. Such cascades are likely to exhibit skewed subcellular distribution of the moonlighting proteins and gradients of their small molecule products. Growing pollen tubes in fact have marked distribution gradients in calcium ions that are independent of cytoplasmic streaming and diffusion (Tuteja and Mahajan, 2007). Advances in spatial and concentration level detection methods will enable demonstration of the skewed distribution of the moonlighting proteins and their products which are restricted to small defined areas possibly due to affinity with other molecules that prevents their diffusion.

We have focused on how PSK signals via the PSKR1 receptor to generate a complex series of cross-talk situations on PSKR1 itself involving phosphorylation, cGMP and calcium ions that also influence other proteins such as CNGC17 present in the receptor complex (**Figure 2**). Ligand activated PSKR1 raises cGMP levels that in turn activate CNGC17 increasing calcium ion influx. The consequences of activating this moonlighting protein system are twofold. Firstly, increases in calcium ion concentration potentiate cGMP production amplifying the signal. Secondly, increases in calcium reduce kinase activity of PSKR1 (and its downstream signal cascades). However, there may be increases in activity of other kinases that are dependent on cGMP and/or calcium ion and/or calmodulin. If this is the case, then the moonlighting action of PSKR1 would be a tuner switch for two or more distinct kinase dependent cascades. How changes in calcium ions and cGMP modulate PSK signaling is not clear and will be subject of future investigation. An area that is particularly worth focusing on is how PSK signaling is modulated and switched from growth promotion, to specialized cell development and/or defense responses and the role of subcellular microenvironments in

### REFERENCES


these pathways. In conjunction with these questions, we need to consider the role of phosphodiesterase and suppressors of other signaling molecules that contribute to changes in cellular microenvironments.

We predict that changes at the intracellular microenvironmental level are likely to affect more than homeostasis of individual proteins and will actually have an important part in initiating cellular signaling pathways to maintain plant function in response to rapidly changing environmental conditions and stresses. Understanding the roles of cellular microenvironments is a current focus in diverse research areas as it is now evident that the location of ribosomes influences the mRNA that will be translated and the post-translation modifications that follow (Shi et al., 2017; Simsek et al., 2017).

### AUTHOR CONTRIBUTIONS

HI and CG conceived the perspective and drafted the manuscript. All authors revised the manuscript, agreed to content, and approved the final version.




**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Irving, Cahill and Gehring. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Magnesium Transporter MGT6 Plays an Essential Role in Maintaining Magnesium Homeostasis and Regulating High Magnesium Tolerance in Arabidopsis

Yu-Wei Yan1,2, Dan-Dan Mao3,4, Lei Yang<sup>3</sup> , Jin-Liang Qi1,3, Xin-Xin Zhang1,5 , Qing-Lin Tang1,6, Yang-Ping Li1,2, Ren-Jie Tang<sup>1</sup> \* and Sheng Luan<sup>1</sup> \*

<sup>1</sup> Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, United States, <sup>2</sup> Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, Maize Research Institute of Sichuan Agricultural University, Chengdu, China, <sup>3</sup> Nanjing University–Nanjing Forestry University Joint Institute for Plant Molecular Biology, State Key Laboratory for Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, Nanjing, China, <sup>4</sup> College of Life Sciences, Hunan Normal University, Changsha, China, <sup>5</sup> Key Laboratory of Saline-Alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Alkali Soil Natural Environmental Science Center, Northeast Forestry University, Harbin, China, <sup>6</sup> Key Laboratory of Horticulture Science for Southern Mountainous Regions, Southwest University, Chongqing, China

### Edited by:

Kai He, Lanzhou University, China

### Reviewed by:

Dai-Yin Chao, Shanghai Institutes for Biological Sciences (CAS), China Caiji Gao, South China Normal University, China

### \*Correspondence:

Ren-Jie Tang rjtang@berkeley.edu Sheng Luan sluan@berkeley.edu

### Specialty section:

This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science

Received: 04 January 2018 Accepted: 16 February 2018 Published: 12 March 2018

### Citation:

Yan Y-W, Mao D-D, Yang L, Qi J-L, Zhang X-X, Tang Q-L, Li Y-P, Tang R-J and Luan S (2018) Magnesium Transporter MGT6 Plays an Essential Role in Maintaining Magnesium Homeostasis and Regulating High Magnesium Tolerance in Arabidopsis. Front. Plant Sci. 9:274. doi: 10.3389/fpls.2018.00274 Magnesium (Mg) is one of the essential nutrients for all living organisms. Plants acquire Mg from the environment and distribute within their bodies in the ionic form via Mg2+ permeable transporters. In Arabidopsis, the plasma membrane-localized magnesium transporter MGT6 mediates Mg2<sup>+</sup> uptake under Mg-limited conditions, and therefore is important for the plant adaptation to low-Mg environment. In this study, we further assessed the physiological function of MGT6 using a knockout T-DNA insertional mutant allele. We found that MGT6 was required for normal plant growth during various developmental stages when the environmental Mg2<sup>+</sup> was low. Interestingly, in addition to the hypersensitivity to Mg2<sup>+</sup> limitation, mgt6 mutants displayed dramatic growth defects when external Mg2<sup>+</sup> was in excess. Compared with wild-type plants, mgt6 mutants generally contained less Mg2<sup>+</sup> under both low and high external Mg2<sup>+</sup> conditions. Reciprocal grafting experiments further underpinned a role of MGT6 in a shoot-based mechanism for detoxifying excessive Mg2<sup>+</sup> in the environment. Moreover, we found that mgt6 mgt7 double mutant showed more severe phenotypes compared with single mutants under both low- and high-Mg2<sup>+</sup> stress conditions, suggesting that these two MGT-type transporters play an additive role in controlling plant Mg2<sup>+</sup> homeostasis under a wide range of external Mg2<sup>+</sup> concentrations.

Keywords: Mg2<sup>+</sup> transporter, Mg2<sup>+</sup> homeostasis, Arabidopsis, MGT6, MGT7

## INTRODUCTION

Magnesium (Mg) is an essential macronutrient for plants. Being the most abundant free divalent cation in living cells, Mg2<sup>+</sup> serves as a counter ion for nucleotides and a central metal for chlorophylls, and acts as a cofactor for many enzymes in catalytic processes. Mg2<sup>+</sup> also contributes to membrane stabilization and active conformation of macromolecules (Shaul, 2002).

**16**

Both low and high levels of Mg present in the soil are deleterious to plant growth, thus affecting crop production. Due to unbalanced application of chemical fertilizers, plants may exhibit Mg deficiency symptoms in the presence of high levels of other cations such as calcium (Ca2+) and potassium (K+) in the soil (Hermans et al., 2013). Moreover, excessive aluminum (Al3+) in acidic soils or other heavy metals severely inhibit the uptake of Mg2+, resulting in Mg deficiency in the plants. These problems lead to reduction in crop yield as well as higher susceptibility to some plant diseases. On the other hand, high levels of Mg are found in serpentine soils featuring a low Ca/Mg ratio (Brady et al., 2005). Genome sequencing of Arabidopsis lyrata plants grown in serpentine or non-serpentine habitats has identified a number of polymorphisms associated with Ca2<sup>+</sup> and Mg2<sup>+</sup> transport (Turner et al., 2010). Although it is critical for plant cells to maintain an optimal Mg2<sup>+</sup> level for normal growth and development, the transport and regulatory mechanisms that govern Mg2<sup>+</sup> acquisition, distribution, and reallocation are poorly understood (Tang and Luan, 2017).

In bacterial cells, there are at least three distinct types of membrane proteins CorA, MgtE, and MgtA/B that are capable of transporting Mg2+. While the MgtE channel and the P-type ATPases MgtA/B do not seem to have any close homologs in plants, there is a major family of Mg2<sup>+</sup> transporters (MGTs) related to bacterial CorA proteins (Li et al., 2001). They are also named as "MRS2s" based on the ability to rescue the yeast mrs2 mutant lacking the Mrs2 protein, a yeast homolog of CorA-type transporter that mediates Mg2<sup>+</sup> transport into the mitochondrial matrix (Schock et al., 2000). The CorA-family proteins feature a unique topology with two closely spaced, C-terminal transmembrane (TM) domains, the first of which contains a conserved GMN (Gly-Met-Asn) tripeptide motif that is essential for Mg2<sup>+</sup> transport (Szegedy and Maguire, 1999). Crystal structure of the Thermotoga maritima CorA establishes the protein as a pentameric cone-shaped ion channel (Eshaghi et al., 2006; Lunin et al., 2006).

Several members of the Arabidopsis MGTs facilitate Mg2<sup>+</sup> transport in bacteria or yeast (Li et al., 2001, 2008; Mao et al., 2008, 2014; Gebert et al., 2009). Genes coding for MGT-type transporters are widely expressed in various plant tissues and cell types in Arabidopsis (Li et al., 2001; Gebert et al., 2009) and the proteins are targeted to plasma membrane or intracellular membranes, implicating MGT members functioning in Mg2<sup>+</sup> transport across multiple cellular membranes. MGT1 is mainly expressed in the root hair and the elongation zone as well as the vascular tissues and leaf trichomes (Gebert et al., 2009), suggesting a potential role in Mg2<sup>+</sup> translocation in these particular cell types. MGT2 and MGT3 are associated with vacuolar membrane and possibly involved in Mg2<sup>+</sup> homeostasis in leaf mesophyll cells (Conn et al., 2011). Quite a few MGTs including MGT4, MGT5, and MGT9 are highly expressed in pollen and anther cells, and are required for plant reproduction, suggesting that active Mg2<sup>+</sup> transport is critical for pollen development (Li et al., 2008, 2015; Chen et al., 2009; Xu et al., 2015). MGT10 is localized in the chloroplast envelope, and is strongly expressed in the rosette and cauline leaves, indicating its possible function in Mg2<sup>+</sup> translocation into chloroplasts (Drummond et al., 2006). Indeed, two recent studies confirmed that mutant plants lacking MGT10 show defects in chloroplast development and plant photosynthesis (Liang et al., 2017; Sun et al., 2017). In rice, OsMGT1 is localized to the plasma membrane and its rapid up-regulation upon Al3<sup>+</sup> stress confers Al3<sup>+</sup> tolerance on rice plants as a result of enhanced Mg2<sup>+</sup> uptake (Chen et al., 2012). Interestingly, OsMGT1 plays a role in rice salt tolerance possibly through activating the transport activity of OsHKT1;5 (Chen et al., 2017).

Among all the MGT-type Mg2<sup>+</sup> transporters in Arabidopsis, MGT6 and MGT7 are thought to be more directly involved in controlling cellular Mg2<sup>+</sup> homeostasis because impairment of MGT6 or MGT7 function renders Arabidopsis plants hypersensitive to low-Mg conditions (Gebert et al., 2009; Mao et al., 2014; Oda et al., 2016). MGT6 appears to be localized to the plasma membrane and mediate the high-affinity Mg2<sup>+</sup> uptake via roots (Mao et al., 2014). Consistent with this role, expression of MGT6 is dramatically up-regulated at the transcriptional level when external Mg2<sup>+</sup> becomes limited (Mao et al., 2014). MGT7 is preferentially expressed in roots, and also plays an important role for plant adaptation to low-Mg conditions although the mechanism is not clear (Gebert et al., 2009). In this study, we showed that MGT6 is equally important for controlling plant Mg2<sup>+</sup> homeostasis under normal and high-Mg conditions. We uncovered a shoot-based mechanism that underlies MGT6 function in detoxifying excessive Mg2+, in addition to its role in root Mg2<sup>+</sup> uptake under Mg-limited conditions. Furthermore, by analyzing the mgt6 mgt7 double mutant, we showed that these two Mg2<sup>+</sup> transporters MGT6 and MGT7 play an overlapping role in maintaining essential Mg2<sup>+</sup> homeostasis under a wide range of external Mg2<sup>+</sup> concentrations.

### MATERIALS AND METHODS

### Plant Materials and Growth Conditions

Arabidopsis thaliana ecotype Col-0 was used in this study. T-DNA insertional mutant lines were obtained from the Arabidopsis Biological Resource Center. The seed stock IDs are as follows: SALK\_205483 (mgt6) and SALK\_064741 (mgt7). The double mutant mgt6 mgt7 was generated by crossing mgt7 to mgt6 mutant, and progeny of F2 generation was screened for double homozygous mutations in MGT6 and MGT7 using a PCR-based genotyping approach.

Wild-type and mutant plants were grown in the soil at 22◦C under the 16-h-light/8-h-dark condition in the greenhouse. Hydroponically grown plants were generally kept in the 1/6 strength MS solution under the short-day condition (8-hlight/16-h-dark) in the greenhouse. Fresh liquid solutions were replaced twice a week.

### Phenotypic Assays

Arabidopsis seeds of different genotypes were sterilized with 10% bleach for 5 min and washed in sterilized water for 3 times. Seeds were sown on the solid plates supplemented with different concentrations of Mg2+. The basal medium contained 1/6 strength of MS salt (Murashige and Skoog, 1962) in which MgSO<sup>4</sup>

was replaced by the K2SO4. Different concentrations of MgCl<sup>2</sup> were added as the Mg2<sup>+</sup> source. After 2-day stratification at 4◦C, plates were vertically grown at 22◦C in the growth chamber.

For the post-germination assay, seeds were first sown on MS medium solidified with 1% phytoagar. After germination, 5-dayold seedlings were transferred onto 1/6 Mg2+-free MS medium (containing 1% sucrose, pH = 5.8, solidified with 0.8% agarose) supplemented with Mg2<sup>+</sup> at the indicated concentrations.

For phenotypic assay in the hydroponics, 7-day-old seedlings were transferred to liquid solutions containing 1/6 MS salts supplemented with 1.25 mM MgSO4. After 2-week culture, the plants were treated with solutions containing different concentrations of Mg2+.

### Functional Complementation

For complementation of the mgt6 mutant, a 3.5-kb genomic fragment including the MGT6 coding region as well as 1.5 kb of the 5<sup>0</sup> flanking DNA upstream of the starting codon was amplified by PCR from Arabidopsis genomic DNA with forward (5<sup>0</sup> -AC GGATAAATGTGGGGATGCTTG-3<sup>0</sup> ) and reverse (5<sup>0</sup> -CCAA ATCAAATCAACCCATAAAC-3<sup>0</sup> ) primers. The PCR product was cloned into the SmaI site of the binary vector pCAMBIA1300. After sequencing, the construct was transformed into Agrobacterium tumefaciens strain GV3101 and introduced into mgt6 mutant plants by the floral dip method (Clough and Bent, 1998). Transgenic seeds were screened on MS medium supplemented with 25 mg/L hygromycin. Resistant seedlings were transplanted to soil and grown in the greenhouse for seed propagation. T3 homozygous transgenic plants were subject to gene expression analysis and phenotypic assays together with wild-type plants and mgt6 mutants.

### RNA Isolation and Gene Expression Analysis

Total RNA was extracted from plant materials using the TRIzol reagent (Invitrogen). After being digested by DNase I (Invitrogen) to decontaminate DNA, cDNA was generated from RNA samples at 42◦C using SuperScript II reverse transcriptase (Invitrogen). The resultant cDNA samples were used for PCR amplification with the gene-specific primers. Quantitative real-time PCR was performed on the DNA Engine Opticon System (MJ Research) using the SYBR Green Realtime PCR Master Mix to monitor double-stranded DNA products. Data were calculated based on the comparative threshold cycle method. The relative expression of each Mgstarvation marker gene was double-normalized using the housekeeping gene ACTIN2 and using the control expression values measured in the wild type when external Mg2<sup>+</sup> is 1.5 mM.

### Grafting Experiments

Reciprocal grafting experiments were performed as previously described with minor modifications (Marsch-Martínez et al., 2013). Seeds were sown on MS medium containing 1% agar and 2% sucrose, and grown vertically in the growth chamber (22◦C, 14-h-light/10-h-dark) after 2-day stratification at 4◦C. Six-day-old Arabidopsis seedlings were transversely cut with a sharp blade in the middle position of the hypocotyl so that each individual seedling was divided into two parts. Subsequently, different parts of each material were re-assembled and grafted on half MS medium supplemented with 1.2% agar, 0.5% sucrose, 3 mg/L Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate], 0.02 mg/L IAA (indole acetic acid) and 0.04 mg/L 6-BA (6-benzylaminopurine). The grafted seedlings were grown vertically in the growth chamber for another 10 days to allow the formation of the graft union. Successfully unified seedlings with the same size and status were then transferred to the hydroponic culture for further experiments.

### Measurement of the Mg and Ca Content

Plant samples were harvested from root and shoot tissues, respectively, and briefly washed with ddH2O for 10 s. The samples were then thoroughly dried up in the oven at 80◦C. The dry matters were collected in the 15 mL centrifuge tubes (ions free) and digested with 1 mL ultrapure HNO<sup>3</sup> (Sigma-Aldrich) in the water bath at 95◦C for 4 h. Digested samples were diluted to the appropriate concentrations with ddH2O, and the elemental concentrations were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES; PerkinElmer, Waltham, MA, United States).

### RESULTS

### Knockout Mutation in MGT6 Leads to Plant Hypersensitivity to Mg Deficiency

In a previous study, we have shown that knock-down of MGT6 in transgenic plants by RNA interference resulted in growth retardation under low-Mg conditions (Mao et al., 2014). To further address the physiological role of MGT6, we isolated a previously unidentified T-DNA insertional mutant from the SALK collection (SALK\_203866), in which the T-DNA insertion is located in the third exon of MGT6, 39 base pair (bp) upstream of the stop codon (**Figure 1A**). RT-PCR analyses showed that full-length MGT6 transcript was not detectable in the mgt6 mutant, while MGT4 gene located in the same chromosome is normally expressed (**Figure 1B**). Consistent with earlier findings, mutation in MGT6 leads to hypersensitivity to Mg limitation in that the mgt6 mutants experienced growth defects at the germination stage (**Figures 1C–F**). When germinated on the medium containing no Mg2<sup>+</sup> or 0.01 mM Mg2+, the mgt6 mutants showed shorter roots and smaller and pale cotyledons (**Figures 1C,D**). In the presence of 0.25 mM Mg2+, mgt6 seedlings appeared more normal, albeit still smaller than the wild-type (**Figure 1E**). Early seedling establishment during germination became comparable between wild-type and mutant plants when external Mg2<sup>+</sup> reached 1.5 mM (**Figure 1F**). Statistical analysis of root length (**Figure 1G**) and seedling fresh weight (**Figure 1H**) verified the hypersensitivity to Mg deficiency in the mgt6 mutant.

Because MGT-type transporters are capable of transporting several divalent cations in bacteria and yeast (Li et al., 2001; Mao et al., 2008), we examined the growth of mgt6 mutant in the absence of other divalent cation nutrients. Whereas mgt6 consistently displayed growth defects in the absence of Mg2+, seedling growth appeared indistinguishable between wild type and mgt6 on the medium lacking other divalent cations including Ca2+, Fe2+, Mn2+, and Zn2<sup>+</sup> (Supplementary Figure S1). These data suggest that under physiological conditions MGT6 may function in plants to cope with variable external Mg status, but is not relevant to other divalent cations.

### MGT6 Is Required for Plant Growth in Arabidopsis Under a Wide Range of External Mg2<sup>+</sup> Concentrations

To extend the phenotypic analysis of the mgt6 mutant, we grew the seedlings of the mutant together with the wildtype plants on the plates containing various levels of Mg2<sup>+</sup> in the post-germination assay. When grown on the low-Mg medium containing 0, 0.01, 0.05, or 0.25 mM Mg2+, the mgt6 mutant plants were clearly stunted as compared with Col-0 (**Figure 2A**); the primary roots were shorter (**Figure 2B**) and the seedling fresh weight was significantly reduced (**Figure 2C**). Because Mg2<sup>+</sup> is the central structural cation for chlorophyll, we analyzed the chlorophyll content in the young leaves and found that the mutant had a lower chlorophyll level under extremely low-Mg conditions (0, 0.01, and 0.05 mM Mg2+; **Figure 2D**). When the medium Mg2<sup>+</sup> levels reached a moderate range (0.75, 1.25, and 3 mM), the growth of mgt6 mutants appeared comparable to that of wild-type (**Figure 2A**), although primary root length or seedling fresh weight was slightly affected (**Figures 2B,C**). Notably, in the presence of 6 mM Mg2<sup>+</sup> that is regarded as high, the mgt6 seedlings exhibited a strong growth defect (**Figure 2A**), with much lower fresh weight (**Figure 2C**) and reduced chlorophyll content (**Figure 2D**) than wild-type plants. These data suggested that the mgt6 mutant is not only

compromised under low-Mg levels but also hypersensitive to high-Mg stress.

To verify the observed phenotypes in the mgt6 mutant resulted from MGT6 mutation, we conducted a complementation test. A genomic fragment of MGT6 was introduced into the mgt6 mutant. Several homozygous transgenic lines with a similar MGT6 transcript level to that in wild type were obtained (Supplementary Figure S2B). Phenotypic analysis of two representative lines showed that seedling growth defects were fully rescued under both low- and high-Mg conditions (Supplementary Figure S2), suggesting MGT6 is indeed required for plant adaptation to Mg deficiency as well as plant tolerance to high-Mg stress.

To assess the function of MGT6 in mature plants, we grew wild-type and mgt6 plants to flowering stage in the hydroponic solutions with defined levels of external Mg2+. We found that the mgt6 plants showed compromised growth in all conditions tested (**Figure 3A**), but the growth difference was much more pronounced between wild-type and mgt6 plants under extremely low (0.01 and 0.05 mM) and high-Mg2<sup>+</sup> (10 mM) conditions, as revealed by the root and shoot biomass (**Figures 3B,C**). These results suggest that MGT6 is essential for plant growth at all developmental stages under a wide range of Mg2<sup>+</sup> concentrations in the environment, and particularly plays an important role in plant adaption to low- and high-Mg2<sup>+</sup> stresses.

### MGT6 Controls Plant Mg2<sup>+</sup> Homeostasis in Both Root and Shoot Tissues

In order to investigate how plant Mg2<sup>+</sup> homeostasis is affected by loss of MGT6 function under various conditions, we measured metal content in the roots and shoots of the wild type and mgt6. We first employed the plant materials cultivated in vitro after 2 weeks' growth on the plates. As expected, compared with wildtype plants, we observed a dramatic decrease in Mg content in both roots and shoots of mgt6 mutants grown under low (0.01 mM) Mg conditions (**Figure 4A**). In the presence of normal (1.5 mM) and high (6 mM) external Mg2<sup>+</sup> levels, mgt6 mutants also contained less Mg in both roots and shoots than wild-type plants, when the seedlings were grown on the plates (**Figure 4A**). Because Ca is usually associated with Mg homeostasis, we also measured Ca content in the plants. While Ca content in the root of mgt6 mutant was slightly higher, we surprisingly found that Mg deficiency resulted in a drastic reduction in shoot Ca compared with wild-type (**Figure 4B**). The Ca content, like other

parameters of plant growth, was comparable between the wildtype and mutant plants grown under 1.5 mM Mg2<sup>+</sup> (**Figure 4B**). The mgt6 mutant retained significantly less Ca in the root and slightly decreased Ca content in the shoot tissue when plants were cultured in 6 mM Mg2<sup>+</sup> (**Figure 4B**).

We further measured the Mg and Ca content in the hydroponically grown mature plants. As the external Mg2<sup>+</sup> levels increased, wild-type plants accumulated elevated amount of Mg in both root and shoot tissues. The mgt6 plants generally showed a significant reduction in root Mg content compared with

wild type, except under 10 mM Mg2<sup>+</sup> (**Figure 4C**). However, the shoot Mg content between wild type and mgt6 is most strikingly different under 10 mM Mg2+, although under some other concentrations of Mg2+, such as 0.05, 0.25, and 1.5 mM, mgt6 mutant also contained lower Mg content in the shoot compared with wild type (**Figure 4C**). Plant Ca contents are negatively correlated with external Mg2<sup>+</sup> levels. Under low-Mg conditions (0.01, 0.05, and 0.25 mM), an obvious elevation in root Ca was observed in the mgt6 mutant (**Figure 4D**), presumably due to the antagonistic interaction between Mg and Ca. These results suggest MGT6 regulates plant Mg2<sup>+</sup> homeostasis in both roots and shoots, and functions in a wide range of external Mg2<sup>+</sup> concentrations at all developmental stages.

### Grafting Assay Uncovers a Shoot-Based Mechanism for MGT6 Function in High-Mg Tolerance

While the low-Mg sensitive phenotype of mgt6 can be explained by impaired Mg2<sup>+</sup> uptake by root under Mg-limited conditions in the mutant, the high-Mg susceptibility of mgt6 remains obscure. Since MGT6 controls both root and shoot Mg2<sup>+</sup> homeostasis, we attempted to further investigate the mechanism by which MGT6 contributes to plant Mg2<sup>+</sup> tolerance. Because MGT6 is widely expressed in plants, we decided to examine the relative contribution of MGT6 in roots versus in shoots through reciprocal grafting experiments between mgt6 mutants and wildtype plants (**Figure 5**). When grown under low-Mg2<sup>+</sup> conditions (0.01 mM), the shoots with wild-type scions and mgt6 rootstocks appeared to be smaller than that of self-grafted wild-type plants, although the root looked similar. The grafted plants with mgt6 scions and wild-type rootstocks were significantly smaller than wild-type self-grafted plants, but generally larger than mgt6 selfgrafted plants. Under the moderate level of Mg2<sup>+</sup> (1.5 mM), both groups of the reciprocal grafted plants grew smaller than wild-type self-grafted plants. However, in the hydroponic culture containing 10 mM Mg2+, which is considered to be a toxic concentration, the grafted plants with mgt6 scions and wildtype rootstocks phenocopied the defects seen in the mgt6 selfgrafts, whereas the grafted plants with wild-type scions and mgt6 rootstocks resembled the phenotype of wild-type selfgrafted plants (**Figure 5A**). We measured root and shoot fresh weight quantitatively, which verified the growth phenotypes (**Figures 5B,C**). These observations suggested that MGT6 is important in both root and shoot tissues when external Mg2<sup>+</sup> is low and moderate. Presumably, MGT6-mediated absorption of external Mg2<sup>+</sup> represents the dominant role under these conditions. When the external Mg2<sup>+</sup> is extremely high, MGT6 function in the shoot becomes critical to detoxify excessive Mg2<sup>+</sup> at the whole plant level. Consistent with this notion, wild-type scions grafted on mgt6 rootstocks lead to significantly lower root Mg2<sup>+</sup> content under 0.01 and 1.5 mM Mg2<sup>+</sup> conditions (**Figures 6A,B**). In the presence of 10 mM external Mg2+, shoots from mgt6 grafted onto wild-type rootstocks retained much less Mg2<sup>+</sup> in the shoot, similar to that observed in self-grafted mgt6 plants (**Figure 6C**). This further supported the idea that MGT6 fulfills a shoot-based mechanism to detoxify excessive Mg2+, which could involve vacuolar Mg2<sup>+</sup> storage based on the observation of lower Mg content in the mutant shoots.

### Functional Synergy of MGT6 and MGT7 in Arabidopsis

Arabidopsis MGT7 encodes a low-affinity Mg2<sup>+</sup> transporter (Mao et al., 2008) and is indispensable for optimal plant growth under low-Mg2<sup>+</sup> conditions (Gebert et al., 2009). To investigate the functional interaction between MGT6 and MGT7, we created a double mutant that lacks both MGT6 and MGT7 transcripts (Supplementary Figure S3A). We found that the mgt6 mgt7 double mutant displayed pronounced growth retardation in the soil (Supplementary Figure S3B). Quantitative analysis indicated that the shoot fresh weight of the double mutant was only half of that of the wild type and single mutants (Supplementary Figure S3C).

We examined the growth phenotype of mgt6 mgt7 double mutant under various external Mg2<sup>+</sup> concentrations, in comparison with wild-type as well as the mgt6 and mgt7 single mutants. While mgt6 single mutants exhibited very strong growth defects under both low- and high-Mg conditions, the phenotype of mgt7 single mutant under the same condition was mild (**Figure 7A**). However, the mgt6 mgt7 double mutant was significantly more sensitive to external Mg2<sup>+</sup> than the mgt6 single mutant (**Figure 7A**). The primary root of mgt6 mgt7 was shorter than that of mgt6 under low-Mg conditions (**Figure 7B**), although seedling fresh weight was comparable (**Figure 7C**). The leaf chlorophyll content in mgt6 mgt7was lower compared with mgt6 when high Mg2<sup>+</sup> is present in the medium (**Figure 7D**).

Gene expression analysis indicated that a handful of gene markers (Kamiya et al., 2012) were more responsive to Mgstarvation in the mgt6 or mgt7 mutant background than in the wild type, suggesting that the mgt6 and mgt7 mutants are impaired in low-Mg2<sup>+</sup> adaptation. Consistent with the more severe phenotype, the mgt6 mgt7 double mutant displayed enhanced expression of Mg-starvation marker genes compared with the single mutants (Supplementary Figure S4). Taken together, these results indicate both MGT6 and MGT7 are important for plant Mg homeostasis and their functions are additive in regulating Mg2<sup>+</sup> transport under a wide range of external Mg2<sup>+</sup> concentrations.

### DISCUSSIONS

In addition to air and water, plant growth and development rely on mineral nutrients taken up by roots and translocated into the shoot tissues through apoplast and symplast pathways, which entail not only transpiration-driven mass flow but also active membrane transport processes facilitated by various ion channels and transporters. Mg is an essential macronutrient in plants with diverse biological functions. However, the molecular mechanisms for Mg transport and homeostasis in plant cells remain largely unknown. Genomes of many plants such as Arabidopsis, rice, and maize, encode homologs of the bacterial CorA-type proteins referred to as MGTs/MRS2s (Li et al., 2001, 2016; Saito et al., 2013). Some members of the MGT family have been functionally

characterized, but the physiological roles of these transporters are not well understood. In Arabidopsis, we previously showed that MGT6 is capable of facilitating high-affinity Mg2<sup>+</sup> uptake from the soil when external Mg2<sup>+</sup> concentration is in the submillimolar range (Mao et al., 2014). Consistent with this role, expression of the MGT6 gene is highly inducible in the root tissues in response to low Mg (Mao et al., 2014). In the present study, we not only corroborated earlier findings regarding the critical role of MGT6 in low-Mg adaptation, but also extended the function of MGT6 in controlling plant Mg2<sup>+</sup> homeostasis within a wide range of external Mg2<sup>+</sup> levels. The mgt6 knock-out mutant displayed obvious phenotype under high-Mg conditions, suggesting MGT6 exerts physiological functions in plants other than Mg2<sup>+</sup> absorption. In higher plants, after absorption from the soil solution by roots, Mg2<sup>+</sup> is believed to be transported to the aerial parts via transpiration stream moving through the xylem vessels. However, little is known about the molecular identity of the transporters involved in this long-distance transport. MGT6 might fulfill such a role in the xylem transport of Mg2+. Considering the negative membrane potential, Mg2<sup>+</sup> is expected to be loaded passively into the pericycle cells. MGT6 may be responsible for Mg2<sup>+</sup> import into pericycle and xylem parenchyma cells. On the other hand, the possibility that MGT6 serves as an "exporter" in this process cannot be excluded. It is generally believed that ion secretion occurs across plasmalemma of the parenchyma cells surrounding the xylem vessels (Clarkson, 1993; Gaymard et al., 1998). Interestingly, MGT5, the closest homolog of MGT6 in Arabidopsis, was shown to be a bidirectional Mg2<sup>+</sup> carrier that operates in a concentration-dependent manner (Li et al., 2008). Therefore, it is possible that MGT6 might also function in Mg2<sup>+</sup> efflux from xylem parenchyma cells, pushing Mg2<sup>+</sup> influx into the xylem vessel. Future electrophysiological analysis of MGT6 conductance is required to test this hypothesis.

Although Mg2<sup>+</sup> is an essential mineral, high levels of Mg2+, such as those in the serpentine soils, could be toxic to plants. Recently we established that vacuolar sequestering of Mg2+, regulated by the tonoplast CBL-CIPK signaling network, is crucial for plants to survive under high-Mg conditions (Tang et al., 2015). In the present work, we uncovered another component mainly fulfilled by MGT6 that underlies high-Mg tolerance at the whole plant level. Previous studies indicate that serpentine-adapted plants appear to efficiently transport Mg2<sup>+</sup> from root to shoot, whereas the serpentine-sensitive counterparts are less capable of driving Mg2<sup>+</sup> entry into the transpiration stream, resulting in a lower Mg2<sup>+</sup> concentration in the shoot (Palm et al., 2012). Consistent with this finding, our physiological analysis of mgt6 mutant under high-Mg conditions showed that mgt6 retained considerably less Mg2<sup>+</sup> in the shoot tissue compared to wild-type, accompanied by the growth retardation upon high-Mg stress. These data support the notion that long-distance Mg2<sup>+</sup> transport mediated by MGT6 may play a critical role in protecting plants from Mg2<sup>+</sup> toxicity at the whole plant level. More importantly, reciprocal grafting test indicated that MGT6 function in the shoot tissue is responsible for the high-Mg2<sup>+</sup> tolerance. Considering the plasma membrane localization of MGT6, it is reasonable to speculate that MGT6 probably facilitates Mg2<sup>+</sup> entry into the cytosol of leaf cells after the Mg2<sup>+</sup> ions in the xylem unload into the apoplastic space. The excessive Mg2<sup>+</sup> in the cytosol is subsequently sequestered into the central vacuole via tonoplastlocalized Mg2<sup>+</sup> transporters. This transport cascade is critical for detoxification of excessive Mg2+, which is reminiscent of a recent model proposed for Ca2<sup>+</sup> detoxification in plants (Wang et al., 2017).

Another notable finding in this study is that another MGTtype transporter MGT7 partially overlaps with the function of MGT6. With a preferential expression in the root, MGT7 was shown to be important for plant adaptation to low-Mg conditions (Gebert et al., 2009). In our study, we found that although MGT6 plays a more dominant role in low-Mg conditions, MGT7 seems to be additive to MGT6 function because mgt6 mgt7 double mutant is more sensitive to low-Mg stress than the mgt6 single mutant, which is also supported by the enhanced activation of Mg-starvation gene expression in the double mutant. Interestingly, under high-Mg conditions, mutation of MGT7 also significantly enhanced the sensitivity of mgt6, although single mutant of mgt7 only exhibited a subtle phenotype under the same condition. These results suggest that MGT7 synergistically works together with MGT6 in the context of Mg2<sup>+</sup> homeostasis at the whole plant level. Further investigations will sort out the mode of action for each of them to explain this functional synergy.

Subcellular localization of the MGT proteins may prove to be difficult to study. For instance, recent studies reported discrepant cellular localizations for MGT6 in the plasma membrane (Mao et al., 2014) and endoplasmic reticulum (ER; Oda et al., 2016), respectively. Several other MGT members such as MGT7 (Gebert et al., 2009) and MGT4 (Li et al., 2015) were also shown to be ER-associated, which needs to be re-evaluated because quite a few membrane proteins tend to be mis-targeted to ER, especially when overexpressed in a transient expression system (Denecke et al., 2012; Quattrocchio et al., 2013; Segami et al., 2014). Future studies using the native promoter, coupled with functional complementation in the mutant background as well as other approaches, are needed to verify the subcellular localization of MGT-type transporters in situ. It will also be interesting to examine if the targeting of MGT6 or MGT7 would be dynamically altered in different subcellular compartments in response to various Mg2<sup>+</sup> concentrations.

As sessile organisms, plants have to cope with fluctuating concentrations of Mg2<sup>+</sup> in nature. How plants maintain a balanced level of Mg2<sup>+</sup> is not well understood. The present study as well as our previous work provides a working model in which MGT6 plays a dual role in controlling Mg2<sup>+</sup> homeostasis. When external Mg2<sup>+</sup> is limited, expression of MGT6 is induced in root epidermal cells and root hairs, making this transporter primarily responsible for Mg2<sup>+</sup> uptake from the soil. When external Mg2<sup>+</sup> is sufficient or becomes excessive, MGT6 mediates Mg2<sup>+</sup> loading into the shoot tissues, where leaf mesophyll cells can subsequently sequester extra amount of Mg2<sup>+</sup> into large vacuoles via yet-unknown transporters. Future efforts

should be made in identifying uncharacterized Mg2<sup>+</sup> transport proteins in plants. Furthermore, establishing the regulators and signaling pathways that fine-tune the expression and function of these transport systems will be a challenging but urgent task, which will ultimately lead to genetic manipulation of plants for precise adaption to the changing Mg2<sup>+</sup> concentrations in the environment.

### AUTHOR CONTRIBUTIONS

Y-WY and R-JT designed and conducted most of the experiments, interpreted the results, and wrote the draft of the manuscript. D-DM, X-XZ, Q-LT, and Y-PL assisted in some experiments and helped analyze the data. LY and J-LQ provided tools and reagents and made helpful discussions. SL supervised and conceptualized the study and finalized

### REFERENCES


the paper. All the authors approved the final version of the manuscript.

### FUNDING

This work was funded by National Science Foundation to SL and National Natural Science Foundation of China (31500200) to D-DM. Y-WY was in part supported by a fellowship from the China Scholarship Council.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.00274/ full#supplementary-material



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Yan, Mao, Yang, Qi, Zhang, Tang, Li, Tang and Luan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# SlCNGC1 and SlCNGC14 Suppress Xanthomonas oryzae pv. oryzicola-Induced Hypersensitive Response and Non-host Resistance in Tomato

#### Xuan-Rui Zhang<sup>1</sup> , You-Ping Xu<sup>2</sup> and Xin-Zhong Cai<sup>1</sup> \*

1 Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China, <sup>2</sup> Center of Analysis and Measurement, Zhejiang University, Hangzhou, China

Mechanisms underlying plant non-host resistance to Xanthomonas oryzae pv. oryzicola (Xoc), the pathogen causing rice leaf streak disease, are largely unknown. Cyclic nucleotide-gated ion channels (CNGCs) are calcium-permeable channels that are involved in various biological processes including plant resistance. In this study, functions of two tomato CNGC genes SlCNGC1 and SlCNGC14 in non-host resistance to Xoc were analyzed. Silencing of SlCNGC1 and SlCNGC14 in tomato significantly enhanced Xoc-induced hypersensitive response (HR) and non-host resistance, demonstrating that both SlCNGC1 and SlCNGC14 negatively regulate non-host resistance related HR and non-host resistance to Xoc in tomato. Silencing of SlCNGC1 and SlCNGC14 strikingly increased Xoc-induced callose deposition and strongly promoted both Xoc-induced and flg22-elicited H2O2, indicating that these two SlCNGCs repress callose deposition and ROS accumulation to attenuate non-host resistance and PAMP-triggered immunity (PTI). Importantly, silencing of SlCNGC1 and SlCNGC14 apparently compromised cytosolic Ca2<sup>+</sup> accumulation, implying that SlCNGC1 and SlCNGC14 function as Ca2<sup>+</sup> channels and negatively regulate non-host resistance and PTI-related responses through modulating cytosolic Ca2<sup>+</sup> accumulation. SlCNGC14 seemed to play a stronger regulatory role in the non-host resistance and PTI compared to SlCNGC1. Our results reveal the contribution of CNGCs and probably also Ca2<sup>+</sup> signaling pathway to non-host resistance and PTI.

Keywords: cyclic nucleotide-gated ion channel (CNGCs), Xanthomonas oryzae pv. oryzicola, PAMP-triggered immunity, non-host resistance, Ca2<sup>+</sup> signaling

### INTRODUCTION

Each pathogen has its own host range. Non-host resistance is triggered when a non-adapted pathogen attempts to infect a plant species outside of its host range. Thus, non-host resistance is widely occurring, durable and broad-spectrum to non-adapted pathogens and is highly potential to be exploited in crop resistance engineering (Schulze-Lefert and Panstruga, 2011; Senthil-Kumar and Mysore, 2013). It has been clear that plant non-host resistance utilizes both preformed and induced defense mechanisms and frequently elicites hypersensitive response (HR)

#### Edited by:

Gerald Alan Berkowitz, University of Connecticut, United States

#### Reviewed by:

Keiko Yoshioka, University of Toronto, Canada Selena Gimenez-Ibanez, Centro Nacional de Biotecnología, Spain

> \*Correspondence: Xin-Zhong Cai xzhcai@zju.edu.cn

#### Specialty section:

This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science

Received: 31 August 2017 Accepted: 19 February 2018 Published: 06 March 2018

#### Citation:

Zhang X-R, Xu Y-P and Cai X-Z (2018) SlCNGC1 and SlCNGC14 Suppress Xanthomonas oryzae pv. oryzicola-Induced Hypersensitive Response and Non-host Resistance in Tomato. Front. Plant Sci. 9:285. doi: 10.3389/fpls.2018.00285

**29**

(Mysore and Ryu, 2004; Senthil-Kumar and Mysore, 2013). The preformed mechanisms generally consist of plant physical and chemical barriers, including antibiotic compounds (Bednarek and Osbourn, 2009; Fan et al., 2011). The induced non-host resistance is elicited after the preformed defense is overcome. As observed for host resistance, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) are often initiated in this layer of defense (Niks and Marcel, 2009; Senthil-Kumar and Mysore, 2013). Efforts to identifiy non-host resistance-related genes resulted in the finding that some genes, such as PLDδ, GOX, SGT1, and NHO1, contribute to both host and non-host resistance in Arabidopsis (Lu et al., 2001; Maimbo et al., 2010; Rojas et al., 2012; Pinosa et al., 2013). Generally speaking, the mechanisms underlying plant non-host resistance are far from well understood.

Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) are two important pathovars of X. oryzae, causing bacterial blight and leaf streak diseases, respectively, in rice (Oryza sativa), which is a staple food in many countries and a model plant for cereal biology (Nino-Liu et al., 2006). It is notable that these two pathovars utilize distinct mechanisms to infect rice leaves. Xoo is a vascular pathogen that enters rice leaves via the hydathodes, while Xoc penetrates rice leaves mainly through stomata or wound sites and colonizes intercellular spaces in the mesophyll (Nino-Liu et al., 2006). During infection of their plant hosts, many strains secrete transcription activator-like (TAL) effectors, which enter the host cell nucleus and activate specific corresponding host genes at effector binding elements (EBEs) in the promoter (Boch et al., 2009; Moscou and Bogdanove, 2009). It has been established that host resistance to Xoo is mostly related to the action of TAL effectors, either by polymorphisms that prevent the induction of susceptibility (S) genes or by executor resistance (R) genes with EBEs embedded in their promoter, and that induce cell death and resistance (Bogdanove et al., 2010; Zhang et al., 2015). Xoc is known to suppress host resistance, and no host R gene has been identified against it (Cai et al., 2017). Compared with the host resistance (Zhang and Wang, 2013), non-host resistance to Xoo and Xoc is much less studied. We have previously identified seven genes required for non-host resistance to Xoo in Nicotiana benthamiana. Among them are a calreticulin and a peroxidase, indicating that oxidative burst and calcium-dependent signaling pathways play an important role in non-host resistance to Xoo (Li et al., 2012). However, molecular mechanisms underlying non-host resistance to Xoc remain largely unknown. Whether the oxidative burst and calcium signaling pathway contribute to the non-host resistance to Xoc as to Xoo awaits examination.

The cyclic nucleotide-gated ion channels (CNGCs) are suggested to be one of the important pathways for conducting Ca2<sup>+</sup> ions in signaling transduction (Talke et al., 2003). They are ligand-gated Ca2+-permeable divalent cation-selective channels that are often localized in plasma membrane, presumptively are activated by direct binding of cyclic nucleotides and are complexly regulated by binding of calmodulin (CaM) to the CaM Binding (CaMB) domain (Chin et al., 2009; Ma et al., 2009; Wang et al., 2013; Gao et al., 2014; DeFalco et al., 2016a,b; Fischer et al., 2017). The plant CNGCs are involved in numerous biological functions varying from plant development and stress tolerance to disease resistance (Kaplan et al., 2007; Qi et al., 2010; Ma and Berkowitz, 2011; Moeder et al., 2011). CNGCs are well conserved among plant species, comprising 20 members in Arabidopsis and 18 members in tomato (Saand et al., 2015a,b). Earlier studies revealed that AtCNGC2, AtCNGC4, AtCNGC11, and AtCNGC12 and their homologs play an important role in disease resistance against various pathogens (Yu et al., 1998; Clough et al., 2000; Balagué et al., 2003; Jurkowski et al., 2004; Yoshioka et al., 2006; Ali et al., 2007; Ma and Berkowitz, 2011; Chin et al., 2013; Fortuna et al., 2015; Saand et al., 2015a). Later, other CNGC members such as SlCNGC1 and SlCNGC14 were also found to contribute to disease resistance (Saand et al., 2015a,b). We found that SlCNGC1 and SlCNGC14 play a negative role in non-host resistance to Xoo in tomato (Saand et al., 2015b). However, whether these SlCNGCs indeed encode functional CNGC channels and whether and how they function in non-host resistance to Xoc in tomato remains further study.

Our data in this study strongly indicate that SlCNGC1 and SlCNGC14 function as Ca2<sup>+</sup> channels and negatively regulate tomato non-host resistance to Xoc through modulating ROS accumulation and callose deposition. Our results provide evidence for the contribution of CNGC-mediated Ca2<sup>+</sup> signaling pathway to non-host resistance.

### MATERIALS AND METHODS

### Plant Growth and Inoculation

Tomato (cv. Money Maker) plants were grown in growth room at 21◦C with16 h light/8 h dark photoperiod. For disease resistance evaluation analyses, tomato plants were inoculated with X. oryzae pv. oryzicola (Xoc). After single colony propagation culture in NA medium, the Xoc bacterial cells were collected and resuspended in ddH2O to 1 × 10<sup>8</sup> cfu mL−<sup>1</sup> . The bacterial suspension was then infiltrated into leaves with sterilized needleless syringe. The infiltration zones were marked immediately after infiltration. The inoculated plants were grown in growth room at 26◦C with16 h light/8 h dark photoperiod.

### Gene Silencing Analyses

The virus-induced gene silencing (VIGS) target fragments of SlCNGC1 (Solyc01g095770.2) and SlCNGC14 (Solyc03g114110.2) were amplified by RT-PCR with gene-specific primers (Supplementary Table S1) and ligated into the TRV-based VIGS vector pYL156, which was subsequently electroporated into Agrobacterium tumefaciens strain GV3101 for VIGS analyses. VIGS analyses were conducted with vacuum infiltration delivery approach as described using recombinant pYL156 with insertion of an eGFP fragment instead of an empty pYL156 as control to alleviate viral symptom (Saand et al., 2015a). At about 3 weeks post agro-infiltration, plants were inoculated with Xoc as described above.

### Detection of Callose Deposition

Callose deposition were stained with aniline blue and visualized in fluorescence microscope. Briefly, the collected leaves were

washed twice with ddH2O and ethanol, respectively, and cleared in acetic acid-ethanol (1:3) for 4 h. After washed twice with ddH2O, the leaves were incubated in aniline blue solution [150 mM KH2PO4, 0.1% (w/v) aniline blue, pH 9.5] for 1 h. The stained leaves were washed with ddH2O and observed in 30% glycerol by fluorescence microscopy.

### Bacterial Number Counting Assays

Bacterial numbers in Xoc-infiltrated leaves of silenced plants were determined as previously reported (Li et al., 2012).

### Detection of ROS

Xoc-inoculated leaves of tomato plants were detached and stained with 3,3-diamino benzidine hydrochloride (DAB) (1mg/mL) as described (Li et al., 2015). Quantitative determination of PAMP-elicited H2O<sup>2</sup> was conducted using a luminol-based approach (Saand et al., 2015a). For each experiment, six tomato leaf disks of 3 mm at diameter from three plants were dipped in distilled water in the light over night. The leaf disks were then transferred into 50 µL of distilled water in a 96-well plate. After addion of the same volume mixture which contains 200 nM luminol (Sigma-Aldrich), 20 µg of horseradish peroxidase and 200 nM flg22, H2O<sup>2</sup> were measured for 35 min as luminescence using a Microplate Luminometer (Titertek Berthold, Germany).

### Calcium Assay

Transient increase of cytosolic Ca2<sup>+</sup> concentration was monitored in the Aequarin transgenic tomato lines. For each experiment, six leaf disks were punched from three plants and vacuum-infiltrated in 12.5 µM coelenterazin h (Sigma) on a 96-well plate for 1 min and then set for 2 h at room temperature. Before measurement, the coelenterazin solution was gently removed and 200 µL of PAMP solution (100 nM flg22) was added to the wells. Luminescence was measured using a Microplate Luminometer (Titertek Berthold, Germany).

### Gene Expression Analyses by qRT-PCR

Five defense-related Ca2<sup>+</sup> signaling genes SlCDPK10 (Solyc11g018610.1), SlCAMTA3 (Solyc04g056270.2), SlCBP60G (Solyc01g100240.2), SlCAM2 (Solyc10g081170.1) and SlCAM6 (Solyc03g098050.2) were subjected to gene expression analyses (Zhao et al., 2013; Rahman et al., 2016b; Wang et al., 2016). Total RNA was isolated by Trizol (TaKaRa, Japan) extraction according to the manufacture's instructions. RNAs were treated with DNase I (TaKaRa, Japan) and then reverse transcribed. Quantitative real time PCR (qRT-PCR) was performed as previously described using the StepOne Real-Time PCR system (Applied Biosystems, United States) with SYBR Green PCR Master Mix (TaKaRa, Japan) (Saand et al., 2015a). 18s rDNA was used as internal control. The accession number and the specific primers for amplification of the analyzed genes were listed (Supplementary Table S1). The expression of target genes relative to control was calculated based on a value of 2−11Ct as recommended by the manufacturer. Meanwhile, semi-qRT-PCR for these genes were conducted in parallel and obtained PCR products were analyzed by agarose gel electrophoresis.

## Statistical Analyses of Data

All experiments were conducted three times independently. The quantitative measurement data were statistically analyzed using SPSS software and represent means ± standard error. Significant difference between mean values was determined with DMRT (p < 0.05).

## RESULTS

### Silencing of SlCNGC1 and SlCNGC14 Enhanced Xoc-Induced Hypersensitive Response and Non-host Resistance

In order to dissect the function of SlCNGC1 and SlCNGC14 in Xoc-induced HR and non-host resistance, we used TRV-based VIGS vector to perform VIGS analyses for these genes. Transcripts of SlCNGC1 and SlCNGC14 genes accumulated to only about 20% of the eGFP silencing control (**Figure 1A**), indicating that SlCNGC1 and SlCNGC14 genes had been effictiently silenced in these plants. Inoculation assays with Xoc in these silencing plants demonstrated that SlCNGC1- and SlCNGC14-silenced leaves showed more severe Xoc-induced HR than the eGFP control leaves. SlCNGC1- and SlCNGC14-silenced leaves showed obvious HR necrosis at 3 h post Xoc inoculation, and exhibited strong HR necrosis at 9 hpi, especially SlCNGC14-silenced leaves, which had turned into desiccative, while the eGFP control plants displayed barely visible and weak HR necrosis at 3 and 9 hpi, respectively (**Figure 1B**). This result indicated that silencing of SlCNGC1 and SlCNGC14 enhanced Xoc-induced HR.

We further counted Xoc bacterial number in the infiltrated leaf areas. The result showed that compared with the eGFP controls, Xoc bacterial number in SlCNGC1- and SlCNGC14-silenced plants decreased significantly by 0.7 orders of magnitude at 9 hpi (**Figure 1C**), indicating that silencing of SlCNGC1 and SlCNGC14 enhanced non-host resistance to Xoc.

Collectively, these results implied that both SlCNGC1 and SlCNGC14 might negatively regulate non-host resistance related HR cell death and non-host resistance to Xoc in tomato.

### Silencing of SlCNGC1 and SlCNGC14 Increased Xoc-Induced Callose Deposition

To probe the mechanisms underlying SlCNGC1- and SlCNGC14 dependent regulation of non-host resistance against Xoc, effect of these CNGC genes on callose deposition upon pathogen infection was examined. Callose deposition was visible in the aniline blue-stained leaves under the fluorescence microscopy. Microscopic observation result showed that SlCNGC1- and SlCNGC14-silenced leaves deposited much more callose than eGFP control leaves (**Figure 2A**). Further quantification revealed that callose deposits in SlCNGC1- and SlCNGC14-silenced leaves was 5.9- and 7.3-fold respectively, as many as in the eGFP control leaves at 4 hpi (**Figure 2B**). This result demonstrates that SlCNGC1 and SlCNGC14 repress the callose deposition upon

plants and the eGFP-control plants was detected by qRT-PCR analyses. (B) Xoc-induced hypersensitive response (HR). Photographs were taken at 3 and 9 hpi. (C) Xoc bacterial number counting. Xoc bacterial numbers were counted in leaf areas that were inoculated with bacterium at 3 and 9 hpi. At least five leaves were tested for each treatment, and the data represent means ± standard error (SE). Significant differences between treatments and the control are indicated by an asterisk (p < 0.05).

pathogen infection to alleviate non-host resistance to Xoc in tomato.

### Silencing of SlCNGC1 and SlCNGC14 Promoted ROS Accumulation

Reactive oxygen species (ROS) is indispensable to X. oryzae pv. oryzae (Xoo)-induced HR and non-host resistance (Li et al., 2015). Thus, we analyzed the effect of SlCNGC1 and SlCNGC14 on the production of hydrogen peroxide (H2O2), one of the primary species of ROS. DAB staining result demonstrated that infiltration areas of SlCNGC1- and SlCNGC14-silenced leaves showed significantly stronger DAB staining than those of control (**Figure 3A**), implying that silencing of SlCNGC1 and SlCNGC14 increased the production of H2O2.

Effect of SlCNGC1 and SlCNGC14 on the PAMP (flg22)-elicited H2O<sup>2</sup> accumulation was further examined using leaf disk assays and was indicated as relative luminescence (RLU). The luminol-based assay showed that flg22-induced H2O<sup>2</sup> in SlCNGC1- and SlCNGC14-silenced leaves culminated to over 4100 RLU, while that in eGFP control leaves was peaked only to 940 RLU (**Figure 3B**), demonstrating that silencing of SlCNGC1 and SlCNGC14 promoted the production of flg22-elicited H2O2.

Together, these results suggest that SlCNGC1 and SlCNGC14 supress the ROS accumulation to attenuate non-host resistance and PTI.

### Silencing of SlCNGC1 and SlCNGC14 Compromised Cytosol Ca2<sup>+</sup> Influx

Some Arabidopsis CNGCs have been proved to be functional Ca2<sup>+</sup> channels (Gao et al., 2014; Wang et al., 2017). To examine whether SlCNGC1 and SlCNGC14 function as Ca2<sup>+</sup> channels,

effect of silencing of these genes on accumulation of cytosolic Ca2<sup>+</sup> elicited by the PAMP flg22 was monitored through leaf disk assays using aequorin transgenic tomato lines. In eGFP control aequorin transgenic plants, flg22-triggered Ca2<sup>+</sup> increased rapidly and peaked to 192 RLU, while those in SlCNGC1 and SlCNGC14-silenced aequorin transgenic plants strongly decreased with the peak value drop to only 97 and 39 RLU, respectively (**Figure 4**). This result indicates that SlCNGC1 and SlCNGC14 function as Ca2<sup>+</sup> channels and negatively regulate non-host resistance and PTI through modulating cytosolic Ca2<sup>+</sup> accumulation.

### Silencing of SlCNGC1 and SlCNGC14 Altered Expression of Defense-Related Ca2<sup>+</sup> Signaling Genes

To obtain a clue to understand how SlCNGC1 and SlCNGC14 genes, as Ca2<sup>+</sup> channel genes, regulate plant disease resistance, we examined effect of silencing of these genes on expression of a set of defense-related Ca2<sup>+</sup> signaling genes to probe the possibility of their involvement in SlCNGC1- and SlCNGC14-mediated resistance regulation. The genes under this expression analysis included two CaM genes SlCaM2 and SlCaM6, a tomato homolog (SlCDPK10) of Arabidopsis calcium-dependent protein kinase gene AtCDPK11, and the tomato homologs (SlCBP60g and SlCAMTA3) of two Arabidopsis CaM-binding transcription factors AtCBP60g and AtCAMTA3. All these genes play an important role in regulating disease resistance (Du et al., 2009; Wang et al., 2009, 2011; Boudsocq et al., 2010; Zhao et al., 2013; Sun et al., 2015; Rahman et al., 2016a,b; Wang et al., 2016). Result of qRT-PCR showed that at 4 h after inoculation with Xoc, SlCNGC1- and SlCNGC14-silenced leaves reduced expression of the negative defense regulatory gene SlCAMTA3 by 2.5-fold, and generally increased expression of the positive defense regulatory genes SlCaM6, SlCDPK10,

was detected using a luminol-based assay in leaf disks of SlCNGC1-, SlCNGC14-silenced plants and the eGFP non-silenced control plants. Data are shown as relative luminol units (RLUs) and represent the mean ± SE of three independent experiments.

and SlCBP60g to different extent, which was much higher in SlCNGC14-silenced leaves than SlCNGC1-silenced leaves. Expression of SlCaM6, SlCDPK10 and SlCBP60g genes in SlCNGC1-silenced leaves was 4.3-, 1.5- and 2.5-fold, respectively, as high as that in the eGFP controls, while these folds were 34.0,

defense-related Ca2<sup>+</sup> signaling genes. Expression of defense-related Ca2<sup>+</sup> signaling genes SlCAM2, SlCAM6, SlCDPK10, SlCAMTA3, and SlCBP60g were examined for SlCNGC1-, SlCNGC14-silenced plants relative to the eGFP non-silenced control plants after inoculation with Xoc at 4 hpi by qRT-PCR with 18s rDNA gene serving as a loading control gene. Data represent the mean ± SE of three independent experiments.

4.5, and 9.1 for that in SlCNGC14-silenced leaves (**Figure 5**). This result implies that SlCNGC1 and SlCNGC14 might modulate the expression of these Ca2<sup>+</sup> signaling genes.

### DISCUSSION

We previously found that two tomato CNGC genes SlCNGC1 and SlCNGC14 play an important role in tomato HR elicited by the rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) (Saand et al., 2015a). Here, we further reveal that these two CNGC genes likely encode functional Ca2<sup>+</sup> channels and negatively affect tomato non-host resistance to the rice bacterial leaf streak pathogen Xoc, through modulating ROS accumulation and callose deposition. The role of Ca2<sup>+</sup> signaling pathway in non-host resistance to Xoc is previously unknown. Our results provide evidence for the contribution of CNGC-mediated Ca2<sup>+</sup> signaling pathway to this non-host resistance.

In plants, only a few CNGCs such as AtCNGC2 and AtCNGC18 have been proved to function as Ca2<sup>+</sup> channels (Gao et al., 2014; Wang et al., 2017). Whether the tomato CNGC genes encode functional Ca2<sup>+</sup> channels remain to be verified. In this study, we demonstrated that silencing of SlCNGC1 and SlCNGC14 significantly reduced or almost abolished cytosolic Ca2<sup>+</sup> accumulation (**Figure 4**). Thus, SlCNGC1 and SlCNGC14 likely function as Ca2<sup>+</sup> channels. Further electrophysiological studies are required to provide more evidences to verify it.

It is recently reported that Arabidopsis cngc2 mutant overaccumulates Ca2<sup>+</sup> in apoplastic compartment and increased HR and resistance (Wang et al., 2017). Similarly, in this study, we found that silencing of SlCNGC1 and SlCNGC14 significantly reduced cytosolic Ca2<sup>+</sup> accumulation and enhanced HR and non-host resistance to Xoc and PTI. Whether silencing of SlCNGC1 and SlCNGC14 results in overaccumulation of Ca2<sup>+</sup> in apoplastic compartment as observed in Atcngc2 mutant (Wang et al., 2017) requires further confirmation.

ROS is the well-known master signal in plant immunity and is indispensable to Xoo-induced HR and non-host resistance (Li et al., 2015). Thus, it is considerable that regulation of ROS accumulation represents one of the essential mechanisms underlying SlCNGC1- and SlCNGC14-dependent regulation of HR and non-host resistance to Xoc and PTI. How SlCNGC1 and SlCNGC14 negatively regulate ROS accumulation remains an intriguing question to be addressed. NADPH oxidase encoded by RBOH genes is the key enzyme to generate ROS during plant-pathogen interactions. Interestingly, Ca2<sup>+</sup> affects the activity of this enzyme which contains EF-hands to bind Ca2+. Moreover, AtCPK28, a calcium-dependent protein kinase gene, negatively regulate ROS accumulation during PTI (Monaghan et al., 2014). In this context, it is notable that silencing of SlCNGC1 and SlCNGC14 strongly represses cytosolic Ca2<sup>+</sup> accumulation and expression of a set of denfense-related Ca2<sup>+</sup> signaling genes including SlCaM6, SlCDPK10 and SlCBP60g (**Figures 4**, **5**). Whether and how these changes affect NADPH oxidase thereby modulate ROS accumulation deserves further investigation.

SlCNGC1 and SlCNGC14 belong to group I and group III of tomato CNGC family. They function similarly in negative modulation of tomato HR and non-host resistance to Xoc. Both of them suppress ROS accumulation and callose deposition and alter expression of a same set of defense-related Ca2<sup>+</sup> signaling genes including SlCaM6, SlCDPK10 and SlCBP60g. However, the degree of effect in resistance responses by silencing SlCNGC1 and SlCNGC14 varies (**Figures 1–5**), indicating the difference of their involvement in this non-host resistance although it can be the consequence of difference of silencing degree. In animal, CNGC often forms homo- or hetero-oligomer to function (Kaupp and Seifert, 2002). Furthermore, some plant CNGC isoforms, such as AtCNGC2 and AtCNGC4, have been reported to be able to homo- or hetero-complex in planta (Chin et al., 2013). In this context, whether SlCNGC1 and SlCNGC14 form oligomer to regulate HR and plant immunity is worth further clarifying. Additionally, cellular localization of plant CNGCs other than long expected plasma membrane have been reported recently (DeFalco et al., 2016b). Whether the cellular localization and accumulation of SlCNGC1 and SlCNGC14 differ awaits further study.

Zhang et al. SlCNGC1/14 Repress Non-host Resistance

Roles of CNGCs in plant disease resistance seem to be complex. Results from previous studies using Ca2<sup>+</sup> channel blockers indicated that cytosolic Ca2<sup>+</sup> elevation in response to PAMPs such as flg22 is required for PAMP induced defense resposnes including ROS burst (Jeworutzki et al., 2010; Ranf et al., 2011; Segonzac et al., 2011), suggesting a positive role of the possibly involved CNGCs in PTI defenses. However, CNGC genes likely play different roles in various types of resistance. For instance, T-DNA insertion knockout mutants for AtCNGC11 and AtCNGC12 exhibit unaffected flg22 induced PTI responses but show a partial breakdown of resistance against avirulent, but not virulent Hyaloperonospora arabidopsidis and Pseudomonas syringae, indicating that both AtCNGC11 and AtCNGC12 might be not involved in PTI but act as positive regulators of R gene-mediated resistance responses (Yoshioka et al., 2006; Moeder et al., 2011). Differently, null mutants for AtCNGC2 and AtCNGC4 display impaired HR cell death, while maintaining resistance against avirulent pathogens, exhibiting enhanced broad-spectrum resistance against virulent pathogens, elevated levels of SA, and constitutive expression of PR genes (Yu et al., 1998; Clough et al., 2000; Yu et al., 2000; Balagué et al., 2003; Jurkowski et al., 2004; Genger et al., 2008), and thus these AtCNGCs seem to play positive role in HR while have negative role in R gene-mediated resistance and basal resistance. Additionally, AtCNGC2 was reported to positively contribute to Pep-elicited immunity (Qi et al., 2010; Ma et al., 2012, 2013) and LPS-triggered Ca2<sup>+</sup> influx that led to nitric oxide production and consequent HR formation (Ali et al., 2007; Ma et al., 2007), but might be not involved in flg22-triggered immunity (Jeworutzki et al., 2010). In this study, we found the negative role of SlCNGC1 and SlCNGC14 in Xoc-induced HR and non-host resistance, adding further complexity to the function of plant CNGCs. It is likely that different members of CNGC family might have different roles in resistance and at least some CNGCs are multifunctional and differentially regulate various types of resistance against different pathogens. SlCNGC1 and SlCNGC14, which exhibit similar function in Xoc-induced HR and non-host resistance, belong to different groups of CNGCs, with SlCNGC1 to group I as AtCNGC11 and AtCNGC12, while SlCNGC14 to group III, indicating that the function of CNGCs is not group-dependent, as we suggested previously (Saand et al., 2015b). Mechanisms underlying plant CNGC-mediated resistance againt various pathogens await further elucidation.

### CONCLUSION

Gene silencing analyses demonstrated that SlCNGC1 and SlCNGC14 negatively regulate non-host resistance related HR cell

### REFERENCES


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### AUTHOR CONTRIBUTIONS

The project was coordinated by X-ZC. X-RZ conducted the gene silencing analyses. X-RZ and Y-PX carried out the gene expression, designed and analyzed all statistical data. X-ZC conceived of the study, and participated in its design and coordination. X-ZC and X-RZ prepared the manuscript. All authors read and approved the final manuscript.

### FUNDING

This work was financially supported by grants from the Zhejiang Provincial Natural Science Foundation of China (No. LZ18C140002), the Genetically Modified Organisms Breeding Major Projects (No. 2014ZX0800905B), the National Natural Science Foundation of China (No. 31672014), and the National Key Research and Development Project (No. 2017YFD020 0602).

### ACKNOWLEDGMENTS

We are grateful to Prof. Gerald Alan Berkowitz, Department of Plant Science and Landscape Architecture, Agricultural Biotechnology Laboratory, University of Connecticut, United States, for providing seeds of aequorin transgenic tomato lines.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.00285/ full#supplementary-material

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Zhang, Xu and Cai. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Mapping of HKT1;5 Gene in Barley Using GWAS Approach and Its Implication in Salt Tolerance Mechanism

Khaled M. Hazzouri 1,2 \*, Basel Khraiwesh<sup>3</sup> , Khaled M. A. Amiri 1,4, Duke Pauli <sup>5</sup> , Tom Blake<sup>6</sup> , Mohammad Shahid<sup>7</sup> , Sangeeta K. Mullath<sup>8</sup> , David Nelson<sup>2</sup> , Alain L. Mansour <sup>9</sup> , Kourosh Salehi-Ashtiani <sup>3</sup> , Michael Purugganan<sup>2</sup> and Khaled Masmoudi <sup>8</sup> \*

*<sup>1</sup> Khalifa Center for Genetic Engineering and Biotechnology, United Arab Emirates University, Al Ain, United Arab Emirates, <sup>2</sup> Center for Genomics and Systems Biology, New York University of Abu Dhabi, Abu Dhabi, United Arab Emirates, <sup>3</sup> Laboratory of Algal and Systems Biology, New York University of Abu Dhabi, Abu Dhabi, United Arab Emirates, <sup>4</sup> Department of Biology, College of Science, United Arab Emirates University, Al Ain, United Arab Emirates, <sup>5</sup> Plant Breeding and Genetics, School of Integrative Plant Science, Cornell University, Ithaca, NY, United States, <sup>6</sup> Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT, United States, <sup>7</sup> International Center for Biosaline Agriculture, Dubai, United Arab Emirates, <sup>8</sup> Department of Arid Land Agriculture, College of Food and Agriculture, United Arab Emirates University, Al Ain, United Arab Emirates, <sup>9</sup> Date Palm Tissue Culture, United Arab Emirates University, Al Ain, United Arab Emirates*

### Edited by:

*Gerald Alan Berkowitz, University of Connecticut, United States*

#### Reviewed by:

*Ahmad Arzani, Isfahan University of Technology, Iran Wei Li, China Agricultural University, China*

#### \*Correspondence:

*Khaled Masmoudi khaledmasmoudi@uaeu.ac.ae Khaled M. Hazzouri hazzourik@nyu.edu; khaled\_hazzouri@uaeu.ac.ae*

#### Specialty section:

*This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science*

Received: *04 September 2017* Accepted: *29 January 2018* Published: *19 February 2018*

#### Citation:

*Hazzouri KM, Khraiwesh B, Amiri KMA, Pauli D, Blake T, Shahid M, Mullath SK, Nelson D, Mansour AL, Salehi-Ashtiani K, Purugganan M and Masmoudi K (2018) Mapping of HKT1;5 Gene in Barley Using GWAS Approach and Its Implication in Salt Tolerance Mechanism. Front. Plant Sci. 9:156. doi: 10.3389/fpls.2018.00156* Sodium (Na+) accumulation in the cytosol will result in ion homeostasis imbalance and toxicity of transpiring leaves. Studies of salinity tolerance in the diploid wheat ancestor *Triticum monococcum* showed that *HKT1;5*-like gene was a major gene in the QTL for salt tolerance, named *Nax2.* In the present study, we were interested in investigating the molecular mechanisms underpinning the role of the *HKT1;5* gene in salt tolerance in barley (*Hordeum vulgare*). A USDA mini-core collection of 2,671 barley lines, part of a field trial was screened for salinity tolerance, and a Genome Wide Association Study (GWAS) was performed. Our results showed important SNPs that are correlated with salt tolerance that mapped to a region where *HKT1;5* ion transporter located on chromosome four. Furthermore, sodium (Na+) and potassium (K+) content analysis revealed that tolerant lines accumulate more sodium in roots and leaf sheaths, than in the sensitive ones. In contrast, sodium concentration was reduced in leaf blades of the tolerant lines under salt stress. In the absence of NaCl, the concentration of Na<sup>+</sup> and K<sup>+</sup> were the same in the roots, leaf sheaths and leaf blades between the tolerant and the sensitive lines. In order to study the molecular mechanism behind that, alleles of the *HKT1;5* gene from five tolerant and five sensitive barley lines were cloned and sequenced. Sequence analysis did not show the presence of any polymorphism that distinguishes between the tolerant and sensitive alleles. Our real-time RT-PCR experiments, showed that the expression of *HKT1;5* gene in roots of the tolerant line was significantly induced after challenging the plants with salt stress. In contrast, in leaf sheaths the expression was decreased after salt treatment. In sensitive lines, there was no difference in the expression of *HKT1;5* gene in leaf sheath under control and saline conditions, while a slight increase in the expression was observed in roots after salt treatment. These results provide stronger evidence that

**38**

*HKT1;5* gene in barley play a key role in withdrawing Na<sup>+</sup> from the xylem and therefore reducing its transport to leaves. Given all that, these data support the hypothesis that *HKT1;5* gene is responsible for Na<sup>+</sup> unloading to the xylem and controlling its distribution in the shoots, which provide new insight into the understanding of this QTL for salinity tolerance in barley.

Keywords: GWAS, barley, salinity tolerance, HKT1;5 gene, sodium transport

### INTRODUCTION

The world's land area affected by salinized soil and water is approximately 7%, and according to the FAO Land and Plant Nutrition Management service, most of the world's land is not cultivated, but a significant proportion of irrigated land is saltaffected. In arid and semi-arid regions, soil salinization is a major threat to agriculture, where water scarcity and inadequate irrigation management will severely reduce crop yield (FAO, 2008). Accumulation of sodium in the cytosol of plants is toxic due to imbalance of ions in the transpiring leaves. There are two major mechanisms of salinity stress at the whole plant level that are advanced so far: a rapid and early osmotic stress which reduces shoot growth, and a slower accumulating ionic stress which accelerates senescence of older leaves (Sahi et al., 2006; Munns and Tester, 2008). Osmotic stress affects the plant's water relations due to reduced availability of water from the soil solution (Munns, 2005), which in turn disturbs the growth of the plant by reducing cell expansion and elongation rates. This mechanism will lead to smaller and thicker leaves, reducing photosynthesis by stomatal closure, and limiting water uptake (Fricke et al., 2004). To minimize the toxic effects of ionic Na<sup>+</sup> stress, plants employ different mechanisms to adapt and tolerate saline conditions. The most important ones to minimize the harmful effects of ionic Na<sup>+</sup> stress include immediate Na<sup>+</sup> exclusion from uptake, limit of xylem Na<sup>+</sup> loading and/or retranslocation from the shoot; efficient compartmentalization of Na<sup>+</sup> mainly into vacuoles, cytosolic K<sup>+</sup> homeostasis and preservation in root and mesophyll cells, efficient osmotic adjustment by a decrease of water loss and an increase of water uptake, and last but not least a ROS detoxification (Blumwald, 2000; Zhu, 2003; Munns and Tester, 2008). To tolerate salinity, plants deploy a variety of traits to control the function and development of the cell that relies on signal perception, signal integration and processing. Therefore, adaptation to salinity stress is a quantitative character, which is controlled by different genetic pathways, where multiple genes are implicated in salinity tolerance (DeRose-Wilson and Gaut, 2011).

Barley (Hordeum vulgare) is the major and most salt tolerant cereal crop worldwide (Munns and Tester, 2008). It can tolerate up to 250 mM NaCl (equivalent to 40% seawater), beyond which the survival rates drop drastically. Cultivated barley, its wild ancestor (Hordeum vulgare subp. Spontaneum) and domesticated barley are originated from the Fertile Crescent and Tibet (Kilian et al., 2006; Dai et al., 2012). Genetic diversity and adaptation of barley to extreme conditions resulted in a rich pool of genetic variation (Nevo and Chen, 2010). However, breeding efforts to select high yield and stability genotype of barley plants in marginal environment have met with limited success (Flowers and Flowers, 2005). Indeed, modern cultivated barely varieties share 15–40% of all alleles within the barley gene pool, which indicate that only a small fraction of barley genetic resource has been used to improve salinity tolerance (Long et al., 2013). Salinity tolerance in plants is under complex polygenic trait controlled by numerous quantitative trait loci (QTLs) and several genes have been proposed to be involved (Flowers, 2004). High-Affinity K<sup>+</sup> Transporter (HKT) genes encode Na<sup>+</sup> and/or K<sup>+</sup> transport systems, active at the plasma membrane (Almeida et al., 2013; Véry et al., 2014). While weakly represented in dicot species genomes (e.g., one single HKT gene in Arabidopsis and poplar), the HKT family comprises more members displaying a large functional diversity in monocots, including cereals. For instance, rice (Oryza sativa) possesses 9 HKT genes (Garciadeblás et al., 2003), and barley (Hordeum vulgare) and wheat (Triticum aestivum) have been deduced from southern blot analyses to possess 5 to 11 HKT genes per genome, respectively (Huang et al., 2008). Based on phylogenetic and functional analyses, plant HKT genes have been divided into two subfamilies (Platten et al., 2006). Subfamily 1 HKT (present in all higher plant species) encode Na+-selective transporters, while subfamily 2 ones (monocot specific) encodes systems permeable to both Na<sup>+</sup> and K<sup>+</sup> (Jabnoune et al., 2009; Munns et al., 2012; Sassi et al., 2012; Ben Amar et al., 2014; Suzuki et al., 2016).

In durum wheat, two QTLs (Nax1 and Nax2) were found to be involved in salinity tolerance. The Nax2 region corresponds to the HKT1;5 locus, which encodes a selective Na<sup>+</sup> transporter. The Nax2 locus on the 5AL chromosome is homologous to Kna1 located on chromosome 4DL carrying the major QTL for Na<sup>+</sup> exclusion in common wheat (Byrt et al., 2007; James et al., 2011; Arzani and Ashraf, 2016). Moreover, HKT genes shown to be associated to QTLs of salt tolerance belong to subfamily 1. They have been shown to play crucial roles in salinity tolerance in different plant species (Munns et al., 2012; Asins et al., 2013, 115; Arzani and Ashraf, 2016).

A wide range of physiological and agronomic traits was used as selection criteria to map QTL for salinity tolerance in barley. Among these, we can enumerate plant survival (Zhou et al., 2012; Fan et al., 2015), stomatal size, frequency, and photosynthesis parameters (Liu et al., 2017), yield and agronomic traits (Xue et al., 2009), seed germination and seedling growth stage (Witzel et al., 2010; Ahmadi-Ochtapeh et al., 2015), Na<sup>+</sup> exclusion (Shavrukov et al., 2010), tissue ion content (Xue et al., 2009), water soluble carbohydrate and chlorophyll content (Siahsar and Narouei, 2010). In previous study, Salinity tolerance in barely was assessed through a combination of plant survival and leaf wilting (Zhou et al., 2012; Fan et al., 2015). These two major symptoms caused by salt stress had been used for evaluating salinity tolerance of barley through QTL mapping.

To uncover and elucidate the genetic basis of complex agronomic traits, genome-wide association studies (GWAS) have been increasingly used. To elucidate the genetic basis of plant height and inflorescence architecture in sorghum, GWAS was performed using genome-wide map of SNP variation that allowed to map several classical loci of plant height, candidate genes for inflorescence architecture (Morris et al., 2013). In rice, GWAS was implemented to identify loci controlling salinity tolerance. Depending on 6,000 SNPs in many stress-responsive genes, Infinium high-throughput assay was used to genotype 220 rice accessions. In addition to saltol, a major QTL, identified to control salinity tolerance at seedling stage, GWAS peaks representing new QTLs was found on chromosome 4, 6, and 7 (Kumar et al., 2015). In wheat, the analysis of the Nax2, which corresponds to TmHKT1;5-type gene, suggests that this gene display distinctive expression pattern in roots.

Here, we present a genome-wide association study (GWAS) in the USDA barley core collection. This population comprising 2,671 lines collected worldwide, selected to represent the entire 30,000 lines of the USDA collection, was evaluated for different agronomic characters performance and yield in the field in order to identify loci associated with salinity tolerance using single nucleotide polymorphism (SNP) data and to study the molecular mechanism underpinning the role of the HKT1;5 ion transporter gene in slat tolerance in barley. Real time PCR experiments allowed to compare the expression patterns of HKT1;5 gene in roots, leaves and leaf sheaths in tolerant and sensitive barley lines.

### MATERIALS AND METHODS

### Barley Germplasm

A total of 2,671 barley accessions collected worldwide, selected to represent the entire 30,000 lines of the USDA barley collection (**Figure 1A**), was evaluated for salinity response to map the locations of genes contributing to low flag leaf Na+/K<sup>+</sup> ratio under saline production environment using association analysis.

### Field Trials and Evaluation of Salinity Tolerance

Field trial was implemented at the International Center for Biosaline Agriculture (ICBA), Dubai, United Arab Emirates (25◦ 05′ 40.8′′ N 55◦ 23′ 23. 5′′ E), from November 2012 to May 2013.

The surface soil texture at ICBA field experimental station is composed of 98% sand, 1% silt, and 1% clay. The native soil is non saline, where the electrical conductivity of its saturated extract is 1.2 dS m−<sup>1</sup> ). It is moderately alkaline (pH 8.22), strongly calcareous, porous (45% porosity), and the organic matter content is very low (<0.5%). The saturation percentage of the soil is 26 and has a high drainage capacity. ICBA soil is classified as Typic Torripsamments, carbonatic, and hyperthermic (Shahid et al., 2014). Soil amendment provided by the company Koblenz compost organic fertilizer (manufactured by Tadweer waste Treatment LLC, Dubai, UAE), was supplemented to the top of the soil at a rate of 40 tons FW. ha−<sup>1</sup> (at 85% moisture) to increase the soil water-holding capacity and to deliver some of the deficient and required nutrients, such as nitrogen (N), potassium (K), sulfur (S), and micro-nutrients. The field was irrigated with fresh water and fertilization with phosphorous was conducted 2 weeks before sowing using a single supply of 45 kg P2O<sup>5</sup> ha−<sup>1</sup> . Granular urea nitrogen (N) fertilizer was applied once at a rate of 30 kg N ha−<sup>1</sup> , 3 weeks after planting. An application of NPK fertilizer (20-20-20) at a rate of 30 kg ha−<sup>1</sup> was also given 6 weeks after planting by fertilization.

The 2,671 barley lines were sown in plots of one row of 2 m length each. Plots were randomized in an augmented design, and salt tolerant check line (58/1A) was sown every 50 plots. Two rows of a local barley cultivar (cv. Omani) were sown around the experimental area to reduce edge effects. Approximately 50 seeds per line were hand sown at 2 cm spacing and 1 cm depth per row. The space between rows was 25 cm as per the drip irrigation system design. As we are dealing with sandy soil with very poor water holding capacity and the evapotranspiration was very high, all plots were irrigated twice per day for 5 min each time. Each plot received 4.4 L water per day. The USDA barley core collection was evaluated for different traits related to agronomic performance and yield, but in this paper we will focus only on Na<sup>+</sup> and K<sup>+</sup> content in the flag leaf as an indice of salinity tolerance. Plants were irrigated with fresh water (< 1 dS m−<sup>1</sup> ), referred to hereafter as the control condition, or with saline ground water (≈23 dS m−<sup>1</sup> ), referred to hereafter as saline condition. Saline plots were irrigated with ground water (≈23 dS m−<sup>1</sup> ) for the entire growing period, starting from the third week after germination and until the physiological maturity of the spikes. The distribution of drippers was homogenous and the distance between drippers allowed the overlapping of wetting fronts. Flag leaf was harvested during the heading time from all lines from the control and saline conditions. The electrical conductivity of the saturated soil extract at harvesting time was 16.8 dS m−<sup>1</sup> . Na<sup>+</sup> and K<sup>+</sup> content was determined from samples of 1 g flag leaf digested with 2% nitric acid in ultrapure Milli Q water. After complete digestion, the filtrate was analyzed by the Inductively Coupled Plasma Emission Optical Spectrophotometer (ICP-OES, Perkin Elmer) to determine Na<sup>+</sup> and K<sup>+</sup> concentrations in the flag leaf.

### Genotyping of the USDA Barley Core Collection

The USDA Barley core collection, a sub-sample of 2,671 lines chosen to represent the genotypic and phenotypic variability within the entire USDA collection of 30,000 accessions, was genotyped using the Illumina 9K single nucleotide polymorphism (SNP) array (Comadran et al., 2012). Briefly, this SNP array was constructed from a combination of the existing Barley Oligo Pool Assay (BOPA) SNPs (Close et al., 2009) and SNPs discovered from RNA-seq analyses as described by Comadran et al. (2012). To generate the called SNPs from the RNA-seq data, short-read data from 10 common cultivars (Barke, Betzes, Bowman, Derkado, Intro, Morex, Optic, Quench,

Sergeant, and Tocada) were aligned onto the Harvest 35 reference sequences (available at http://harvest-web.org/hweb/ mainmenu.wc). These putative SNPs were then further processed through quality control measures including removing minor alleles, selecting those SNPs with adequate read depth, and removing duplicate SNPs so that there was only a single SNP per unigene. From the initial 31,616 SNP candidates, 5,010 SNPs were selected for their coverage, information content, and technical performance. These SNPs were combined with the existing 2,832 BOPA SNPs to construct the Illumina 9K array. Of the 7,842 SNPs, 3,968 have been genetically mapped using 360 individuals from a F<sup>6</sup> RIL population derived from the F<sup>1</sup> progeny of a cross between Morex and Barke (complete information regarding the SNP assay development can be found in Comadran et al. (2012) and at http://bioinf.hutton.ac.uk/iselect/app/, verified 8 Aug. 2017. All genotypic data used in this work is available from The Triticeae Toolbox (https://triticeaetoolbox.org/, verified 8 Aug. 2017).

### Population Structure and Kinship Analysis

Population structure was investigated using a Bayesian modelbased approach of clustering implemented in STRUCTURE (Pritchard et al., 2000) to assign individuals to subpopulations. A clustering was run from K = 2 to K = 10 with 20 iterations with 10,000 burning period and 10,000 MCMC (Markov Chain Monte Carlo) for each value of K clusters and the logarithm of the data was estimated using an admixture model with correlated allele frequency. The optimal K was determined based on the likelihood model across 20 runs using the Evanno method (Evanno et al., 2005). The membership coefficient for the optimal K was permutated to match the various replicates for that value of K using CLUMPP (Jakobsson and Rosenberg, 2007). For visualization, we used the plotting function implemented in DISTRUCT software (Rosenberg, 2004). Using the method of Ritland (1996), we estimated kinship (K) using the software SPAGeDi (Hardy and Vekemans, 2002).

### Genome Wide Association Analysis

The genome-wide association study was performed on the content of flag leaf sodium, potassium and their ratio using 3,968 genome-wide SNP markers. The association analysis was performed using the Genome Association and Prediction Integrated Tool package (GAPIT) (Zhang et al., 2010; Lipka et al., 2012), using the optimum compression mixed linear model and P3D options to increase speed and statistical power. To control for population structure and relatedness, the mixed model incorporated principal components (Price et al., 2006) and a kinship matrix (Ritland, 1996). The amount of phenotypic variation explained by the model was assessed using the R<sup>2</sup> statistics. To correct for multiple testing problem, the procedure by Benjamini and Hochberg (1995) was used at a false discovery rates (FDRs) of 5%. A neighboring joining (NJ) tree was generated using the R package ape (Paradis et al., 2004). Principal component was plotted with the R package scatterplot3D (Ligges and Mächler, 2003). Linkage disequilibrium analysis was performed using HAPLOVIEW v.4.2 (Barrett et al., 2005) and the candidate genes located within and/or adjacent to the associated SNPs were identified using the website Barleymap (http://floresta.eead.csic.es/barleymap/) using the Morex genome annotation data.

### Evaluation of Salinity Tolerance of Selected Sensitive and Tolerant Lines in Hydroponics

Based on the field trial screening of the USDA barley core collection for salinity tolerance and ICP for the estimation of sodium and potassium content in the flag leaf, our results showed that the maintenance of K<sup>+</sup> with exclusion of Na<sup>+</sup> from the flag leaf was highly correlated with salt tolerance. The relationship to discriminate between sodium and potassium and for which a simple index, the Na+/K<sup>+</sup> ratio was strong enough to be exploited as a selection tool to screen for salinity tolerance. In fact, Saltol QTL was reported as a major association with Na+/K<sup>+</sup> ratio measured at reproduction stage in mapping salinity tolerance in rice. Using this index, we selected five barley lines showing the lowest Na+/K<sup>+</sup> ratio and accumulate less Na<sup>+</sup> in the flag leaf, along with five sensitive lines for deep characterization of the maintenance of K<sup>+</sup> acquisition with exclusion of Na<sup>+</sup> from the flag leaf, which has been found highly correlated with plant salt tolerance in the field trial. The experiment was designed to conduct plant growth in hydroponic culture conditions onto half-strength Hoagland's solution (Davenport et al., 2005) under greenhouse conditions and in triplicate for each treatment. Salt treatment (0, 7, and 15 dS m−<sup>1</sup> ) was applied at the early three-leaf stage and was maintained until the end of the plant growth cycle. Leaf, leaf sheath and root samples were harvested after 2 weeks of performing salt treatment for Na<sup>+</sup> and K<sup>+</sup> analysis. Scoring symptoms of survival, wilting, chlorosis, senescence, and death of the plants assessed salinity tolerance.

### Molecular Cloning of the HKT1;5 Alleles

DNA was isolated from the flag leaf of the panel of 5 tolerant and 5 sensitive barely accessions, using the CTAB method. PCR amplification of the HKT1;5 gene was accomplished with Pfu DNA polymerase (Promega) and specific primers (forward: 5′ -CTAGCGCAGCTGTCGCTCTT-3′ ; and reverse: 5 ′ -ACGTTGAAGTTGAGTGGGTC-3′ ). The PCR protocol consisted of an initial denaturation at 95◦C for 3 min, followed by 30 cycles comprising a first step at 95◦C for 30 S, an annealing step at 56◦C for 30 S and an elongation step at 72◦C for 3 min, and a final extension at 72◦C for 5 min. Purified amplified products were cloned into the PCR cloning vector pSC-Aamp/kan from StrataClone, according to the manufacturer's protocol, and sequenced. The sequences were polished and aligned to each other, to other Hordeum and Triticum species.

### Real Time PCR

Total RNA from 2 tolerant and 2 sensitive barely accessions under control and salt treatment (200 mM NaCl) was isolated with RNeasy plant mini kit (Qiagen). The remaining genomic DNA was removed by treating RNA with DNase-RNase free (Promega). The first strand cDNA was synthesized from 1 µg of total RNA, using the SuperScript kit (Invitrogen), according to the manufacturer's protocol.

Real-time PCR was performed in 384-well plates with the Light Cycler <sup>R</sup> 480 Real-Time PCR System (Roche) using SYBR Green I (Roche). Oligonucleotides were designed using Primer 3. Primers were used for HKT1;5 gene (forward, 5′ -tcgtgcatagccatcttcgt-3′ , and reverse 5′ - GATGCTGAGGACGTTGAAGT-3′ ). Actin gene from barley was used as a housekeeping gene, to calculate a normalization factor. Primers used for the actin gene were (forward: 5′ -CAATGTTCCTGCCATGTACG-3′ , and reverse: 5 ′ -ATGAGGAAGGGCGTATCCTT-3′ ). PCR reactions were performed in a 10 µl final volume, containing 3 µl cDNA (40 ng of cDNA), 0.5 µl of each primer (at 10µM), 5 µL 2x SYBR Green I master mix and 1 µl of RNasefree water (Sigma). The reaction consisted of an initial denaturation at 94◦C for 10 min followed by 45 cycles at 94◦C for 10 s, 60◦C for 10 s, and 72◦C for 15 s. three biological repetitions were performed to calculate the expression level.

## RESULTS

### Salinity Tolerance of the USDA Barley Core Collection

The barley core collection tested in the field exhibited significant difference in salinity tolerance. Traits were mapped at the heading stage for flag leaf length, width, weight, Na<sup>+</sup> and K<sup>+</sup> content, and at the maturity stage for grain yield. The scores were presented in **Supplementary Figure 1** and show the density distribution of the phenotypic variation for the different traits measured.

Seeds from the five tolerant and five sensitive lines were sown in perlite and the experiment was designed to conduct plant growth in hydroponics system with half-strength Hoagland's solution under greenhouse conditions and in triplicate for each treatment. Salt treatment (0, 7, and 15 dS m−<sup>1</sup> ) was applied at the stage of three leaves and was maintained until the end of the plant cycle. The sensitive lines exhibited severe symptoms of toxicity and senescence when plants are challenged with 15 dS m−<sup>1</sup> of salt, while the tolerant lines exhibited moderate symptoms of toxicity and senescence (**Figure 2A**).

The tolerant and sensitive lines showed no significant difference in unidirectional root uptake of sodium under control and salt stress conditions at 7 and 15 dS m−<sup>1</sup> (**Figure 2B**). However, the capacity of the leaf sheath in the tolerant lines to extract and sequester sodium as it entered the leaf was higher when the plants were challenged with salt stress. The sensitive lines were leaking sodium to the upper shoots and accumulate more sodium in their flag leaf. In contrast, the tolerant lines showed the lowest Na+/K<sup>+</sup> ratio in their flag leaf, suggesting a lower rate of transfer of sodium from the roots to the shoots (**Figure 2B**). Statistical analysis was done using the Dunn (1964) Kruskal-Wallis multiple comparison with p-value adjusted with the false discovery rate method using an R package (see **Supplementary Table 1**). Our results are consistent with the finding of James et al. (2006), where the lower rates of net Na<sup>+</sup> loading of the xylem is due to lower rates of Na<sup>+</sup> transport from

roots to shoots, and not to lower rates of net uptake of Na<sup>+</sup> from the soil solution or higher rates of retranslocation in the phloem.

### Population Structure

The population structure analysis assigned the genotypes to five subpopulations K = 5 with some admixture individuals in each subpopulation (**Figure 1B**), using a probability of membership P ≥ 0.8. This result is consistent with previous studies using the same USDA barley core collection (Muñoz-Amatriaín et al., 2014). At the contrary, this is not consistent with Fan et al. (2016), may be due to an arbitrary threshold of membership they consider in their analysis. Our structure analysis is consistent with the 3D principal Components analysis as well as the neighboring joining tree (NJ) that shows five clusters representing the five subpopulations (**Figures 1C,D**).

### Genome Wide Association Analysis

The genetic basis of salt tolerance in Hordeum vulgare was examined using a unified mixed model that controls for population structures and kinship. At least four SNPs were most significant for the Na<sup>+</sup> trait as well as the ratio K+/ Na<sup>+</sup> at a genome-wide Bonferroni-corrected threshold that was estimated based on the effective number of independent tests (**Table 1**). No significant SNPs were found for the potassium K <sup>+</sup> trait. Given the complex architecture of the salt tolerance trait, the most significant SNPs using this unified mixed model for both Na<sup>+</sup> and K+/Na<sup>+</sup> were located within 2 Mb region on the distal part of chromosome four (**Figures 3A,B**, **Table 2**). There are four SNPs that are significant (**Table 1**). Two of the SNPs (11\_11186 and 11\_20272) were significantly associated with the low accumulation of Na<sup>+</sup> in the leaves. On average, the lines carrying the major frequency allele of the peak SNPs (11\_11186 and 11\_20272) were accumulating 15.5 mmoles more than with the alternative allele, and their effects were in the opposite direction. The other two SNPs (11\_10610 and 12\_30476) are associated with the trait and the allelic effect estimate is in the same direction (**Supplementary Figure 2**). The SNPs individually in both traits accounted from 16 to 28% of the total phenotypic variation elucidated by the traits and they are in linkage disequilibrium (LD) (r <sup>2</sup> = 0.72–0.83) with each other (**Figure 3C**).

### Co-localization of Candidate Genes Associated with SNPs

Four significant SNPs were shown to be around an important HKT 1;5 gene, which is a known major player in salt tolerance in wheat, rice and Arabidopsis. The SNP 12\_30476 is around 0.5 Mb away from this transporter. There are multiple genes (**Table 2**) that are in the region that are part or adjacent to those significant SNPs. Genes contained in this region with significant SNPs association with reduce accumulation of Na<sup>+</sup> from the Manhattan location also have some predicted functions such as lipid-transfer protein, flavin-containing monooxygenase family protein, zinc finger protein, Protein NRT1/ PTR FAMILY 6.3 and expansin B2. The ortholog of these genes was shown to play a role in salt tolerance in other species (Chen et al., 2012; Jülke and Ludwig-Müller, 2015; Kong et al., 2016; Zhang et al., 2016).

### Molecular Cloning of the HKT1;5 Alleles

Isolation of the barley HKT1;5 transporter gene from the tolerant and sensitive lines was attempted, considering the availability of the full length HKT1;5 gene sequence in the Genbank


*<sup>a</sup> Minor allele frequency*

*<sup>b</sup> percent phenotypic variation explained by the trait*

*<sup>c</sup> False discovery rate adjusted p-value*

(accession # DQ912169.1). The reference sequence was used to design primers for amplification by PCR of the barley HKT1;5 genomic DNA. Eight alleles of the HKT1;5 gene were amplified, cloned, sanger sequenced and aligned. A midpoint rooted phylogenetic tree showing the relationship between the different lines (tolerant and sensitive) along with the HKT1;5 reference sequence for barley and other species was generated (**Supplementary Figure 3**). The assembled sequences from the tolerant and sensitive lines did not show any allelic variation that is linked to function. We could not find any nucleotide substitution in the coding region that differentiate the tolerant from the sensitive ones. Moreover, the analysis of cis-elements of 1 kb fragment from the HKT1;5 promoter sequence (using http://bioinformatics. psb.ugent.be/webtools/plantcare/html/) gene between the tolerant (1\_kb\_PI138711.2\_tolerant; 1\_kb\_PI21378\_tolerant) and sensitive (1\_kb\_Chlo6091.2\_sensitive; 1\_kb\_PI46735 8\_sensitive) lines did not show any differences. A total of 28 cis-elements were distributed along the sequence (**Supplementary Table 3**). These cis-elements don't show any direct relation to regulation of genes under salt stress. However, we can't rule out that these cis-elements could modulate in a positive and indirect way the expression of HKT1;5 gene in the leaf sheath of the tolerant compared to the sensitive lines. Further analysis is needed in this perspective.

### Real Time PCR

Expression of the HKT 1;5 gene in the salt sensitive barley leaf compared to the control showed after 72 h of salt treatment a significant increase (p < 0.05) in the expression than the control plants that are not subjected to salt stress. In contrast, the salt tolerant barley leaf showed a significant decrease in expression (p < 0.05) than the control plants (**Figure 4**). Expression of the HKT 1;5 gene in the salt sensitive barley roots as well as in the salt tolerant ones compared to the control showed a significant (p < 0.05) increase in the expression level (**Figure 4**). Expression of the HKT 1;5 gene in the salt sensitive barley leaf sheaths compared to the control plants showed no significant increase in the expression. At high concentration of salt (200 mM NaCl), the reduction of the expression level of HKT1;5 gene in leaf sheath of the tolerant line is correlated with increases in Na<sup>+</sup> accumulation in this tissue. This is reflecting that HKT1;5 gene play an important role in restricting the transport of Na<sup>+</sup> from leaf sheath to the upper leaves. The range of expression level (means ± SD, n = 3 biological replicates) in each line and tissue was analyzed with a t-student test with reported P-value (<0.05) (**Supplementary Table 2**).

### DISCUSSION

Salinity tolerance involves a complex of responses at molecular, cellular and whole plant levels and is governed by the action of multiple genes that are highly affected by the environment, and genotype-by-environment (G × E) interactions (Arzani and Ashraf, 2016). Sodium exclusion play a major contributor of the mechanism conferring salt tolerance (Munns et al., 2006). Salt tolerance in plants was shown to depend on HKT transporters, which mediate Na+-specific transport or Na+- K<sup>+</sup> co-transport and illustrate a key role in regulation of Na<sup>+</sup> homeostasis (Rodríguez-Navarro and Rubio, 2006). For instance, a source of sodium exclusion named Nax2, found in the diploid ancestral wheat relative, Triticum monococcum, confers a decreased rate of Na<sup>+</sup> transport from roots to shoots by retrieving Na<sup>+</sup> from the root xylem into xylem parenchyma cells (Davenport et al., 2005; James et al., 2006). A candidate gene in the Nax2 locus from T. monococcum (TmHKT1;5-A) and a major salt tolerance locus from bread wheat Kna1 (TaHKT1;5-D), encode a Na+ selective transporter, expressed in stellar root cells surrounding xylem vessels, and therefore can limit the amount of Na<sup>+</sup> that is translocated in the xylem to the leaf tissues (Munns et al., 2012; Byrt et al., 2014).

A new QTL for salinity tolerance in barley was identified through GWAS study (Fan et al., 2016). Association mapping for salinity tolerance was performed on 206 barley accessions and 408 DArT markers. Only two significant marker-trait associations for one QTL were detected on 4H. The authors showed that QTL on 4H with the nearest marker of bPb-9668 was the most significant, consistently detected in all methods. In the present study, using GWAS we mapped important SNPs that are located within 0.5 Mb from an important HKT1;5 gene

in barley, which may play a significant role in reducing Na+ concentration in the leaves. Since we don't have the sequence that expand this region in all the lines, more sequencing could be done across the majority of the barely accession (tolerant and sensitive) for better quantification. Other genes co-localizing in this region that have orthologs involved in salinity stress tolerance in other species could also play potential candidates for future validation. The application of linked SNP molecular markers for salinity tolerance in barley would be suitable to relevant crosses within the barley breeding program. They have the ability to cost effectively enable breeders to select germplasm and breeding lines using a simple DNA test without the need of a lot of phenotypic variation.

In rice and Arabidopsis, allelic variation for OsHKT1;5 and AtHKT1;5 was shown to be linked to the function of the gene (Ren et al., 2005; Rus et al., 2006). Our result in barley for


the plants under salt stress condition (200 mM NaCl). A house keeping gene actin from barely was used for control and the data was normalized to 0 mM NaCl. The range of expression level (means ± SD, *n* = 3 biological replicates) in each line and tissue was analyzed with a *t*-student test and significance was reported (\**P* < 0.05; \*\**P* < 0.005; \*\*\**P* < 0.0005).

5 tolerant and 5 sensitive lines showed no allelic variation for the HKT1;5 gene that is correlated with the phenotype, which is similar to what has been identified in wheat (Byrt et al., 2007). This result seems to suggest that difference in leaf Na<sup>+</sup> concentration between the tolerant and sensitive lines, is mostly due to expression variation of this gene, rather than allelic variation.

The exclusion of Na<sup>+</sup> is an important strategy employed by plants to prevent any shoot damage induced by Na<sup>+</sup> accumulation. In barley, Na<sup>+</sup> exclusion is an important salt tolerance strategy (Chen et al., 2007) as in wheat, however barley can tolerate more Na<sup>+</sup> accumulated in the shoot than wheat (Munns and James, 2003; Colmer et al., 2005). This will suggest that tissue tolerance of Na<sup>+</sup> in barley is a unique feature for salt tolerance. As an evidence of this, a recent study showed a constitutive overexpression of HKT2;1 gene that increased salt tolerance in barley, showed an increase in the level of Na<sup>+</sup> concentration in the shoots (Mian et al., 2011).

HKT1;5 gene expression in the sensitive leaf sheath was the highest and did not change upon salt stress. However, Na<sup>+</sup> accumulation in leaf sheath was much lower than in the tolerant line and there is no evidence of removal of Na<sup>+</sup> from xylem in the leaf sheath of the sensitive line and Na<sup>+</sup> is leaking to the upper shoots. In the tolerant line, HKT1;5 gene expression was decreased upon salt treatment and Na<sup>+</sup> accumulation in this tissue was higher than in the sensitive line, reducing its transport to the upper shoots. Similarly, reduced expression level of Na<sup>+</sup> transporter, TaHKT1;5 gene by gene silencing in transgenic bread wheat lines, increases Na<sup>+</sup> accumulation in the leaves which reflecting that TaHKT1;5 has in important role in restricting the transport of Na<sup>+</sup> from the root to the leaves in bread wheat and showed the potentiality to increase salinity tolerance in bread wheat by the manipulation of HKT1;5 gene (Byrt et al., 2014). In roots, there is an increase in the expression of HKT1;5 gene in both tolerant and sensitive lines and no significant effect on root Na<sup>+</sup> accumulation was observed. The precise control of Na+/K<sup>+</sup> selective accumulation in leaf, leaf sheath and root tissues is an essential mechanism to maintain cellular homeostasis in the presence of imposed high salt concentration in the growth solution. It was shown in bread wheat that TaHKT1;5 confers the essential salinity tolerance mechanism associated with the Kna1 locus via shoot Na<sup>+</sup> exclusion and is critical in maintaining a high K+/Na<sup>+</sup> ratio in the leaves (Byrt et al., 2014). In rice, it was shown that OsHKT1;5 plays a major role in the removal of Na<sup>+</sup> from the xylem sap into the surrounding xylem parenchyma cells, thereby protecting leaves from Na<sup>+</sup> toxicity (Ren et al., 2005). Possible roles of known HKT transporters in controlling Na<sup>+</sup> flux in barley could function as an Na<sup>+</sup> uptake system in the epidermal cortical cells and their expression could be down-regulated in conditions of salinity. This was shown to be the case for OsHKT2;1, which mediates the transport of Na<sup>+</sup> into roots of K+-starved plants and was down-regulated when plants were exposed to salinity (Horie et al., 2007). In addition, comparative analysis using salt-tolerant and sensitive rice varieties have led to the hypothesis that OsHKT1;4 restricts leaf-sheath-to blade Na<sup>+</sup> transfer in rice plants under salinity stress (Cotsaftis et al., 2012). This mechanism is consistent with tissue specific expression of this gene that is regulating the transport of Na<sup>+</sup> from root to leaves. In previous reports, it was demonstrated that HKT genes from Arabidopsis thaliana and rice, AtHKT1;1 and OsHKT1;5, reduce transport of Na<sup>+</sup> to the upper shoot and increase tolerance of the plant to salinity (Møller et al., 2009; Plett et al., 2010). Moreover, Munns et al. (2012) reported that TmHKT1;5 encoding a Na <sup>+</sup>-selective transporter located on the plasma membrane of root cells surrounding xylem vessels, was perfectly localized to withdraw Na<sup>+</sup> from the xylem and therefore reduce its transport to leaves. In accordance with the expression results, our data related to Na<sup>+</sup> and K<sup>+</sup> content analysis showed that the ratio Na+/K<sup>+</sup> was associated with a higher root Na<sup>+</sup> concentration in both tolerant and sensitive lines, while in leaf sheath there was a higher Na<sup>+</sup> concentration than in leaf blade of the tolerant lines.

Our results support what has been known about HKT 1;5 transporter gene and salt tolerance in barely, but it is the first time, that this gene is mapped using a GWAS approach. The question next is to test the function of this gene and its transport properties by performing electrophysiological recordings in Xenopus oocytes. We will design in the future an RNA interference experiment to knock down the gene in a tissue specific manner, study the transcript accumulation and see if the transgenic lines will accumulate more Na<sup>+</sup> in their leaves. Similar work has been done in bread wheat, where TaHKT1;5-D expression was predominantly observed within the stele, particularly within xylem parenchyma and pericycle cells, which are adjacent to the xylem vessels, whereas TaHKT1;5-D transcripts was reduced in roots of transgenic lines containing an

RNAi construct, increasing leaf Na<sup>+</sup> concentration (Byrt et al., 2014).

Few data allowing to understand the different wheat and rice salt tolerance QTL linked to HKT1;5 transporter genes are available so far. Indeed, the Kna1 QTL involving the bread wheat D genome TaHKT1;5 gene could certainly be explained by the low level of expression of the durum wheat HKT1;5 genes as compared to TaHKT1;5-D (Byrt et al., 2014). The Nax2 QTL, which has a similar mechanism to Kna1 in bread wheat, involving T. monococcum TmHKT1;5-A gene might be similarly explained by higher expression of the TmHKT1;5 gene as compared to durum wheat ones (Byrt et al., 2007, 2014; Munns et al., 2012). In rice, it was demonstrated that Na<sup>+</sup> exclusion in the leaf was influenced by OsHKT1;5 transcript abundance and that functional differences between the OsHKT1;5 allele from salttolerant rice variety, Nona Bokra, and an allele from the saltsensitive variety, Koshihikari, were referred to four amino acid substitutions (Ren et al., 2005; Cotsaftis et al., 2012). So far, 15 different OsHKT1;5 alleles in rice showed a strong association with leaf Na<sup>+</sup> concentrations (Negrão et al., 2013; Platten et al., 2013).

The present data of this work indicates that HKT1;5 gene from barley has a higher expression in roots and therefore limiting the amount of Na<sup>+</sup> transported in the xylem to the leaf tissues. Moreover, in previous work we demonstrated that alleles in the Nax1 locus from durum wheat, TdHKT1;4-1 and TdHKT1;4-2 encode Na+ selective transporters and are effective in limiting Na<sup>+</sup> accumulation in the leaves (Ben Amar et al., 2014).

### CONCLUSION

New insights into understanding the role of HKT1;5 transporter gene in salt tolerance in barley was achieved. Using GWAS approach, we mapped for the first time HKT1;5 gene from barley. Important SNPs markers were mapped and are located within 0.5 Mb from the HKT1;5 gene and may play a significant role in reducing Na<sup>+</sup> concentration in the leaves. Sequence analysis of HKT1;5 alleles from tolerant and sensitive barley lines did not show any allelic variation that is linked to function and correlated with the phenotype. This is similar to what was found in wheat (Byrt et al., 2007). This seems to suggest that difference in leaf Na<sup>+</sup> concentration between tolerant and sensitive lines, is mostly due to expression variation of HKT1;5 gene, rather than allelic variation. The expression pattern of HKT1;5 gene in barley revealed through real time PCR suggests that exclusion of Na<sup>+</sup> from the roots is happening in the salt tolerant line due to the higher expression of the HKT1;5 gene in the roots, but the accumulation of Na<sup>+</sup> in the leaf sheath in the tolerant lines is consistent with the decrease in expression of HKT1;5 gene in the leaf sheath and leaf to compensate the increase of expression in roots, which imply that the Na<sup>+</sup> is going to accumulate less in the leaves of the tolerant lines than the sensitive. This mechanism is consistent with tissue specific expression of this gene that is regulating the exclusion of Na<sup>+</sup> from the leaves. Ultimately, to study the function of HKT1;5 gene in barley, knock down experiment via CRISPR-Cas9 technology or RNA interference will be applied in a tissue specific manner to study the accumulation of Na<sup>+</sup> in leaves and the tolerance phenotype.

### AUTHOR CONTRIBUTIONS

KH, KA, and KM did the design of the paper and the analysis. BK did the real time PCR. DP and TB provided the SNP arrays and MS did the field testing. SM and DN did some laboratory work related to real time PCR. AM and KS-A discussed the manuscript. MP improved the manuscript.

### FUNDING

This research work was supported by funding from the United Arab Emirates University to KM under grant number 31F096. We are grateful to the International Center for Biosaline Agriculture team for phenotyping the barley USDA core collection in their field experimental station and evaluation of the salinity tolerance.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018. 00156/full#supplementary-material

Supplementary Figure 1 | Histogram plot of the phenotypic variation of the different traits (grain yield, average flag leaf width, average flag leaf length) measured. Wide dashed line represents the density distribution, while narrow ones represent the normal fitted distribution.

Supplementary Figure 2 | Phenotypic differences between lines carrying different alleles of the four SNPs (11\_10610; 12\_30476; 11\_11186; 11\_20272) associated with salt concentration (Na+) showing allelic estimate and directionality. The boxplot shows the differences of Na<sup>+</sup> concentration for the four SNPs showing the different alleles of the SNP locus. The box shows the first, second and third quartile. The width of the box is proportional to the square root of the number of individuals for each allele. The difference of mean (1m) and the Pearson correlation (r) between the genotype and phenotype values as well as the P value of the correlation is shown on the side of the boxplots.

Supplementary Figure 3 | A midpoint rooted phylogenetic tree showing the relationship between the different lines (tolerant and sensitive) along with the *HKT1;5* reference sequence for barley and other species was generated. The accession numbers are as follows: *Oryza\_indica* (HQ162137.1), *Oryza\_glaberrima* (JQ695813.1), *Oryza\_japonica* (AP014957.1), *Oryza\_rufipogon* (JQ695808.1), *Oryza\_sativa* (JQ695818.1), *Triticum monococcum* (DQ646332.1), *Triticum\_aestivum\_cultivar Kharchia* (KU212875.1),

*Hordeum\_marinum\_Subsp\_marinum* (KF606928.1),

*Hordeum\_marinum\_Subsp\_gussoneanum* (KF606929.1), *Horedum\_vulgare* (DQ912169.1).

Supplementary Table 1 | Statistical analysis using the Dunn (1964) Kruskal-Wallis multiple comparison with *p*-value adjusted with the false discovery rate method for Na+/K<sup>+</sup> ratio in roots, leaf sheaths and leaves at 0, 7, and 15 dSm−<sup>1</sup> .

Supplementary Table 2 | Statistical analysis using the *t*-student test comparing the range of expression level (means ± SD, *n* = 3 biological replicates) in tolerant and sensitive lines and tissues (roots, sheaths and leaves) and *P*-value was reported (<0.05).

Supplementary Table 3 | A summary of the putative 28 cis-elements along the 1 Kb sequence promoter of the *HKT1;5* gene in two tolerant and two sensitive lines using the PlantCARE database.

### REFERENCES


HKT transporter in barley improves salt tolerance. Plant J. 68, 468–479. doi: 10.1111/j.1365-313X.2011.04701.x


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Hazzouri, Khraiwesh, Amiri, Pauli, Blake, Shahid, Mullath, Nelson, Mansour, Salehi-Ashtiani, Purugganan and Masmoudi. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Auxin and Gibberellins Are Required for the Receptor-Like Kinase ERECTA Regulated Hypocotyl Elongation in Shade Avoidance in Arabidopsis

### Edited by:

Kai He, Lanzhou University, China

#### Reviewed by:

Joshua Blakeslee, The Ohio State University, United States Yingfang Zhu, Purdue University, United States

#### \*Correspondence:

Junbo Du junbodu@hotmail.com Wenyu Yang mssiyangwy@sicau.edu.cn

†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science

Received: 30 June 2017 Accepted: 23 January 2018 Published: 07 February 2018

#### Citation:

Du J, Jiang H, Sun X, Li Y, Liu Y, Sun M, Fan Z, Cao Q, Feng L, Shang J, Shu K, Liu J, Yang F, Liu W, Yong T, Wang X, Yuan S, Yu L, Liu C and Yang W (2018) Auxin and Gibberellins Are Required for the Receptor-Like Kinase ERECTA Regulated Hypocotyl Elongation in Shade Avoidance in Arabidopsis. Front. Plant Sci. 9:124. doi: 10.3389/fpls.2018.00124 Junbo Du1,2,3 \* † , Hengke Jiang1,2,3† , Xin Sun1,2,3† , Yan Li1,2,3† , Yi Liu<sup>1</sup> , Mengyuan Sun1,2,3 , Zhou Fan<sup>1</sup> , Qiulin Cao<sup>1</sup> , Lingyang Feng1,2,3, Jing Shang1,2, Kai Shu1,2,3, Jiang Liu1,2,3 , Feng Yang1,2,3, Weiguo Liu1,2,3, Taiwen Yong1,2,3, Xiaochun Wang1,2,3, Shu Yuan<sup>4</sup> , Liang Yu1,2,3, Chunyan Liu1,2,3 and Wenyu Yang1,2,3 \*

<sup>1</sup> College of Agronomy, Sichuan Agricultural University, Chengdu, China, <sup>2</sup> Sichuan Engineering Research Center for Crop Strip Intercropping System, Sichuan Agricultural University, Chengdu, China, <sup>3</sup> Key Laboratory of Crop Ecophysiology and Farming System in Southwest China – Ministry of Agriculture, Sichuan Agricultural University, Chengdu, China, <sup>4</sup> College of Resources, Sichuan Agricultural University, Chengdu, China

Plants use shade avoidance strategy to escape the canopy shade when grown under natural conditions. Previous studies showed that the Arabidopsis receptor-like kinase ERECTA (ER) is involved in shade avoidance syndrome. However, the mechanisms of ER in modulating SAR by promoting hypocotyl elongation are unknown yet. Here, we report that ER regulated hypocotyl elongation in shade avoidance requires auxin and gibberellins (GAs). The T-DNA insertional ER mutant er-3 shows a less hypocotyl length than that in Col-0 wild type. Promoter::GUS staining analysis shows that ER and its paralogous genes ERECTA-LIKE1 (ERL1) and ERECTA-LIKE2 (ERL2) are differentially expressed in the seedlings, of which only ER is most obviously upregulated in the hypocotyl by shade treatment. Exogenous feeding assay by using media-application with vertical-grown of Arabidopsis seedlings showed that the hypocotyl length of er-3 is partially promoted by indol-3-acetic acid (IAA), while it is relatively insensitive of er-3 to various concentrations of IAA than that of Col-0. Hypocotyl elongation of er-3 is promoted similar to that of Col-0 by high temperature in the white light condition, but the elongation was not significantly affected by the treatment of the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA). Exogenous GA3 increased the hypocotyl elongation of both er-3 and the wild type in the shade condition, and the GA3 biosynthesis inhibitor paclobutrazol (PAC) severely inhibits the hypocotyl elongation of Col-0 and er-3. Further analysis showed that auxin biosynthesis inhibitors yucasin and L-kynurenine remarkably inhibited the hypocotyl elongation of er-3 while yucasin shows a more severe inhibition to er-3 than Col-0. Relative expression of genes regulating

**51**

auxin homeostasis and signaling, and GA homeostasis is less in er-3 than that in Col-0. Furthermore, genetic evidences show that ER regulated hypocotyl elongation is dependent of PHYTOCHROME B (PHYB). Overall, we propose that ER regulated shade avoidance by promoting hypocotyl elongation is PHYB-dependent and requires auxin and GAs.

Keywords: ERECTA, receptor-like kinase, shade avoidance, auxin, GA

### INTRODUCTION

fpls-09-00124 February 5, 2018 Time: 14:54 # 2

Light is one of the most important factors for plant survival and production. In a natural environment, plants always grow closely to one another. Under these conditions, red light wavelengths is been absorbed while the far red light wavelengths is reflected by the leaves of the neighboring plants, resulting in reduction of red:far red (R:FR) light ratio and light intensity, referred to as shade condition. Plants have evolved sophisticated mechanisms which involves architecture and physiological process including increase of the hypocotyl and stem length, hyponasty, early flowering and yield reduction, which is referred to as shade avoidance syndrome (SAR) (Casal, 2013; Wit et al., 2016). In the shade, plants must accelerate their growth in order to maintain their height at least as tall as their neighboring plants to succeed in light sensing competition conditions (Ballaré, 1999). In this process, plants have to expend more energy to support their elongation growth at the expense of leaf development, seed number and yield reduction. Arabidopsis and most crops show typical phenotypes of SAR, while some other plants display shade tolerance phenotype mimicking the phenotypes of the plants in the white light condition (Valladares and Niinemets, 2008; Carriedo et al., 2016).

Upon sensing the canopy shade by the neighboring plants, phytochromes in plant cells would perceive the changes of R:FR light condition and rapidly evoke cascades of actions. Of all the five phytochromes, phytochrome A (PHYA), phytochrome B (PHYB), phytochrome C (PHYC), phytochrome D (PHYD), and phytochrome E (PHYE) in Arabidopsis, PHYB was found to play dominant roles in shade avoidance (Ballaré, 1999; Ruberti et al., 2012; Casal, 2013; Wit et al., 2016). When plants were under shade conditions, PHYB releases the binding of phytochrome interacting factor (PIF) transcription factors and facilitates their entry into the nucleus to bind the promoters of the target genes to trigger the expression of genes in regulating phytohormone levels and signaling pathways (Lorrain et al., 2008; Casal, 2013). Most phytohormones which are found to participate in several aspects of the shade avoidance signaling pathways, auxin and gibberellins (GAs) are best established to be essential for elongation-promoting of plant hypocotyls, stems and petioles (Wit et al., 2016; Yang and Li, 2017). In Arabidopsis and Brassica rapa seedlings, auxins are biosynthesized in the cotyledon when suffered to shade and cotyledon-synthesized auxins are then transported to promote the hypocotyl elongation (Tao et al., 2008; Procko et al., 2014). In these processes, genes related to plant growth and development, and adaptation are largely expressed.

Indol-3-acetic acid (IAA) is the predominant naturally occurring auxin in plants (Zhao, 2010, 2012). In higher plants, auxin biosynthesis is likely extremely complex in plants, which includes de novo auxin production and the release from auxin conjugates (Zhao, 2010, 2012, 2014). IAA exists in two forms, the free IAA and conjugated IAA, the free IAA can be converted from the conjugated IAA, which is considered as the storage forms or the intermediates for degradation (Woodward and Bartel, 2005; Ludwig-Müller, 2011; Zhao, 2014). Previous isotope-labeling experiment and genetic evidence demonstrated that auxin principally biosynthesized via tryptophan (Trp)-dependent and Trp-independent pathways to coordinately regulate plant growth and development (Wright et al., 1991; Normanly et al., 1993; Woodward and Bartel, 2005; Wang et al., 2015). More evidence showed that several Trp-dependent auxin biosynthesis pathways contribute predominantly to IAA levels referring to the indole-3-acetaldoxime (IAOx) pathway, indoleacetamine (IAM) pathway, and the indole-3-pyruvic acid (IPA) pathway, of which the IPA pathways is the well studied pathway up to date (Korasick et al., 2013; Tivendale et al., 2014; Zhao, 2014). IAA biosynthesized from the Trp by using the IPA as intermediate by a two-step pathway is the best completely established pathway (Zhao, 2012, 2014). In this pathway, Trp is first converted to IPA by TAA1/TARs and IPA is subsequently catalyzed by YUCCAs (YUCs) into IAA (Zhao et al., 2001; Tao et al., 2008). In recent years, more and more compelling evidence showed that, in addition to auxin biosynthesis, auxin transport and metabolism are also essential to hypocotyl elongation in shade avoidance (Pierik et al., 2009; Keuskamp et al., 2010; Zhao, 2010; Yang and Li, 2017). Several studies have demonstrated that auxin transport is important in hypocotyl elongation in etiolated growth, photomorphogenesis, and phototropism similar to shade avoidance response (Jensen et al., 1998; Wu et al., 2016).

When plants are exposed to the adverse environment, the external stimuli will activate the cell membrane-located receptor molecules and initiate the changes of conformation of the receptors. Receptor-like kinases (RLKs) are a set of single transmembrane proteins located on the plasma membrane which involve in sensing the environmental changes including cell-to-cell and cell-to-environment communications (Becraft, 2002; Li, 2010). A typical RLK contains an extracellular domain for signal perception, a transmembrane domain for membrane anchoring and an intercellular Ser/Thr/Tyr kinase domain for signal transduction via phosphorylation (Walker and Zhang, 1990; Shiu and Li, 2004; Oh et al., 2009; Li, 2010; Oh et al., 2010). The first plant RLK was identified from maize by using degenerate PCR primers to the protein kinase domain

(Walker and Zhang, 1990). More than 610 RLKs have been found in Arabidopsis in recent years (Shiu and Bleecker, 2001; Shiu et al., 2004). Up to date, more and more RLKs have been found to function in many aspects of plant growth and development, cell death and defense (Li and Chory, 1997; Li et al., 2002; Nam and Li, 2002; Zipfel et al., 2006; Chinchilla et al., 2007; He et al., 2007; Du et al., 2012). For instance, the BRI1 was found as a receptor of brassinosteroids (BRs) (Li and Chory, 1997), BAK1 is a co-receptor of BRI1 in BR signaling pathways in regulating plant growth and development (Li et al., 2002; Nam and Li, 2002). Furthermore, BAK1 was also found as a co-receptor of FLS2 and EFR in pathogen perception and defense pathways (Zipfel et al., 2006; Chinchilla et al., 2007). ERECTA (ER) was firstly found in 1957 by using X-ray irradiated Arabidopsis Landsberg ecotype (Rédei et al., 1992). ER and its functional paralogs ERL1 and ERL2 not only control multiple aspects of plant morphology, but also regulate plant responses to environmental changes (Shpak et al., 2004). Genetic analysis has shown that the er mutant displays compact inflorescence and short blunt silique phenotypes due to the decrease in cell proliferation and growth (Shpak et al., 2003). In addition, ERL1 and ERL2 were found to play a redundant role in cell proliferation of organ growth and patterning (Shpak et al., 2004). Furthermore, ER was found to regulate transpiration efficiency in Arabidopsis (Masle et al., 2005). Overexpression of truncated Arabidopsis ER in tomato decreased water loss and enhanced drought tolerance (Villagarcia et al., 2012). Overexpression of Arabidopsis ER in Arabidopsis, rice and tomato increased plant biomass and improved thermal tolerance independent of water content (Shen et al., 2015). Single nucleotide polymorphism (SNP) analysis has shown that an ER homologous gene might be associated with drought adaptation between wild and common bean (Blair et al., 2016).

In recent studies, Patel et al. demonstrated that ER regulates petiole angle and elongation in the shade particularly at cool temperatures in Landsberg ecotype (Patel et al., 2013). It is possible that ER stimulate petiole elongation by promoting the cell expansion in the petioles. Another study also showed that ER makes an important contribution to the shade avoidance syndrome in some stages during plant development against light fluctuations (Kasulin et al., 2013). However, the mechanisms of ER in modulating the SAR are not clear yet, which needs further investigation.

Here, we report our identification of ER in regulating Arabidopsis hypocotyl elongation in the shade in Col-0 background. The results show that loss-of-function of ER displays a shorter hypocotyl length than that of Col-0 wild type in the shade condition. Promoter::GUS analyses show that the expression of ER is remarkably induced in the hypocotyl by shade treatment. Further investigation show that ER regulated hypocotyl elongation is probably via actions of auxin and GAs, and is dependent of PHYB. Our data supplied a new identification of ER in shade avoidance and detailed a possible mechanism of ER regulated shade avoidance underlying the involvement of auxin homeostasis and signaling pathways, as well as GA homeostasis, which provides new evidence and mechanisms for ER regulated shade avoidance.

### MATERIALS AND METHODS

### Plant Materials and Growth Conditions

All the Arabidopsis seeds used in these studies were Col-0 ecotype. Seeds of er-3 (SALK\_044110) (Durbak and Tax, 2011), phyB (SALK\_022035C) were ordered from Arabidopsis Biological Resource Center (ABRC). Plants were grown at 22◦C in a long-day growth condition (16 h of light and 8 h of dark) in a greenhouse except those for special treatments.

Arabidopsis seeds were surface-sterilized and grown in the soil or on the 1/2 Murashige and Skoog (MS) media (pH 5.7) supplemented with 1% sucrose and 0.8% agar. For shade treatment of soil-grown plants, 10-day-old Arabidopsis Col-0 and er-3 single mutant grown in a normal light condition (16 h of light and 8 h of dark) with PAR of 65 µmol m−<sup>2</sup> s −1 and R/FR of 1.3) in a greenhouse and then transferred to a green filter (type No. 122 with a transmittance of 45.6%<sup>1</sup> , England) with PAR of 31 µmol m−<sup>2</sup> s −1 and R/FR of 0.5 for another 15 days. For shade treatment of media-grown plants, plants were grown in normal light condition for 3 days and then treated with the green filter for another 5 days. Plants were then used for further analyses. Unless it is specially stated that, the plants were grown on slightly vertical 1/2 MS media.

### Semi-quantitative Reverse Transcription (RT)-PCR and Quantitative PCR (qPCR) Analyses

Two micrograms of total RNA were extracted by using an RNAprep pure Plant Kit (Tiangen Biotech) used for reverse transcription with M-MLV (Invitrogen). The first strand of cDNAs was used for semi-quantitative reverse transcription PCR (RT-PCR) analyses with ExTaq DNA polymerase (TaKaRa) according to previous studies (Du et al., 2012). Real-time PCR was employed with SYBR <sup>R</sup> Premix Ex TaqTM II (TaKaRa) and relative expression of genes compared to ACT2 was calculated using 11Ct method. Primers used in this study are listed in **Supplementary Table S1**.

### Promoter::GUS Construction of ER Family Genes and GUS Staining

Promoters of ER, ERL1 and ERL2 with 1.5 kb were amplified from genomic DNA and cloned with a Gateway <sup>R</sup> Cloning technology (Invitrogen). The genes were recombined into a binary vector pBASTA-GWR-GUS and the destination plasmids were then overexpressed in Col-0 with Agrobacterium-mediated transformation. Surface-sterilized seeds of homozygous transgenic plants harboring promoter-GUS were grown on 1/2 MS media. After stratification for 2 days at 4◦C, the plates were grown vertically at 22◦C in white light for 3 days, and then transferred to white light or shade conditions for another 5 days for GUS staining. For GUS staining, ER, ERL1, or ERL2 promoter-GUS transgenic seedlings were stained according to previous studies (Guo et al., 2010). The stained plants were observed and for photo-capture under a Leica M165C digital

<sup>1</sup>www.q-max.net

stereo microscope, the images were subsequently arranged by using the Adobe Photoshop CS6 software.

### Treatment of Phytohormones and the Biosynthesis Inhibitors

Surface-sterilized seeds were grown on 1/2 MS plates supplemented with 1% sucrose, 0.8% agar and different concentrations of IAA (Sangon), GA3 (Sangon), the auxin biosynthesis inhibitors yucasin and L-kynurenine (kyn) (Sigma-Aldrich), and the GA biosynthesis inhibitor paclobutrazol (PAC) (Sangon). The seedlings were grown at a slight angle with the hypocotyl touching the media. Stock solutions of the phytohormones and the inhibitors were dissolved as follows: 20 mM of IAA were dissolved in ethanol, 20 mM of GA3 in methanol, 500 mM of yucasin in DMSO, 100 mM of kyn in 0.5 M HCl and 5 mM PAC in methanol, respectively. ddH2O was used for dilution for the working concentrations. The hypocotyl length was measured with ImageJ 1.6 and analyzed with Graphpad 5.0.

### RESULTS

### Loss-of-Function of ER Is Shade Insensitive in Hypocotyl Elongation

To understand whether ER regulates hypocotyl elongation in shade response, er-3 (Durbak and Tax, 2011), a T-DNA insertional loss-of-function mutant of ER, was used to assess the shade response. After 3-day white-light growth, Col-0 and er-3 were then moved to the shade condition for another 5 days. The results showed that the hypocotyl length of er-3 were significantly less sensitive to shade treatment than that of Col-0 in our shade condition (**Figures 1A,B**). To further confirm the shade response phenotypes of er mutant, we grew the Col-0 and er-3 seeds in the soil and covered a green filter for shade treatment. In the shade condition, both soil-grown Col-0 and er showed a typical shade avoidance response including petiole elongation (**Figures 1C,D**). Nevertheless, the petiole elongation was shorter in er-3 single mutant than that in Col-0 in the shade, indicating that petiole elongation of er-3 is relatively insensitive to shade, which is consistent with other er alleles in previous studies (Patel et al., 2013). These results suggest that ER in Col-0 ecotype makes a contribution to shade avoidance syndrome.

### Expression of AtER in Hypocotyl Is Upregulated by Shade

To examine whether the transcript level of ER in the hypocotyl of Arabidopsis responds to shade, we performed RT-PCR analysis to check gene expression by the time-course shade treatment. The results showed that the shade inducible gene PIL1 was upregulatedly expressed in the hypocotyls of Col-0 after 30 min by shade treatment and increased as time rises (**Supplementary Figure S1**), indicating that our shade condition is reliable for study on the shade avoidance syndrome. Under this shade condition, we further found that the expression of ER was upregulated in the hypocotyls of Col-0 after 30 min and reached

FIGURE 1 | Phenotypes of Col-0 and er-3 single mutant in shade avoidance. (A) Phenotypes of Col-0 and er-3 grown on MS media under the shade conditions. Scale bar represents 5 mm. (B) Statistical data of hypocotyl length of Col-0 and er-3 under white light and shade conditions. At least 30 seedlings were measured for each genotype. Error bars represent SE. (C) Phenotypes of Col-0 and er-3 grown in the soil under white light and shade conditions. Scale bars represent 10 mm. (D) Statistical data of petiole length of Col-0 and er-3 grown in the soil under white light and shade conditions. At least 30 seedlings were measured for each genotype. Error bars represent SE. Student's t-test indicated the differences are statistically significant (∗∗∗P < 0.001).

at a highest level after 2 h of shade treatment (**Supplementary Figure S1**). These results indicate that ER is responsive to shade at the transcription level, suggesting that ER might function in shade avoidance pathways in the hypocotyl of Arabidopsis.

### ER Family Genes Are Differentially Responsive to Shade

There are three ER family genes, ER and its paralogous genes ERL1 and ERL2 in the Arabidopsis genome. To investigate whether ER family members are responsive to shade stress, we constructed expression vectors harboring a GUS reporter gene driven by the native promoters of ER family genes. As shown in **Figure 2**, ER is principally expressed in the young tissues and the hypocotyls when grown in the white light, whereas slightly expressed in the cotyledons. However, ER shows a higher expression level in the cotyledon, petiole and hypocotyl in the shade condition. ERL1 is slightly expressed on the leaf margin, petiole and meristem, and its expression is remarkably induced in the meristem by shade but only slightly induced in the hypocotyl. The expression of ERL2 is mainly distributed on the leaf margin and the meristem of the seedling, whereas downregulated by shade in the leaves and obviously upregulated in the meristem. These results indicate that ER family genes differentially respond to shade stress and only the expression of ER is most obviously

increased in the hypocotyls, suggesting that ER might contribute more to shade avoidance rather than ERL1 and ERL2 in the hypocotyls.

### Exogenous Auxin Promoted the Hypocotyl Length of er-3 in Shade Response

To examine whether er-3 inhibited hypocotyl elongation in the shade is impaired in auxin biosynthesis, we determined the response of er-3 to indole-3-acetic acid (IAA), the main natural auxin. As shown in **Figure 3**, 0.1 and 0.5 µM of IAA remarkably promoted the hypocotyl elongation of both Col-0 and er-3, and 0.5 µM of IAA fully rescued the hypocotyl length of er-3 than that of Col-0 without IAA treatment. The results indicate that IAA is essential for ER regulated hypocotyl elongation and IAA biosynthesis might be impaired in er-3 in the shade. Further, higher concentrations of IAA inhibit the elongation of both Col-0 and er-3, but er-3 shows a shorter hypocotyl phenotype than that of Col-0 by the same concentration of IAA treatment (**Figures 3A,B**), implying that auxin signaling is also diminished in the er-3 mutant. These results suggested that both auxin biosynthesis and signaling pathways might be impaired in the er-3 mutant in the shade condition.

Genetic and physiological evidences show that IAA is principally biosynthesized from the precursor tryptophan via TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS/SHADE AVOIDANCE 3 (TAA1/SAV3), which catalyzes the production of IPA from L-tryptophan (L-Trp), then the IPA is as the substrate of YUC proteins to produce IAA (Zhao et al., 2001; Tao et al., 2008; Dai et al., 2013). Chemical library screening assay showed that kyn is a Trp analog as an effective competitive inhibitor of TAA1/TARs (TRYPTOPHAN AMINOTRANSFERASE RELATEDs) in Arabidopsis, which can effectively block the steps of Trp to IPA (He et al., 2011). In IPA to IAA steps, yucasin was recently found as a potent inhibitor of YUC enzymes in the major IAA biosynthesis pathways (Nishimura et al., 2014). To further understand whether ER regulated shade avoidance is deficient in auxin biosynthesis and in which step of auxin biosynthesis pathways, the effects of kyn and yucasin on Col-0 and er-3 single mutant were investigated. In the shade condition, the results showed that the hypocotyl elongation of both vertically grown Col-0 and er-3 at a slight angle are inhibited by both kyn and yucasin treatment (**Supplementary Figures S2A,B**). However, elongation of er-3 shows less sensitive to kyn and yucasin than that of the wild type (**Supplementary Figures S2A,B**), suggesting that ER regulated hypocotyl elongation might depend on conversion of both Trp to IPA and IPA to IAA. It is reported that in some growth conditions, the plant appears thigmotropic response (Meng et al., 2012). To clarify that whether the inhibition of hypocotyl elongation by kyn and yucasin is resulted from the thigmotropism, we also test the horizontal growth of the plants on agar plates (0.5% agar) feeding by IAA biosynthesis inhibitors in the shade condition. Plants showed a similar inhibition of the hypocotyl elongation of Col-0 and er-3 by both kyn and yucasin to that grown vertically at a slight angle (**Supplementary Figures S2A,C**). The inhibition of the hypocotyl elongation of Col-0 and er-3 by IAA biosynthesis inhibitor therefore seems to be independent of thigmotropism. These results indicate that auxin biosynthesis is essential for the hypocotyl elongation of Col-0 and er-3 stimulated by shading.

### High Temperature Increases Shade-Stimulated Hypocotyl Elongation of er-3

Previous studies demonstrated that high temperature can increase the endogenous free auxin levels to promote the hypocotyl elongation (Gray et al., 1998). To test whether er-3 regulated hypocotyl elongation in the shade is dependent on endogenous auxin levels, we tested the hypocotyl elongation

SE. Scale bars represent 0.5 cm.

response of er-3 to a high temperature of 30◦C. The results showed that, similar to shade avoidance syndrome, hypocotyl elongation of both Col-0 and er-3 is enhanced by high temperature even in the white light condition, and hypocotyl length of er-3 is rescued similar to that of Col-0 by the same high temperature treatment, indicating that endogenous auxin level is essential for ER-modulated hypocotyl elongation. To further investigate that whether auxin transport is also essential for promoting the hypocotyl elongation of er-3 by endogenous IAA increase, we use a polar auxin transport inhibitor 1-Nnaphthylphthalamic acid (NPA) treatment at 30◦C. As shown in **Supplementary Figure S3**, NPA could slightly inhibit the hypocotyl length of both Col-0 and er-3 that increased by high temperature in the white light, while the hypocotyl length shows no significance between Col-0 and er-3 by 5 µM of NPA treatment. These results suggest that ER-mediated hypocotyl elongation is dependent on endogenous auxin levels but not remarkably depends on auxin transport.

### Exogenous GA3 Increased the Hypocotyl Length of er in the Shade

Gibberellins are another important group of phytohormones required to regulate the hypocotyl elongation in shade avoidance. To understand whether ER modulated hypocotyl elongation is deficient in GA biosynthesis, the effects of various concentrations of GA3 on the hypocotyl elongation of er-3 and Col-0 were investigated (**Figure 4**). The results showed that lower concentrations of GA3 can promote the hypocotyl elongation of both Col-0 and er-3, and the promotion of the hypocotyl length in er-3 is less than that in Col-0 by concentrations of 0.5 and 1 µM of GA3 (**Figures 4A,B**). However, 2 µM of GA3 can stimulate the hypocotyl elongation of er-3 similar to that of Col-0 (**Figures 4A,B**), indicating that the ER regulated hypocotyl elongation is partially dependent of GAs. Furthermore, the hypocotyl elongation of both Col-0 and er-3 are impaired to the similar length by the GA biosynthesis inhibitor PAC

(**Figures 4A,B**), indicating that GAs are important, but not only specific, for ER-mediated shade avoidance. These results suggest that GA level is an important regulator for hypocotyl growth for both the wild type and er-3 mutant in the shade.

### Auxin- and GA-Related Genes Are Differentially Regulated by ER in the Shade

To further elucidate the molecular mechanisms of ER modulated hypocotyl growth, genes related to auxin and GA biosynthesis and signaling pathways were examinated. Auxin biosynthesis gene YUC9 and auxin-responsive genes, IAA29 and SAUR68, whose expression is also rapidly upregulated by shade (Tao et al., 2008; Galstyan et al., 2011), are upregulated in both Col-0 and er-3 under shade condition, and that the expression level is higher in Col-0 than that in er-3, suggesting that ER mutation impairs the expression level of auxin biosynthesis gene YUC9 and auxin response might be impaired in er-3 in the shade. Previous studies revealed that VAS2, encoding an IAA-amido synthetase Gretchen Hagen 3 (GH3).17, is expressed predominantly in the hypocotyl and plays important role in conversion of free IAA to IAA-Glu (IAA-glutamate) independent of IPA-mediated IAA biosynthesis (Zheng et al., 2016). To test whether ER regulated hypocotyl elongation in the shade is dependent on VAS2, we checked the expression level of VAS2 in Col-0 and er-3. The results showed that relative expression of VAS2 is decreased in Col-0 by shading compared to that in the white light condition. While VAS2 is significantly upregulated in shade-treated er-3 compared to that in er-3 grown in the white light, and is much higher in expression than that of Col-0 in the shade condition. These results indicate that VAS2 might also contribute to ER-regulated hypocotyl elongation in the shade.

The GA biosynthetic genes GA20OX1, GA3OX1, and GA1 were induced in the shade treated both Col-0 and er-3, respectively. While expression of GA3OX1 is less in er-3 than that in Col-0, indicating that GA biosynthesis might be diminished in the conversion of GA9/GA20 to GA4/GA1. Expression of GA1 is induced in both Col-0 and er-3 by shade, whereas the expression level is lower in er-3 than that in Col-0 (**Figure 5**). However, the expression of the GA catabolic gene, GA2OX1 is sharply decreased in both shade treated Col-0 and er-3 than that in white light-treated plants. GA2OX1 exhibit a slightly less expression in shade treated er-3 than that in shade-treated Col-0, suggesting that conversion of copalyl diphosphate (CPP) from geranylgeranyl diphosphate (GGPP) in GA biosynthesis might be impaired in er-3. However, significant changes in expression of GA biosynthesis and catabolic genes were not more remarkable than that of genes in auxin homeostasis and signaling pathways, implying that auxin might contribute more to ER-regulated hypocotyl elongation than GA in the shade.

### ER Regulated Hypocotyl Elongation in Response to Shade Is Dependent of PHYB

Pants use red and far-red light-absorbing phytochromes A and B to sense the changes of R:FR ratio, of which phytochrome B (PHYB) play dominant role in shade avoidance inhibition. To determine whether ER-controlled hypocotyl elongation is via PHYB, we crossed er-3 with a T-DNA insertional mutant phyB to generate an er-3 phyB double mutant (**Figure 6A**). In the white light condition, Col-0 and er-3 single mutant show a hypocotyl inhibition phenotype, and phyB single mutant shows a hypocotyl elongation phenotype compared with that of Col-0 and er-3 single mutant. However, er-3 phyB double mutant displays a hypocotyl elongation phenotype mimicking phyB single mutant but ER mutation significantly reduced hypocotyl elongation of phyB (**Figures 6A,B**). These results indicate that PHYB inhibited hypocotyl elongation in the white light is ER dependent. In the shade condition, er-3 single mutant shows a shade avoidance phenotype in the hypocotyl, but elongation of the hypocotyl of er-3 was significantly insensitive to shade compared with Col-0. Hypocotyls of both phyB and er phyB mutants also elongated in the shade condition. However, er-3 phyB is less sensitive to shade compared to the phyB single mutant, but er-3 phyB double mutant shows more sensitive in hypocotyl elongation compared to that of er single mutant, indicating that ER promoted hypocotyl elongation depends on PHYB (**Figures 6A,B**).

### DISCUSSION

Hypocotyl growth is stimulated by various factors from internal signals and surrounding environment and controlled by complicated signaling networks. Plant receptor-like kinases play critical roles in perception of environmental signals. However, researches on RLK-mediated shade avoidance are seldom reported. Previous studies demonstrated that the leucine-rich repeat receptor-like kinase ER not only regulates Arabidopsis growth and development, but also plays a role in response to environmental stimuli (Torii et al., 1996; van Zanten et al., 2009; Shpak, 2013). Our study demonstrated that a T-DNA insertional mutation of Arabidopsis ER in Clo-0 background inhibits hypocotyl elongation in shade avoidance. Previous studies showed that Arabidopsis ER regulates petiole elongation and leaf hyponasty response in shade avoidance in a temperature-dependent manner in the Arabidopsis Landsberg ecotype (Patel et al., 2013). Another study from an independent group showed that ER contributes to hypocotyl, petiole and lamina elongation, hyponastic growth and flowering time in response to end-of-day far-red (R/FR) light in a genetic background-dependent manner (Kasulin et al., 2013). However, mechanisms of ER controlled shade avoidance are unknown yet.

Our physiological evidence revealed that ER regulates hypocotyl elongation in shade avoidance probably via auxin homeostasis and signaling pathways, and GA homeostasis pathways. Firstly, expression pattern analyses showed the expression of ER, but not its homologs ERL1 and ERL2, which is specifically induced by shade in the Arabidopsis hypocotyl, suggesting that ER might play roles in hypocotyls in shade avoidance distinct from ERL1 and ERL2. Moreover, a cis-acting regulatory element prediction of the ER promoter region by PlantCARE showed that 14 light responsive elements are found

in the promoter of ER (**Supplementary Figure S4**), providing a clue that ER might involve in light responsiveness. Secondly, exogenous IAA feeding promoted the hypocotyl elongation of both Col-0 and er-3 in the shade, while exogenous IAA only partially promoted the hypocotyl elongation of er-3 comparing to that of the wild type, suggesting that ER-mediated hypocotyl elongation might be partially dependent of auxin biosynthesis. Nevertheless, er-3 was relatively insensitive to IAA treatment than the wild type in the same concentration of IAA in the shade (**Figure 3**), implying that the auxin signaling pathways might be also impaired in the er-3 mutant compared to the wild type in the shade. Previous studies showed that indole-3-pyruvic acid (IPyA) pathway is a major IAA biosynthesis pathway from tryptophan in Arabidopsis, in which the tryptophan aminotransferase SAV3/TAA1 and YUC enzymes play principal roles (Zhao et al., 2001; Tao et al., 2008; Dai et al., 2013). By using a chemical biology approach, Nishimura et al. discovered a potent IAA biosynthesis inhibitor, 5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (yucasin), which can effectively inhibit the activities of YUC thus impaired IAA production in planta (Nishimura et al., 2014). Another study showed that kyn is a tryptophan analog which can effectively inhibit Arabidopsis SAV3/TAA1/TARs identified from a chemical library screen (He et al., 2011). Our results showed that both yucasin and kyn can effectively inhibit the hypocotyl elongation of Col-0 and er-3, suggesting that both yucasin and kyn are potent inhibitors for Arabidopsis in the shade. Thirdly, it is known that high temperature can increase the endogenous auxin levels to promote the hypocotyl elongation of Arabidopsis and the expression of auxin biosynthesis gene YUC9 is also induced by shade (Gray et al., 1998; Müller-Moulé et al., 2016), our results revealed that high temperature can enhance the hypocotyl

elongation of er-3 similar to that of Col-0 in the white light condition (**Supplementary Figure S3**) and auxin biosynthesis gene YUC9 is less induced by shade in er-3 than that in Col-0 (**Figure 5**), indicating that endogenous auxin homeostasis makes an important contribution to ER-mediated hypocotyl elongation in the shade. This conclusion also supported by another recent study which revealed that auxin biosynthesis is essential for ER regulated cell elongation in the hypocotyl under white light (Qu et al., 2017). It is reported that VAS2 controls the endogenous free IAA levels by conversion from IAA-Glu in the hypocotyl epidermis to promote the hypocotyl elongation (Zheng et al., 2016). Our qPCR results showed that relative expression of VAS2 is down-regulated in Col-0 but upregulated in er-3 by shading (**Figure 5**), suggesting that auxin metabolism might also contribute to ER-mediate shade avoidance in hypocotyl elongation. Auxin transport also plays roles in plant hypocotyl elongation in the white light condition (Jensen et al., 1998; Wu et al., 2016), our results indicated that auxin transport might be not essential for ER-regulated hypocotyl elongation, in that NPA treatment cannot cause the significant reduce in hypocotyl elongation of er-3 compared to that of Col-0 in white light with high temperature conditions. These results suggested that auxin transport seems to be less important than auxin homeostasis in ER-mediated hypocotyl elongation in shade avoidance.

To date, the mechanisms of actions for auxin transport inhibitors have remained poorly understood (Teale and Palme, 2017). Although our physiological assays suggested that auxin transport inhibitors did not directly significantly affect the hypocotyl growth of er-3 than that of Col-0, previous studies reported that long-term application of high concentrations of auxin to the roots leads to changes of auxin transport probably through auxin-dependent transcriptional control (Sieberer et al., 2000; Vieten et al., 2005; Vanneste and Friml, 2009). Moreover, high temperature is able to impact on membrane fluidity vesicular trafficking, and other hormonal responses (Kim and Portis, 2005; Asensi-Fabado et al., 2013; Hanzawa et al., 2013). For this reason, we speculate that it is also probably that high temperature induced auxin maxima in the roots or other processes and thus resulted in changes of auxin transport. In addition, several evidences has revealed that NPA probably binds either directly to the auxin efflux carrier including PIN and ABC-transporters (Rubery, 1979; Sussman and Goldsmith, 1981) or to auxin efflux-related regulatory proteins and cytoskeleton (Cox and Muday, 1994; Bailly et al., 2008) to inhibit auxin transport, but it has also been proposed that NPA may have other effects independent of auxin transport (Hössel et al., 2005). Altogether, we could not rule out that auxin balance in the hypocotyl of er-3 might be neither affected by a loss or accentuation of an auxin maxima in the root, leading to alteration of auxin transport, nor affected by other biological processes, which might finally result in changes of ER-mediated hypocotyl elongation. In the future, more molecular and physiological evidences are required to uncover whether and how auxin transportation involving auxin transporters contributes in ER-regulated hypocotyl growth in shade avoidance.

Our exogenous GA3 feeding increased the hypocotyl length of er-3 to the length of the wild type in the shade, suggesting that GA3 biosynthesis might be diminished in er-3 under shade condition. Relative expression of genes in GA biosynthesis and catabolism pathways in er-3 are less than that in the wild type, suggesting that ER modulates hypocotyl elongation might depend on GA homeostasis. Nevertheless, more genetic and biochemical evidences need to be done to elucidate the detailed mechanisms underlying the crosstalk between auxin and GA in ER regulated shade avoidance in the future. PHYB is the main photoreceptor in perception of changes of light quality of red to far-red light wave length. Previous studies show that ER modulates low light intensity induced petiole elongation independent of PHYB (van Zanten et al., 2010). Our genetic evidence shows that loss-of-function of PHYB can promote the hypocotyl elongation of er-3, whereas the hypocotyl length of the double mutant of er-3 phyB is shorter than that of phyB single mutant, suggesting that ER regulated hypocotyl elongation depends on PHYB, and phyB-mediated promotion of hypocotyl elongation is partially dependent of ER. However, the detailed molecular mechanisms of how does PHYB function in ER-mediated shade avoidance pathways needs further investigation. Interestingly, protein–protein interaction analyses by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING<sup>2</sup> ) show that ER and PHYB are in the same protein complex (**Supplementary Figure S5**), thus we speculate that it is probably ER and PHYB function together in hypocotyl

<sup>2</sup>http://string-db.org

elongation in a same protein complex. Furthermore, extensive evidence showed that RLKs play roles in plant growth and development, and defense with their co-receptors as receptor complex in previous studies. For instance, the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) interacts with its co-receptors SOMATIC EMBRYOGENESIS RECEPTOR KINASEs (SERKs) to perceive and transduce BR signals to regulate plant growth and development (Li et al., 2002; Nam and Li, 2002; Gou et al., 2012). EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2) interacts with SERKs to sense the flagellin22 in plant defense signaling pathways (Zipfel et al., 2006; Chinchilla et al., 2007; Roux et al., 2011; Stegmann et al., 2017). ER family receptors interact with a receptor-like protein TOO MANY MOUTH (TMM) to form a homo- and heterodimer receptor complex to regulate stomatal development (Shpak et al., 2005). Receptor-like kinases FERONIA and THESEUS1 were found to control the shoot elongation of Geranium pyrenaicum identified by transcriptome analyses (Gommers et al., 2017). We therefore hypothesize that ER may function with unknown co-receptors to regulate hypocotyl elongation in shade avoidance by regulating the auxin homeostasis and signaling and GA homeostasis related networks. Further studies will focus on disclosing the detailed molecular mechanisms, including the crosstalk of auxin and GA in ER regulated hypocotyl elongation in shade avoidance.

### AUTHOR CONTRIBUTIONS

JD and WY designed the experiments. JD, HJ, XS, YaL, MS, YiL, ZF, QC, LF, JS, KS, JL, WL, FY, TY, XW, SY, LY, and CL performed the experiments. JD, HJ, XS, and YaL analyzed the data. JD, HJ, XS, and YaL wrote the manuscript.

### FUNDING

This work was supported by the National Natural Science Foundation of China grants 31401308, 31671445, and 31371555.

### REFERENCES


### ACKNOWLEDGMENTS

The authors thank Dr. Tomokazu Koshiba at Tokyo Metropolitan University for generously providing yucasin and Mr. George Bawa and Muhmmad Ahsan Asghar for reading and commenting the manuscript. They are also grateful to the Arabidopsis Biological Resource Center (ABRC) for providing some of the T-DNA insertional mutant seeds.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.00124/ full#supplementary-material

FIGURE S1 | Time course of gene expression in Col-0 induced by shade. ER and the shade inducible genes PIL1 and XTR7 were upregulated in the shade. ACT2 was amplified for 23 cycles as an internal control.

FIGURE S2 | Exogenous feeding of kyn and yucasin can inhibit shade stimulated hypocotyl elongation. (A) Phenotypes of Col-0 and er-3 seedlings vertically with a slight angle or horizontally grown with treatment of different concentrations of kyn and yucasin in the shade. Scale bar represents 1 cm. (B) Statistical data of hypocotyl length of vertically grown Col-0 and er-3 with a slight angle treated by kyn and yucasin under shade condition. (C) Statistical data of hypocotyl length of horizontally grown Col-0 and er-3 treated by kyn and yucasin under shade condition. Student's t-test indicated the differences are statistically significant ( ∗∗∗P < 0.001). At least 15 seedlings were measured for each genotype. Error bars represent SE.

FIGURE S3 | High temperature promoted hypocotyl elongation of both Col-0 and er-3. (A) Phenotype of Col-0 and er-3 treated with 22◦C and 30◦C in the while light or shade conditions. 5 µM of NPA feeding were used at 30◦C in the shade. (B) Statistical data for Col-0 er-3 with/without NPA treatment grown at 22◦C and 30◦C in the while light or shade conditions.

FIGURE S4 | Motif prediction of the promoter of ER by PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The yellow boxes show the motifs are light responsive elements.

FIGURE S5 | Prediction of interacting proteins of PHYB and ER in Arabidopsis by STRING (http://string-db.org/).

TABLE S1 | Primers used in this study.


brassinosteroid-independent actions in Arabidopsis thaliana. J. Integr. Plant Biol. 54, 388–399. doi: 10.1111/j.1744-7909.2012.01124.x


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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Du, Jiang, Sun, Li, Liu, Sun, Fan, Cao, Feng, Shang, Shu, Liu, Yang, Liu, Yong, Wang, Yuan, Yu, Liu and Yang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fpls-09-00124 February 5, 2018 Time: 14:54 # 12

# Activation of the LRR Receptor-Like Kinase PSY1R Requires Transphosphorylation of Residues in the Activation Loop

#### Christian B. Oehlenschlæger<sup>1</sup> , Lotte B. A. Gersby<sup>1</sup> , Nagib Ahsan<sup>2</sup>† , Jesper T. Pedersen<sup>1</sup>† , Astrid Kristensen<sup>1</sup> , Tsvetelina V. Solakova<sup>1</sup> , Jay J. Thelen<sup>2</sup> and Anja T. Fuglsang<sup>1</sup> \*

<sup>1</sup> Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark, <sup>2</sup> Christopher S. Bond Life Sciences Center, Department of Biochemistry, University of Missouri, Columbia, MO, United States

PSY1R is a leucine-rich repeat (LRR) receptor-like kinase (RLK) previously shown to act as receptor for the plant peptide hormone PSY1 (peptide containing sulfated tyrosine 1) and to regulate cell expansion. PSY1R phosphorylates and thereby regulates the activity of plasma membrane-localized H+-ATPases. While this mechanism has been studied in detail, little is known about how PSY1R itself is activated. Here we studied the activation mechanism of PSY1R. We show that full-length PSY1R interacts with members of the SERK co-receptor family in planta. We identified seven in vitro autophosphorylation sites on serine and threonine residues within the kinase domain of PSY1R using mass spectrometry. We furthermore show that PSY1R autophosphorylation occurs in trans and that the initial transphosphorylation takes place within the activation loop at residues Ser951, Thr959, and Thr963. While Thr959 and Thr963 are conserved among other related plant LRR RLKs, Ser951 is unique to PSY1R. Based on homology modeling we propose that phosphorylation of Ser951 stabilize the inactive conformation of PSY1R.

Keywords: receptor kinase, phosphorylation, mass spectrometry, PSY1 peptide, signaling peptides, LRR, activation loop

## INTRODUCTION

As multicellular organisms, plants rely on a fine-tuned cell-to-cell communication system to coordinate growth responses. At the core of this system are members of the receptor-like kinase (RLK) superfamily. This massive family of plant proteins, which is implicated in both plant development (De Smet et al., 2009; Murphy et al., 2012) and plant innate immunity (Schwessinger and Ronald, 2012; Tang et al., 2017), maintains an appropriate balance between growth and defense. Plant RLKs belong to the RLK/Pelle class of protein kinases, which is composed of over 600 members in Arabidopsis (Gish and Clark, 2011), and are related to the mammalian Receptor Tyrosine Kinases (RTKs) (Shiu and Bleecker, 2001; Belkhadir et al., 2014). Most plant RLKs share the overall architecture of RTKs, with an extracellular ligand-binding domain, a single transmembrane-spanning domain, and a conserved intracellular kinase domain (KD).

Until recently, it was believed that most plant cell-to-cell communication was mediated by small lipophilic compounds, such as phytohormones and steroids, only. However, plant peptide hormones, which are expressed as precursor peptides (pre-pro-peptides) and undergo

### Edited by:

Yi Ma, University of Connecticut, United States

#### Reviewed by:

Jian Huang, University of Wisconsin–Milwaukee, United States Yukihiro Ito, Tohoku University, Japan

### \*Correspondence:

Anja T. Fuglsang atf@plen.ku.dk

#### †Present address:

Nagib Ahsan, Division of Biology and Medicine, Brown University, Providence, RI, United States; Center for Cancer Research Development, Proteomics Core Facility, Rhode Island Hospital, Providence, RI, United States Jesper T. Pedersen, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

#### Specialty section:

This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science

Received: 01 September 2017 Accepted: 10 November 2017 Published: 27 November 2017

#### Citation:

Oehlenschlæger CB, Gersby LBA, Ahsan N, Pedersen JT, Kristensen A, Solakova TV, Thelen JJ and Fuglsang AT (2017) Activation of the LRR Receptor-Like Kinase PSY1R Requires Transphosphorylation of Residues in the Activation Loop. Front. Plant Sci. 8:2005. doi: 10.3389/fpls.2017.02005

post-translational modification followed by secretion via the secretory pathway, are emerging as important players in this process (Matsubayashi, 2011; Murphy et al., 2012). Plant peptide containing sulfated tyrosine 1 (PSY1; Amano et al., 2007) and phytosulfokine (PSK; Matsubayashi and Sakagami, 1996) are plant peptide hormones responsible for cell elongation activity in the elongation/differentiation zone of the root (Sauter et al., 2009; Matsubayashi et al., 2010) and hypocotyl (Stührwohldt et al., 2011; Fuglsang et al., 2014). The receptors for PSY1 and PSK, termed PSY1R and PSKR1 and PSKR2, respectively, form a small family of redundant RLKs (Matsubayashi et al., 2002; Amano et al., 2007) that are members of the leucine-rich repeat (LRR) RLK subclass of plant RLKs, which is encoded by 216 genes in Arabidopsis. The downstream signaling events of PSY1R and PSKR have not been fully characterized; however, PSKR contains a guanylate cyclase catalytic center in its KD, which enables signaling through the formation of the secondary messenger cyclic guanosine monophosphate (cGMP) (Kwezi et al., 2011). PSY1R was recently shown to regulate cell expansion by phosphorylating the plant plasma membrane H+-ATPase (AHA2) and thereby promoting proton pumping (Fuglsang et al., 2014). Proton pumping is observed upon addition of PSY1 peptide and is dependent on the presence of PSY1R (Fuglsang et al., 2014). Interestingly, the growth-promoting effects of PSK do not require extracellular acidification by plasma membrane H+-ATPases (Stührwohldt et al., 2011) and PSKR1 does not interact with AHA2 in a Bimolecular Fluorescence Complementation (BiFC) assay (Fuglsang et al., 2014), but the two proteins do co-localize in the plasma membrane (Ladwig et al., 2015).

The plant RLK activation mechanism differs from the activation mechanism of mammalian RTKs. In the classical RTK activation known from the mammalian field, ligandinduced RTK homodimerization brings the intracellular KDs into proximity, which thereby allows for intermolecular transphosphorylation and concomitant activation of the KDs and downstream signaling (Lemmon and Schlessinger, 2010). Transphosphorylation within the activation loop is also a central regulatory element of plant RLKs, as demonstrated in phosphorylation studies (Oh et al., 2000; Karlova et al., 2009) and further supported by crystal structures of plant RLK KDs containing phosphorylated residues (Yan et al., 2012; Bojar et al., 2014). Homodimerization, in some cases ligand-induced, has also been observed for a number of plant RLKs, including the S-locus Receptor Kinase (SRK) (Giranton et al., 2000), Brassinosteroid Insensitive 1 (BRI1) (Wang et al., 2005b), Flagellin Sensing 2 (FLS2) (Sun et al., 2012), and the CLV/BAM family (Guo et al., 2010).

However, about a decade ago, an interaction partner of BRI1 was identified and named BRI1-Associated Kinase 1 (BAK1) (Li et al., 2002; Nam and Li, 2002). Sequential transphosphorylation within the BRI1/BAK1 complex was demonstrated, showing that BRI1 is fully activated by BAK1 transphosphorylation within the juxtamembrane and C-terminal domains (Wang et al., 2008). BAK1 is also known as Somatic Embryogenesis Receptor-like Kinase 3 (SERK3) and belongs to a small family of SERK proteins consisting of five members (SERK1–5), each containing five LRRs in the extracellular domain (Hecht et al., 2001). SERK proteins are implicated in a range of diverse processes, including plant cell differentiation, growth, and immunity (Ma et al., 2016).

Additionally it has been found that SERKs acts as co-receptors for a range of LRR-RLKs. Each ligand-binding receptor seems to only interact with a limited number of SERK proteins (Ma et al., 2016). For example, BRI1 is regulated by SERK1, SERK4, and BAK1 (SERK3), whereas PSKR1 is regulated by SERK1, SERK2, and BAK1 (Wang et al., 2015). SERK proteins positively modulate the activity of its interaction partners by transphosphorylation (Schulze et al., 2010).

In this study, we investigated the transactivation mechanism of PSY1R and identified its in vitro autophosphorylation sites. By homology modeling to known structures of BRI1 and SIRK1 we propose how these residues are involved in stabilization of the activation loop in the active and inactive state, respectively. We furthermore showed that PSY1R interacted with members of the SERK family in planta.

### MATERIALS AND METHODS

### DNA Cloning

A list of primers and plasmids used in this study can be found in the Supplementary Tables 1 and 2. Full-length SERK2, SERK4, and SERK5 cDNAs were amplified from an Arabidopsis thaliana Col-0 cDNA preparation. A cDNA clone of SERK1 was kindly provided by Professor S. C. de Vries (University of Wageningen). The PCR fragments were subcloned into Gateway pENTR/D-TOPO vector (Invitrogen Life Technologies). The BAK1 cDNA clone in the pCR8/GW/TOPO vector was obtained from Arabidopsis Biological Resource Center (ABRC) (clone CIW00115). The SERK, and BAK1 genes were cloned into Gateway-compatible BiFC vectors by LR recombination using LR Clonase II Enzyme Mix (Invitrogen Life Technologies) to yield C-terminal fusions to cCFP or nYFP expressed from the 35S promoter. The PSY1R BiFC constructs were used in a previous study (Fuglsang et al., 2014) and the PSY1R K831A mutation, corresponding to the invariant lysine residue present in the protein kinase catalytic domain (Carrera et al., 1993), was generated through QuikChange Site Directed Mutagenesis (Agilent Technologies).

To generate constructs for expression in E. coli, the region of PSY1R encoding the entire intracellular domain of PSY1R was amplified with gene-specific primers carrying a 5<sup>0</sup> CACC overhang for subcloning in the Gateway pENTR/D-TOPO vector (Invitrogen Life Technologies). Mutations and stop codons were introduced through QuikChange Site Directed Mutagenesis. The constructs were transferred into pDEST15 and pDEST17 (Invitrogen Life Technologies) through the LR reaction. All constructs were sequenced by Eurofins MWG Operon.

### Transient Expression in Nicotiana benthamiana

Transformed Agrobacterium tumefaciens strain C58C1 was grown overnight in liquid YEP medium containing 25 µg/mL gentamicin and 50 µg/mL spectinomycin. Cells were washed and resuspended in infiltration solution (10 mM MgCl2, 100 µM acetosyringone), and diluted to an OD<sup>600</sup> of 0.05, before mixing the transformed cells in the combinations to be tested. N. benthamiana leaves were infiltrated with the A. tumefaciens mix using a needleless syringe. Fluorescence was monitored approximately 48 h after infiltration.

### Confocal Microscopy

fpls-08-02005 November 24, 2017 Time: 16:12 # 3

A Leica SP5 confocal laser-scanning microscope with a 20 × 0.7 numerical aperture water-immersion objective was used to examine the lower epidermis of the infiltrated tobacco leaves. The complemented YFP/CFP fluorescence was excited at 448 nm and emission was detected at 515–540 nm. The gain was fixed in all samples to ensure that the emission intensity was comparable. Interaction was tested using both combinations of fusion proteins with similar results.

### Expression and Purification of Recombinant Protein in E. coli

GST-tagged proteins were expressed either from the pGEX-4T-1 vector (GE Healthcare Life Sciences) in BL21(DE3) cells or from the pDEST15 vector in BL21-AI cells (Invitrogen Life Technologies). Briefly, LB medium was inoculated with an overnight LB + 100 µg/mL ampicillin culture to a start OD<sup>600</sup> of 0.1 and grown at 37◦C until the OD<sup>600</sup> reached 0.5–0.7. Expression from the pDEST15 plasmid in BL21-AI cells was induced by the addition of L-(+)-arabinose to a final concentration of 0.2% (w/v) and the culture was incubated at 20◦C overnight. Expression from the pGEX-4T-1 vector in BL21(DE3) cells was induced by the addition of 100 µM IPTG and the culture was incubated at 28◦C for 3–4 h. In both cases, the cells were harvested at 14,000×g for 10 min, washed in cold H2O, and pelleted again. The cells were resuspended in P-buffer (50 mM Tris–HCl, 150 mM NaCl, pH 7.5) containing 1 mM PMSF, 0.01% (w/v) DNase I, and 0.01% (w/v) lysozyme and lysed by sonication. The cell debris was collected by centrifugation at 15,000×g for 30 min and the lysate was incubated with Glutathione Sepharose 4B (GE Healthcare) for 2 h at 4◦C. The resin was washed three times with P-buffer before eluting the protein with 50 mM L-glutathione in P-buffer, adjusted to pH 8.0. His-tagged proteins were expressed from the pDEST17 plasmid in BL21-AI cells as described above. The cells were harvested as described above, resuspended in lysis buffer (50 mM Na-phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0) containing 1 mM PMSF, 0.01% (w/v) DNase I, and 0.01% (w/v) lysozyme and opened by sonication. The cell lysate was collected as described above and incubated with Ni-NTA agarose (Qiagen) for 2 h at 4◦C. The resin was washed three times with wash buffer (as lysis buffer, but containing 60 mM imidazole) before eluting the protein with elution buffer (as lysis buffer, but containing 250 mM imidazole).

### Phosphorylation Assays

In a radiometric assay, purified kinase was incubated for 30 min at 30◦C in kinase assay buffer (50 mM HEPES-NaOH pH 7.2, 150 mM NaCl, 10 mM MgCl2, 1 mM DTT) with 50 µM ATP and 5 µCi γ-<sup>32</sup>P-ATP (PerkinElmer) in a volume of 50 µL. The reaction was stopped by the addition of 10 µL 100% (w/v) TCA. The TCA-precipitated protein was dissolved in twofold concentrated Laemmli buffer and proteins were separated on a 10% SDS-PAGE gel. After the gel was stained and dried, it was exposed to a phosphor screen for 3 days and phosphorylation was detected using a Storm 860 scanner (Molecular Dynamics).

For detection of phosphorylated proteins with phosphoamino acid-specific antibodies, purified kinase was incubated for 1 h at 30◦C in kinase assay buffer with 500 µM ATP. As above, the reaction was stopped by TCA precipitation and proteins were separated by SDS-PAGE. The protein bands were blotted onto a nitrocellulose membrane and the membrane was blocked with 3% (w/v) BSA. Rabbit polyclonal phosphoserine (Invitrogen Life Technologies, 61-8100, 1:200) and phosphothreonine (Invitrogen Life Technologies, 71-8200, 1:1000) antibodies were used as primary antibodies and polyclonal goat anti-rabbit alkaline phosphatase-coupled immunoglobulin (DAKO, D0487, 1:2000) were used as secondary antibody. The immunoblot was developed using NBT/BCIP substrate (Promega, S3771).

### Homology Model of PSYR1 Kinase Domain

Modeller v9.19 software<sup>1</sup> (Sali and Blundell, 1993) was used to generate the homology models with maize SIRK1 KD (PDB ID: SIRK1) and BRI1 KD (PDB ID: 5LPV) as templates. The templates were alignment with the KD of PSY1R (residue 781–1079) individually and as a multiple alignment with both templates. Models were initially evaluated using discrete optimized protein energy (DOPE) score and using energy plots that show problematic regions. The generated models were further evaluated and energy refined using the SAVES<sup>2</sup> and GALAXY (Heo et al., 2013) server, respectively. Final models were visualized using PyMOL (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC) and the inhibitor AMP-PNP and Mg2<sup>+</sup> were modeled into the homology models to visualize the catalytic ATP binding site. The energy plots were visualized using Gnuplot 5.0.

### MS/MS Detection of Phosphosites

Auto- and transphosphorylated sites of the intracellular domain of PSY1R were detected using a LTQ Orbitrap XL ETD mass spectrometer (Thermo Fisher Scientific, San Jose, CA, United States). Full-length recombinant proteins were digested in-solution and/or in-gel with sequencing grade trypsin (Promega, Madison, WI, United States). Freeze-dried tryptic peptides were dissolved by adding 40 µL of 0.1% formic acid and subjected to MS analysis. Ten microliters of each sample was resolved using a Finnigan Surveyor liquid chromatography system interfaced with the mass spectrometer. Tryptic peptides were fragmented using either collision-induced dissociation (CID) or "decision tree" methods that utilize both CID and electron-transfer dissociation (Swaney et al., 2008).

<sup>1</sup>http://www.salilab.org

<sup>2</sup>https://services.mbi.ucla.edu/SAVES/

The MS RAW files were searched against the TAIR10 database combined with a decoy database containing the randomized sequences of the original database. The search parameters, described in detail previously (Ahsan et al., 2013), were briefly as follows: the mass type, average precursor plus fragment; dynamic modifications, phosphorylation of Ser/Thr/Tyr (+79.9799 Da) and oxidation of Met (+15.9994 Da); and the static modification, Cys-carboxyamidomethylation.

Identification data were evaluated using the XCorr function of SEQUEST, and phosphorylation-site localization was accomplished using phosphoRS (Proteome Discoverer, v. 1.0.3, Thermo Fisher Scientific). The XCorr values for each charge state were set to default, and no decoy hits were allowed. Peptide mass deviation was 10 ppm and two peptides/protein were used to further filter the data. Phosphopeptides with a pRS score of ≥50 and a pRS site probability of ≥50% were considered as high-confidence phosphosites. For final validation, each spectrum was inspected manually and accepted only when the phosphopeptide had the highest pRS site probability, pRS score, XCorr value, and site-determining fragment ions allowed for unambiguous localization of the phosphorylation site.

### RESULTS

### Identification of PSY1R in Vitro Autophosphorylation Sites

We set out to investigate the autophosphorylation mechanism of PSY1R. The cytosolic KD of PSY1R (residues 741–1095; kPSY1R) were expressed in E. coli as GST-fused protein (**Figure 1**). An inactive variant of the kinase were generated by mutating the lysine residue in the catalytic domain to an alanine (kPSY1R K831A). Recombinant kPSY1R were incubated in an in vitro kinase assay in the absence or presence of ATP and phosphorylated amino acids were detected with phosphoamino acid-specific antibodies. As seen in **Figure 1**, phosphoserine (pSer) and phosphothreonine (pThr) antibodies reacted with the active kPSY1R protein, but weakly or not at all with the inactive kPSY1R K831A. This implies that PSY1R autophosphorylates on Ser and Thr residues. The addition of ATP to the kinase reaction assay increased the phosphorylation level especially of Thr residues. The presence of phosphorylated residues in samples without ATP indicates the kinase is active and autophosphorylated when expressed in E. coli. The detected phosphorylation of kPSY1R is, however, true autophosphorylation, since phosphorylation of the inactive kPSY1R K831A was nearly undetectable.

We then mapped the autophosphorylation sites of the intracellular domain of PSY1R. We incubated kPSY1R in the absence or presence of ATP to allow autophosphorylation. Afterward the proteins were digested with trypsin. The tryptic peptides were analyzed by LTQ-Orbitrap high-resolution mass spectrometry (MS/MS) to map phosphorylation events. Examples of MS/MS spectra for two of the determined autophosphorylation sites are shown in **Figure 2A**. These spectra presented evidence for phosphorylation of the peptides AKHENLVALQGYCVHDsAR and DIKSsNILLDGNFK

(lowercase letters denote phosphorylated residues), which contain the Ser870 and Ser933 phosphosites, respectively. We unambiguously identified seven total autophosphorylation sites (**Figure 2B**), including three threonine and four serine residues, each of which were located within the KD. Detailed information on the identified phosphopeptides is listed in **Table 1**. The PSY1R intracellular domain contains 12 possible phosphorylation targets within the juxtamembrane domain (JMD) (8 Ser, 2 Thr, and 2 Tyr), 35 possible targets within the KD (13 Ser, 11 Thr, and 11 Tyr), and 1 possible target within the C-terminal domain (1 Thr). However, autophosphorylation of JMD or C-terminal domain residues was not detected in our MS analysis. Spectral

FIGURE 2 | Mapping of autophosphorylation sites in recombinant kPSY1R. (A) Upper panel: The MS/MS spectrum of the doubly charged phosphopeptide DIKSsNILLDGNFK provides evidence of phosphorylation of Ser933, which is found in the Ser/Thr protein kinase active site signature. Lower panel: The MS/MS spectrum of the triple charged peptide AKHENLVALQGYCVHDsAR provides evidence for phosphorylation of Ser870 within the kinase domain. N-terminal and C-terminal peptide sequence ions are indicated with b<sup>i</sup> and y<sup>i</sup> , respectively. In the spectra, lines and stars indicate the neutral loss of the phosphate on fragments or precursor ions and the phosphorylated residues, respectively. (B) Linear domain structure of the intracellular domain of PSY1R revealing the localization of the seven autophosphorylation sites in the kinase domain identified by MS. (C) Quantitative MS analysis of the autophosphorylation sites. Spectral count data (peptide spectral matches, PSM) are the means of four replicates. Percentages above the data bars indicate the pRS site probability for each amino acid. Solid and empty bars represent the presence and absence of ATP, respectively. Error bars represent means ± SE of four independent replicates.


<sup>a</sup>pRS score. This peptide score is based on the cumulative binomial probability that the observed match is a random event. The value of the pRS score strongly depends on the data scored, but usually scores of above 50 give good evidence for a good PSM. <sup>b</sup>pRS site probabilities. For each phosphorylation site this is an estimation of the probability for the respective site being truly phosphorylated.

count analyses revealed that the addition of ATP during the in vitro kinase assay enhanced autophosphorylation of Ser870, whereas the remaining six phosphosites were also identified in the control lacking ATP (**Figure 2C**).

### Autophosphorylation of PSY1R Occurs in Trans

Since the full-length LRR RLK PSY1R protein was previously shown to form homodimers, we were prompted to ask whether the intracellular kinase activation mechanism involved inter- or intramolecular phosphorylation, i.e., trans- or cis-phosphorylation. To investigate this, we constructed a hexahistidine-tagged inactive variant of the intracellular domain of PSY1R (H6-kPSY1R K831A, **Figure 3A**) and incubated it with the active kPSY1R protein. Interestingly, the inactive intracellular domain was phosphorylated in the presence of the active kPSY1R, but not when it was incubated alone (**Figure 3B**, phosphorimage), which demonstrates that in vitro autophosphorylation of the intracellular domain occurs in trans. Transphosphorylation of the inactive H6-kPSY1R K831A by the active kPSY1R was detected when using the pThr antibody, but not with the pSer antibody (**Figure 3B**).

### First Phase Transphosphorylation Sites Include Residues within the Kinase Activation Loop

To examine the transphosphorylation mechanism of kPSY1R in detail, we mapped the transphosphorylation sites of kPSY1R by MS. We incubated the inactive H6-kPSY1R K831A with ATP in the presence or absence of active kPSY1R (GST-tagged) to allow for transphosphorylation. MS analysis identified three phosphorylated sites, Ser951, Thr959, and Thr963, within the KD of H6-kPSY1R K831A after incubation with kPSY1R (**Figure 3B**). No phosphorylated sites were detected in H6-kPSY1R K831A incubated without kPSY1R. Detailed data for the three phosphopeptides AYVADFGLsR, tHVTTELVGTLGYIPPEYGQAWVATLR, and LILPYRTHVTtELVGTLGYIPPEYGQAWVATLR harboring Ser951, Thr959, and Thr963, respectively, are provided in **Table 2**. Interestingly, all three transphosphorylation sites are located within the kinase activation loop, which was previously shown to carry phosphosites important for catalytic kinase activity in other kinases (Huse and Kuriyan, 2002). Spectral count analysis demonstrated that the phosphorylation level of Thr959 and Thr963 was higher than that of Ser951 (**Figure 3C**). This explains why transphosphorylated Ser951 is not detected by the pSer antibody (**Figure 3B**), as this residue has low phosphorylation levels compared to Thr959 and Thr963. Alignment of the activation loops from a number of plant LRR RLKs, showed that residues corresponding to Thr959 and Thr963 are conserved within in the activation loop whereas the Ser951 residue is unique for PSY1R (**Figure 3D**).

To verify the transphosphorylation sites identified by MS, we created a new construct (H6-kPSY1R K831A-3x) in which all three proposed transphosphorylation sites (Ser951, Thr959, and Thr963) were mutated to alanine. We then asked whether the active kPSY1R could transphosphorylate H6-kPSY1R K831A-3x. As shown in **Figure 3B**, transphosphorylation of threonine residues was completely abolished in H6-kPSY1R K831A-3x. This indicates that Thr959 and Thr963 are exclusive first-phase transphosphorylation sites.

### Residue Thr968 Is Indispensable for kPSY1R Kinase Activity, While Ser951 Is a Possible Negative Regulatory Phosphorylation Site

To evaluate the in vitro function of each possible autophosphorylation site on the activity of kPSY1R, we generated a series of point mutations. Each of the autophosphorylation sites identified by MS as well as all possible phosphorylation sites within the activation loop were mutated individually to alanine residues in wild-type kPSY1R, thereby preventing phosphorylation at the corresponding residue. As shown in **Figure 4A**, only mutation of residue T968 in the activation loop affected the autophosphorylation severely, resulting in a nearly inactive kinase. By contrast, mutation of S951 led to an increased autophosphorylation level, suggesting that phosphorylation of S951 negatively regulates kPSY1R activity (**Figure 4**).

To visualize and to identify a possible structural explanation for this positive and negative regulation by Ser/Thr phosphorylation we used Modeller 9.19 to predict a homology model of PSY1R KD (**Figure 5**). PSY1R was modeled in the active conformation using the crystal structure of phosphorylated BRI1 KD as template (Bojar et al., 2014). To predict PSY1R in the

FIGURE 3 | First phase transphosphorylation sites of kPSY1R. (A) Linear domain structure of the intracellular domain of the H6-kPSY1R K831A inactive mutant revealing the localization of the three transphosphorylation sites identified by MS. Juxtamembrane domain (JMD) is indicated by gray and C-terminal domain by dark gray shading. (B) The GST-tagged kPSY1R is able to transphosphorylate the inactive His-tagged kPSY1R K831A in an in vitro kinase assay with radiolabeled ATP. Transphosphorylation of H6-kPSY1R K831A studied using a combination of phosphoserine and phosphothreonine antibodies and radiolabeled ATP. Mutation of the three transphosphorylation sites (3x = S951A, T959A, and T963A) identified by MS prevents transphosphorylation on threonine residues while overall transphosphorylation is reduced markedly as detected with radiolabeled ATP. (C) Average phosphopeptide spectral matches (PSM) identified for each phosphosite. The data represent the means ± SE of two independent replicates. (D) Amino acid sequence alignment of the kinase activation loop of selected plant LRR receptor-like kinases. The activation loop begins at the DFG motif and ends with the A/PPE motif. Serine and threonine residues are shaded in gray. The location of the transphosphorylated sites in PSY1R are indicated above the alignment. Asterisks denote conserved residues, while colons denote partially conserved or similar residues at the indicated position.

TABLE 2 | Mass spectrometry data on the peptides carrying the PSY1R in vitro transphosphorylation sites.


<sup>a</sup>pRS score. This peptide score is based on the cumulative binomial probability that the observed match is a random event. The value of the pRS score strongly depends on the data scored, but usually scores of above 50 give good evidence for a good PSM. <sup>b</sup>pRS site probabilities. For each phosphorylation site this is an estimation of the probability for the respective site being truly phosphorylated.

inactive conformation the recently published crystal structure of the maize SIRK1 KD was used as template (Aquino et al., 2017). However, as the sequence identity between the KDs of SIRK1 and PSY1R is relatively low (34%) and the energy plot (Supplementary Figure 1) indicated several problematic regions the crystal structure of BRI1 was included as template. The model based on the two templates showed a significantly better energy plot than models build on SIRK1 alone as seen in Supplementary Figure 1. The inactive conformation of LRR-RLKs is characterized by a structured activation loop

(**Figure 5A**) (Aquino et al., 2017). As seen in **Figure 5A**, the homology model based on the two templates shows a much more structured activation loop than the model based on BRI1 alone indicating that the model is in the inactive conformation. We next added a phosphate group to Ser-951, Thr-963, and Thr-968 to see whether phosphorylation stabilizes either the active or inactive conformation. As seen in **Figure 5B**, addition of phosphate to the two threonine residues results in two possible salt bridges that can stabilize the active conformation whereas phosphorylation in the inactive conformation did not give rise to any stabilizing interactions (Supplementary Figure 2). In contrast, phosphorylation of Ser-951 seems to stabilize the inactive conformation only as the phosphate group can interact with both the backbone of Thr-963 and Arg-923. These observations support that phosphorylation of Thr-968 leads to increased autophosphorylation whereas Ser-951 seems to be a negative regulatory phosphorylation site.

### kPSY1R Interacts with Members of the SERK Family

BAK1 and other members of the SERK family were previously shown to act as co-receptors for LRR-RLKs such as BRI1 and FLS2. To test whether SERK proteins could regulate PSY1R activity through phosphorylation, we created GST-tagged constructs of the intracellular domain of the SERK proteins (kSERK and kBAK) and tested whether these were able to transphosphorylate the inactive H6-kPSY1R K831A protein. In this case, we used the pThr antibody to detect transphosphorylation. As shown in **Figure 6A**, the intracellular domains of SERK1, SERK2, BAK1, and SERK4 were all able to transphosphorylate H6-kPSY1R K831A, whereas kSERK5 exhibited weak autophosphorylation activity and did not transphosphorylate H6-kPSY1R K831A. In the Col-0 accession, SERK5 harbors an R401L mutation in the otherwise conserved HRD motif of the KD, which could explain the lack of autophosphorylation activity observed in our assay. To test the ability of kPSY1R to transphosphorylate a SERK protein, we constructed an inactive H6-tagged version of the intracellular domain of BAK1, H6-mBAK1 K317A. As seen in **Figure 6B**, kPSY1R was able to transphosphorylate H6-BAK1 K317A. Taken together, these results suggest that one or more of the SERK proteins act as co-receptors for PSY1R via a trans-activation mechanism.

### PSY1R Can Form Homo- and Heterodimers in Planta

Using a BiFC assay, we evaluated the ability of full-length PSY1R and PSY1R mutants to dimerize in planta. As seen in **Figure 6C**, full-length PSY1R forms homodimers in planta, as we reported previously (Fuglsang et al., 2014). PSYR1 protein, harboring a K831A mutation in the KD (psy1rK831A) resulting in loss of kinase activity, also forms homodimers in planta (**Figure 6C**).

We further investigated whether PSY1R also forms complexes with SERK proteins. As shown in **Figure 6D**, PSY1R interacts with SERK5, even though SERK5 seems inactive, and to a lesser extent with BAK1, SERK1, and SERK2, whereas PSY1R barely interacted with SERK4 in the employed BiFC assay. All the interactions take place at the plasma membrane as expected. We did not detect homodimerization of any of the SERK members. Furthermore, PSY1R K831A forms a strong complex with BAK1.

### DISCUSSION

Taken together, we identified three residues within the activation loop of PSY1R, which are part of the first phase transphosphorylation mechanism. By homology modeling to known structures of BRI1 and SIRK1 we demonstrated how these residues are involved in stabilization of the activation loop in the active and inactive state, respectively. Finally we demonstrated

that PSY1R interacts with co-receptors from the SERK family and that kPSY1R are target for SERK kinases.

Mapping of the PSY1R autophosphorylation sites by MS revealed a total of seven autophosphorylation sites, four serine and three threonine residues, all located within the KD, including a single phosphosite (Ser951) within the activation loop. It is surprising that we did not detect any phosphorylation sites within the JMD previously reported to be important for the function of plant RLKs (Yoshida and Parniske, 2005; Xu et al., 2006; Petutschnig et al., 2010; Meyer et al., 2013). The in vivo phosphorylation site database PhosPhAt 4.0 (Durek et al., 2010) contains two PSY1R phosphorylation sites, Ser8 and Ser10, which are probably not authentic PSY1R autophosphorylation sites, as they are located within the predicted signal sequence.

Here we have demonstrated that PSY1R first-phase transphosphorylation occurs within the activation loop, specifically at residues Ser951, Thr959, and Thr963. Phosphorylation of Thr959 and Thr963 were only found in the transphosphorylation experiments and not in the initial mapping of phosphosites. This indicates that these sites are only transiently phosphorylated during the catalytic cycle and therefore caught in the inactive mutant; alternatively a full-length receptor protein is required in order to maintain the conformation with these sites phosphorylated. As seen from an alignment of the activation loops of a number of plant LRR RLKs (**Figure 3D**), the Ser951 residue is not conserved among the aligned RLKs and is unique for PSY1R. Based on homology modeling we propose that the phosphorylated Ser951 stabilizes the inactive conformation of PSY1R. In addition to Ser951, PSY1R contains four threonine residues, Thr959, Thr962, Thr963, and Thr968, which are conserved among the plant RLKs shown here. The residues within the activation loop of plant RLKs are key phosphorylation sites that are important for kinase activity (Oh et al., 2000; Shah et al., 2001; Wang et al., 2005a; Karlova et al., 2009). In the crystal structure of the BAK1 KD, all four threonine residues within the activation loop are phosphorylated and are involved in electrostatic interactions that maintain the conformation of the activation and catalytic loop (Yan et al., 2012). Structurally, the most important residue in BAK1 is Thr450, as it plays a role analogous to the single threonine residue (Thr197) within the activation loop of the mammalian kinase Protein Kinase A (Johnson et al., 1996). Interestingly, we found that the residue in PSY1R that corresponds to BAK1 Thr450, the Thr963 residue, was one of the initial transphosphorylation sites. Based on homology modeling, we speculate that the phosphorylated PSY1R Thr963 residue plays an important role in maintaining the active conformation of an activation loop.

Plant RLKs are categorized into two groups based on whether they are activated through intermolecular trans-phosphorylation or intramolecular cis-phosphorylation. Members of the former group are able to transphosphorylate an inactive version of the KD and show second order kinetics with respect to the kinase concentration and include BRI1 (Wang et al., 2005b), CLV1 (Williams et al., 1997), SERK1 (Shah et al., 2001), and HAESA (Horn and Walker, 1994; Taylor et al., 2013). Members of the latter group do not transphosphorylate an inactive KD and exhibit first order kinetics with respect to kinase concentration and include AtACR4 (Meyer et al., 2011), CrRLK1 (Schulze-Muth et al., 1996), and Xa21 (Liu et al., 2002). In our study, we observed that the KD of PSY1R transphosphorylated the inactive mutant PSY1R K831A. Therefore, we speculate that transphosphorylation is an important event in PSY1R signaling analogous to the proposed activation mechanism of BRI1 (Wang et al., 2008). BRI1 is first partially activated through transphosphorylation within the activation loop. The partially activated BRI1 receptor interacts with and phosphorylates BAK1, which in turn phosphorylates BRI1 on residues within the JMD and C-terminal domain, which fully activates BRI1 (Oh et al., 2014).

It is tempting to speculate that PSY1R is fully activated by SERK phosphorylation, analogous to BRI1. The requirement for a co-receptor would also explain the lack of detected phosphosites

in the PSY1R JMD, as this would require the presence of a co-receptor in the in vitro assay applied for MS studies. In the crystal structure of the closely related PSK receptor PSKR1, a co-receptor is not required for ligand binding but the potential involvement of a co-receptor for the complex activation was suggested and it was found that PSK induces the heterodimerization of PSKR1 with SERK1, SERK2, and SERK3 in planta (Wang et al., 2015).

Additionally, we demonstrated in planta interactions between PSY1R and members of the SERK family, which act as co-receptors and regulate distinct signaling pathways (Chinchilla et al., 2009; Li, 2010). SERK proteins are implicated in controlling the balance between growth and defense, as they regulate the activity of receptors involved in plant development and in plant immunity. Our results suggest that SERK proteins also regulate PSY1R signaling. Based on BiFC analyses, we observed differences in the fluorescence intensity of the signal when testing for interactions between PSY1R and different SERK members. This might indicate differences in the stability of the interaction. Interestingly we observe a very strong interaction between the inactive PSY1R K831A mutant and BAK1 as well as between the inactive SERK5 and PSY1R. This shows that protein kinase activity is not required for the interaction, as previously observed within this family (Karlova et al., 2009). Also it could indicate that the interaction between the two kinases is less transient, compared to a situation with two active kinases, which therefore results in formation of more fluorescent molecules. Future studies will reveal whether the interactions between PSY1R and members of the SERK family are of functional relevance in vivo.

### CONCLUSION

Our study has revealed details of the regulation of the kinase activity of PSY1R. Future studies will reveal the in vivo function of the identified autophosphorylation sites and how the interaction with the SERK co-receptor proteins fine-tunes PSY1R signaling.

### AUTHOR CONTRIBUTIONS

CO and AF designed the study. CO, LG, and TS performed the kinase phosphorylation analysis. CO and AK performed the BiFC analysis. NA performed the MS analysis. JP performed the structural analysis. CO, JT, and AF supervised the project.

CO and AF wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.

### FUNDING

This work was supported by the UNIK research initiative of the Danish Ministry of Science, Technology, and Innovation through the "Center for Synthetic Biology" at University of Copenhagen

### REFERENCES


and by the Danish National Research Foundation through the PUMPKIN Center of Excellence.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2017.02005/ full#supplementary-material



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Oehlenschlæger, Gersby, Ahsan, Pedersen, Kristensen, Solakova, Thelen and Fuglsang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Excessive Cellular S-nitrosothiol Impairs Endocytosis of Auxin Efflux Transporter PIN2

#### Min Ni 1†, Lei Zhang1†, Ya-Fei Shi 1†, Chao Wang2†, Yiran Lu<sup>1</sup> , Jianwei Pan1, 2 and Jian-Zhong Liu<sup>1</sup> \*

*<sup>1</sup> College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua, China, <sup>2</sup> Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China*

*S*-nitrosoglutathione reductase (GSNOR1) is the key enzyme that regulates cellular levels of *S*-nitrosylation across kingdoms. We have previously reported that loss of GSNOR1 resulted in impaired auxin signaling and compromised auxin transport in Arabidopsis, leading to the auxin-related morphological phenotypes. However, the molecular mechanism underpinning the compromised auxin transport in *gsnor1-3* mutant is still unknown. Endocytosis of plasma-membrane (PM)-localized efflux PIN proteins play critical roles in auxin transport. Therefore, we investigate whether loss of GSNOR1 function has any effects on the endocytosis of PIN-FORMED (PIN) proteins. It was found that the endocytosis of either the endogenous PIN2 or the transgenically expressed PIN2-GFP was compromised in the root cells of *gsnor1-3* seedlings relative to Col-0. The internalization of PM-associated PIN2 or PIN2-GFP into Brefeldin A (BFA) bodies was significantly reduced in *gsnor1-3* upon BFA treatment in a manner independent of *de novo* protein synthesis. In addition, the exogenously applied GSNO not only compromised the endocytosis of PIN2-GFP but also inhibited the root elongation in a concentration-dependent manner. Taken together, our results indicate that, besides the reduced PIN2 level, one or more compromised components in the endocytosis pathway could account for the reduced endocytosis of PIN2 in *gsnor1-3*.

Keywords: endocytosis, nitric oxide, PIN-FORMED (PIN) proteins, polar auxin transport, S-nitrosoglutathione reductase

### INTRODUCTION

Nitric oxide (NO) is a reactive free radical gaseous molecule that is involved in battery of biological processes both in animals and plants (Wendehenne et al., 2014). In plants, NO participates in biological processes such as stomatal closure, cell death and disease resistance, abiotic stress, flowering, and many other processes (Durner et al., 1998; Klessig et al., 2000; Neill et al., 2002; Lamattina et al., 2003; He et al., 2004, 2012; Wendehenne et al., 2004, 2014; Zeidler et al., 2004; Lee et al., 2008; Xuan et al., 2010; Fan and Liu, 2012; Lin et al., 2012; Ye et al., 2012; Mur et al., 2013).

S-nitrosylation, adding NO moiety to a protein, is a novel mechanisms by which NO regulates protein functions (Hess and Stamler, 2012; Wendehenne et al., 2014). This non-enzymatic reversible protein modification is analogous to protein phosphorylation (Stamler et al., 1992; Hess et al., 2005). Many proteins have been identified as targets of S-nitrosylation and their functions

#### Edited by:

*Kai He, Lanzhou University, China*

### Reviewed by:

*Frantisek Baluska, University of Bonn, Germany Norbert Rolland, Centre National de la Recherche Scientifique (CNRS), France*

> \*Correspondence: *Jian-Zhong Liu jzliu@zjnu.cn*

*† These authors have contributed equally to this work.*

#### Specialty section:

*This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science*

Received: *06 July 2017* Accepted: *03 November 2017* Published: *23 November 2017*

#### Citation:

*Ni M, Zhang L, Shi Y-F, Wang C, Lu Y, Pan J and Liu J-Z (2017) Excessive Cellular S-nitrosothiol Impairs Endocytosis of Auxin Efflux Transporter PIN2. Front. Plant Sci. 8:1988. doi: 10.3389/fpls.2017.01988*

**75**

are regulated by this modification (Lindermayr et al., 2005; Forrester et al., 2009; Hess and Stamler, 2012; Yang et al., 2015). In plants, the target cysteine residues of some S-nitrosylated proteins have been identified and the functional importance of this modification is unraveled (Lindermayr et al., 2006, 2010; Belenghi et al., 2007; Romero-Puertas et al., 2007; Serpa et al., 2007; Tada et al., 2008; Chen et al., 2009; Wang et al., 2009, 2015; Yun et al., 2011; Astier et al., 2012; Feng et al., 2013; Yang et al., 2015; Hu et al., 2017; Liu et al., 2017).

The level of cellular protein S-nitrosylation is dynamic and governed by NO levels and de-nitrosylation catalyzed by Snitrosoglutathione reductase (GSNOR) (Liu et al., 2001; Feechan et al., 2005) and thioredoxin (Tada et al., 2008; Benhar et al., 2009; Sengupta and Holmgren, 2012). GSNOR is the key enzyme controlling S-nitrosoglutathione (GSNO) levels by reducing GSNO to oxidized GSH and NH<sup>3</sup> and thus indirectly controls the cellular levels of S-nitrosylated proteins (Liu et al., 2001, 2004; Feechan et al., 2005).

Auxin is one of mostly studied plant hormone that plays diverse roles in development (Teale et al., 2006). Auxin gradients, which are created and maintained by groups of transporters localized on plasma membrane (PM) are critical to auxin functions in the regulation of stem cell differentiation, the initiation of lateral organs and gravitropic responses (Woodward and Bartel, 2005; Leyser, 2006; Petrásek and Friml, 2009). One of the most important transporters is the PIN-FORMED (PIN) family of auxin efflux proteins (Chen et al., 1998; Galweiler et al., 1998; Muller et al., 1998; Geldner et al., 2001; Blilou et al., 2005; Wisniewska et al., 2006; Pan et al., 2009).

Clathrin-mediated endocytosis (CME) is an evolutionally conserved pathway that plays a critical role in determining protein abundance at the PM and/or the trans-Golgi network (TGN) during signaling transductions and retargeting/degradation of proteins at PM (Chen et al., 2011; McMahon and Boucrot, 2011; Wang et al., 2013). CME is the predominant pathway for the internalization of numerous membrane-localized proteins including PINs (Paciorek et al., 2005). By inhibiting the endocytosis of PIN, auxin increases levels of various PINs at the PM (Paciorek et al., 2005). As a result, auxin promotes its own efflux by vesicle-traffickingdependent mechanism (Paciorek et al., 2005). In addition to CME, a BFA-insensitive and clathrin-independent endocytic route has also been reported for PM resident proteins (Beck et al., 2012).

We have previously reported that loss of GSNOR1 function in Arabidopsis impairs both auxin signaling and polar auxin transport and thereby the gsnor1-3 mutant displays multiple auxin-related morphological defects including short and highly branched statures, short primary roots, and lack of lateral roots. The compromised polar auxin transport in gsnor1-3 is due to universally reduced levels of auxin efflux transporters PIN proteins at the plasma membrane (PM) (Shi et al., 2015). However, whether loss of GSNOR1 inhibits polar auxin transport exclusively through reducing the abundance of PINs at PM or additional mechanisms are also involved, are largely unknown. Here, we showed that loss of GSNOR1 inhibited the internalization of either the transgenically expressed PIN2-GFP or the endogenous PIN2 independent of de novo protein synthesis and this inhibition could be recapitulated by exogenously applied GSNO. Furthermore, similar to loss of GSNOR1, exogenously applied GSNO inhibited the root elongation in a concentration dependent manner. Together, our results reveal an additional layer of complex roles of NO in regulating plant growth and development through modulating internalization of auxin efflux transporter.

## RESULTS

### Loss of GSNOR1 Results in Reduced Internalization of Transgenically Expressed PIN2-GFP

To examine the effect of NO signaling on the internalization of the PM-associated PIN proteins, we used Brefeldin A (BFA; 50µM), a vesicle trafficking inhibitor (Geldner et al., 2001), to visualize the PIN2-GFP internalization in the wild-type and gsnor1-3 seedlings that express the PIN2-GFP driven by its native promoter (ProPIN2:PIN2-GFP). Consistent with our previous report (Shi et al., 2015), the intensity of the PIN2-GFP fluorescence at the PM was significantly reduced in gsnor1-3 mutants relative to the wild-type cells under mock conditions (compare **Figures 1A,D;** and **Figure 1G**). As expected, both the numbers and the relative intensities of the PIN2-GFPlabeled BFA bodies were also significantly reduced in the gsnor1- 3 compared to the wild-type cells (compare white arrowpointed BFA bodies in **Figures 1B,E**; and see statistical data in **Figures 1H,I**), indicating that the internalization of the PIN2- GFP was reduced in the gsnor1-3 mutants. To dissect whether the reduced numbers and intensities of the BFA-induced PIN2- GFP fluorescent bodies in gsnor1-3 is exclusively resulted from the reduced levels of PIN2-GFP at the PM, we further analyzed the ratios of GFP signals in BFA bodies to those at the PM both in Col-0 and gsnor1-3, respectively, after BFA treatment. As shown in **Figure 1J**, the GFP signal ratio of BFA bodies/PM was significantly lower in gsnor1-3 mutants than in the wild-type cells, indicating that, besides the reduced level of the PIN2-GFP at the PM, PIN2-GFP internalization itself is also compromised in the gsnor1-3 mutant seedlings.

Auxin inhibits internalization of PM proteins (Paciorek et al., 2005). To address whether auxin inhibitory effect on PIN2 endocytosis is altered in the gsnor1-3 mutants, we treated the transgenic seedlings expressing the PIN2-GFP both in the Col-0 and the gsnor1-3 firstly with 10µM 2,4-D for 30 min and followed by treatment with 10µM 2,4-D plus 50µM BFA for additional 60 min as described (Wang et al., 2013). As shown in **Figure 1**, 2,4-D similarly blocked the PIN2-GFP internalization both in the wild-type and the mutant cells (compare 1C and 1F), indicating that auxin inhibition of PIN2 internalization is not significantly impaired in the gsnor1-3 mutant.

**Abbreviations:** 2,4-D, 2,4-dichloropheenoxyacetic; CME, Clathrin-mediated endocytosis; CHX, cycloheximide; NO, Nitric oxide; GSNO, S-nitrosoglutathione; GSNOR1, S-nitrosoglutathione reductase 1; SNO, S-nitrosothiol; TGN, Trans-Golgi network.

FIGURE 1 | The internalization and not the auxin inhibition of internalization of the transgenically expressed PIN2-GFP is impaired in *gsnor1-3* mutant. Six-day-old *ProPIN2*:*PIN2-GFP*-expressing transgenic seedlings either in Col-0 or in *gsnor1-3* background were treated with Mock (A,D), 50µM BFA for 60 min (B,E), and with 10µM 2, 4-D for 30 min, followed by treatment with 50µM BFA+ 10µM 2, 4-D for additional 60 min (C,F). Images were captured by confocal laser scanning microscopy (CLSM, Leica TCS SP5 AOBS). The numbers of BFA bodies (see white arrows) were counted and the fluorescence intensities both at the BFA bodies and at the PM were measured, respectively, using Image J (http://rsb.info.nih.gov/ij) and the statistical data were summarized (G–J). (G) GFP intensity at the PM; (H) Number of BFA bodies per cell; (I) Relative GFP intensities of the BFA bodies; (J) The GFP intensity ratios of the BFA bodies/PM. Bar = 50µm. \*\*\*Indicates significant differences between Col-0 and *gsnor1-3* by Student's *t*-test at 0.001 level.

### Loss of GSNOR1 Results in Reduced Internalization of the Endogenous PIN2

Next, to test whether the internalization of endogenous PIN2 is similarly impaired as PIN2-GFP in the gsnor1-3 mutant, we performed immunofluorescence microscopy analysis using affinity-purified anti-PIN2-specific antibodies (Wang et al., 2013). Similar to the PIN2-GFP shown in **Figure 1**, the level of the PM-localized endogenous PIN2 was significantly reduced in the gsnor1-3 cells compared to the wild-type cells under the mock conditions (compare **Figures 2A,D** and **Figure 2G**). Similarly, the numbers and the relative intensities of PIN2 labeled BFA bodies (compare the white arrow-pointed BFA bodies in **Figures 2B,E**; also see statistical data in **Figures 2H,I**) and the fluorescence intensity ratio of BFA bodies/PM of the endogenous PIN2 (**Figure 2J**) were all significantly decreased in the gsnor1-3 mutant compared to the wild-type cells. Again, the inhibition of the PIN2 internalization in the presence of 2,4-D was not significantly altered in the gsnor1-3 mutant compared to the Col-0 cells (**Figures 2C,F**, no visible BFA bodies). These results confirmed the conclusions drawn from the studies using the ProPIN2:PIN2-GFP-expressing transgenic seedlings (**Figure 1**), suggesting that the transgenic expressed PIN2-GFP driven by its own promoter can recapitulate the endogenous PIN2.

### Loss of GSNOR1 Results in the Reduced Endocytosis of PIN2-GFP in the Absence of de novo Protein Synthesis

To accurately assess the effect of loss of GSNOR1 on the internalization of PM-localized PIN2-GFP, the interference of the newly synthesized PIN2-GFP on the level of PM-localized PIN2- GFP must be excluded. To do so, we firstly treated the 6-dayold seedlings with cycloheximide (CHX; 50µM), an inhibitor of de novo protein synthesis, and followed by washout with CHX plus BFA. As shown in **Figure 3**, the GFP intensity on the PM, the number of BFA bodies per cell and the relative GFP intensity of BFA bodies were all significantly reduced in gsnor1- 3 mutant seedlings relative to the wild-type cells after treatment with CHX for 30 min and followed by washout with CHX and BFA for additional 15 min or 60 min (Compare **Figure 3A** and **Figure 3D**; **Figure 3B** and **Figure 3E**; **Figure 3C** and **Figure 3F**; and see statistical data shown in **Figures 3G–I**). Consistent with the results obtained without CHX treatment (**Figure 1I**), the GFP intensity ratio of BFA bodies/PM was significantly reduced in the gsnor1-3 mutant cells relative to the wild-type cells after CHX and BFA co-treatment (**Figure 3J**). Accordingly, the relative level of the PM-localized GFP signal was higher in the gsnor1- 3 than in Col-0 after CHX and BFA co-treatment (**Figure 3K**). These results again indicate that the PIN2-GFP internalization is impaired in the gsnor1-3 even without de novo protein synthesis.

### The Exogenously Applied GSNO Recapitulates the Loss of GSNOR1 in Inhibiting PIN2-GFP Internalization

We reasoned that if the impaired internalization of the PIN2-GFP or PIN2 observed in the gsnor1-3 (**Figures 1–3**) is indeed resulted from over-accumulation of cellular SNO, the exogenously applied GSNO should have a similar inhibitory effect on PIN2 internalization. To test this hypothesis, we tested the effect of exogenous GSNO treatment on the PIN2-GFP internalization in the wild-type cells. Consistent with the results obtained using the gsnor1-3 mutant (**Figures 1**, **2**), exogenously applied GSNO not only reduced the level of the PM-localized PIN2-GFP (**Figures 4A–C**) but also inhibited the PIN2-GFP internalization in the presence of BFA in a concentration-dependent manner (**Figures 4E–G**, and statistical data in **Figures 4I,J**). These results suggest that the impaired polar auxin transport observed in the gsnor1-3 mutant seedlings (Shi et al., 2015) could be resulted at least partially from the reduced internalization of PIN2 (**Figures 4D,H**–**J**).

### Exogenous GSNO Inhibits Root Elongation in a Concentration Dependent Manner

Our previous studies have shown that loss of GSNOR1 impairs auxin signaling and transport and the gsnor1-3 mutant plants display a wide range of auxin-related morphological defects including a short root phenotype (Shi et al., 2015). We postulated that if the short root phenotype observed in the gsnor1-3 mutant seedlings is indeed a consequence of cellular SNO overaccumulation, the exogenously applied GSNO could mimic the effects of the loss-of-function mutant of GSNOR1. To test this postulation, we treated the Col-0 seedlings with different concentrations of GSNO and used the gsnor1-3 mutant seedlings as a control. As expected, the exogenously applied GSNO inhibited the root elongation of the wild type seedlings in a concentration-dependent manner (**Figures 5A–C**), confirming that the excessive cellular SNO is at least partially, if not fully, responsible for the short root phenotype of the gsnor1-3 mutant seedlings (**Figures 5D,E**, and Shi et al., 2015).

## DISCUSSION

It is not an uncommon phenomenon that phytohormones play roles in regulating CME. It has been reported that strigolactones affect shoot branching by modulating the endocytosis of PIN1 (Shinohara et al., 2013), and salicylic acid (SA) represses endocytosis of different PM-associated proteins by blocking clathrin recruitment at the PM (Du et al., 2013). Auxin inhibits the CME of several PM-localized proteins, including several PIN proteins (Paciorek et al., 2005). As a result, auxin promotes its own efflux by inhibiting the internalization of PINs and increases various PIN levels at the PM (Paciorek et al., 2005). In this report, we provided evidence that NO, the other phytohormone, also play a role in the regulation of PIN2 endocytosis.

Our previous results have shown that even though the transcript level of PIN2-GFP was higher in the gsnor1-3 mutant than in the wild type Col-0, the intensity of PIN2-GFP at the PM was significantly reduced in the gsnor1-3 mutant relative to the wild-type plants (Shi et al., 2015). Likely, the reduced accumulation of PIN2 at the PM in the gsnor1-3 could be partially resulted from compromised protein synthesis and/or stability and the reduced levels of the various PIN proteins in the gsnor1-3 could be the primary cause of the compromised polar auxin transport (Shi et al., 2015). However, our present data uncover that, in addition to the reduced level of PIN2 at the PM, the internalizations of PIN2 was also compromised in the gsnor1-3 mutant seedlings (**Figures 1**–**3**). Post-translational modifications play critical roles in regulating endocytosis. Both mono- and poly-ubiquitylation of single lysine is associated with cargo internalization and the intracellular sorting and targeting of PM proteins to the vacuole/lysosome rely on K63-linked ubiquitylation (Luschnig and Vert, 2014). PIN2 is modified by K63-linked poly-ubiquitin chains, which dependent on a class of ring-domain E3 ligase (RGLGs) (Yin et al., 2007; Leitner et al., 2012). Protein phosphorylation has been identified as a major determinant of PIN sorting and the sorting decision is dependent on the phosphorylation status of PINs and the activity of the serine/threonine protein kinase PINOID (PID) and its related proteins impact polar PIN distribution (Friml et al., 2004). As NO can also regulate protein functions by S-nitrosylation, it is highly possible that NO regulates internalization either directly by Snitrosylating PINs or indirectly by S-nitrosylating the other key

FIGURE 2 | The internalization and not the auxin inhibition of internalization of the endogenous PIN2 is impaired in *gsnor1-3* mutant. The immunolocalizations were performed using PIN2-specific antibodies (Wang et al., 2013) and Cy3-labeled anti-rabbit secondary antibodies (Sigma-Aldrich) on the primary roots of 6-day-old seedlings treated with Mock (A,D), 50µM BFA for 60 min (B,E) or with 10µM 2, 4-D for 30 min, followed by treatment with 50µM BFA+ 10µM 2, 4-D for additional 60 min (C,F). Images were captured using CLSM (Leica TCS SP5 AOBS). The numbers of BFA bodies (see white arrows) were counted and the fluorescence intensities (FI) at the BFA bodies and at the PM were measured, respectively, using Image J (http://rsb.info.nih.gov/ij) and the statistical data were summarized (G–J). (G) Fluorescence intensity (FI) at the PM; (H) Number of BFA bodies per cell; (I) Relative fluorescence intensities (FI) of the BFA bodies; (J) The fluorescence intensity (FI) ratios of the BFA bodies/PM. Bar = 50µm. \*\*\*Indicates significant differences between Col-0 and *gsnor1-3* by Student's *t*-test at 0.001 level.

proteins in the endocytic pathways. This statement is supported by the facts that the activities of many PM-resident ion channels in animals, including Na<sup>+</sup> and Ca2<sup>+</sup> channels, are regulated by S-nitrosylation (Xu et al., 1998; Renganathan et al., 2002) and Snitrosylation is invloved in modification of an outward-rectifying K <sup>+</sup> channel in Vicia faba (Sokolovski and Blatt, 2004).

Theoretically, the relatively enhanced level of PIN2-GFP at PM could relatively enhance the auxin polar transport (Paciorek et al., 2005). However, we observed a much reduced polar auxin transport in the gsnor1-3 mutant primarily because of the universally reduced levels of PINs (Shi et al., 2015). One possibility is that the positive contribution of the relatively enhanced PM-localized PIN2, as a result of impaired internalizations, to polar auxin transport is masked by the inhibition resulted from the overall reduced level of PIN2 (**Figures 1**, **2**; Shi et al., 2015). The other possibility is that, even though the relative level of PM-localized PIN2 is enhanced in the gsnor1-3 mutant relative to WT, the function of the PM-localized PIN2 is impaired under the excessive SNO condition, probably by S-nitrosylation. The functional endocytosis/exocytosis is required for replenishing the non-functional PM-localized PIN2 with newly synthesized functional PIN2 at PM. As a result, the reduced level of PIN2 and the compromised internalization of PIN2 (**Figures 1**–**3**) could contribute additively to the impaired polar auxin transport in the gsnor1-3 mutant and thus its auxinrelated morphological phenotypes.

FIGURE 3 | The internalization of PIN2-GFP is reduced in *gsnor1-3* in the absence of *de novo* protein synthesis. *ProPIN2*:*PIN2-GFP* transgenic Col-0 and *gsnor1-3* mutant seedlings (6-day-old) were treated with cycloheximide (CHX) for 30 min first, followed by CHX alone for 15 min (A,D) or co-treatment with CHX+BFA for 15 (B,E) or 60 min (C,F). Images were captured by confocal laser scanning microscopy (CLSM, Leica TCS SP5 AOBS). The numbers of BFA bodies (see white arrows) were counted and the fluorescence intensities at the BFA bodies as well as at the PM were measured, respectively, using Image J (http://rsb.info.nih.gov/ij) and the statistical data were summarized (G–K). (G). GFP intensity at the PM; (H) Number of BFA bodies per cell; (I) Relative GFP intensities of the BFA bodies; (J) GFP intensity ratios of the BFA bodies/PM; (K) GFP intensity ratios on PM (*gsnor1-3*/Col-0). Bar = 50µm. \*\*\*Indicates significant differences between Col-0 and *gsnor1-3* by Student's *t-*test at 0.001 level. a, b, and c Indicate significant differences between the treatments by Student's *t*-test.

Even though the auxin signaling is significantly impaired in the gsnor1-3 (Shi et al., 2015), the auxin inhibition of either the transgenically expressed PIN2-GFP or the endogenous PIN2 internalization is not affected in the gsnor1-3 (**Figures 1C,F**, **2C,F**), suggesting that auxin signaling does not play a critical role in the inhibition of PIN2 internalization. In agreement with this, it has been shown that differential auxin regulation of CLC and CHC membrane association is ABP1-dependent but TIR1/AFBindependent (Wang et al., 2013) and the role of ABP1 in auxin signaling is questionable (Gao et al., 2015; Liu, 2015). Given that the clathrin are required for the auxin inhibition of the PIN2- GFP internalization (Wang et al., 2013) and the auxin inhibition of PIN2-GFP is not affected in gsnor1-3 mutant (**Figures 1C,F**, **2C,F**), it is suggested that the reduced internalization of PIN2- GFP or endogenous PIN2 (**Figures 1**, **2**) may not be clathrindependent. In supporting this, it has been reported previously that the same PM protein could traffic through distinct endocytic routes (Beck et al., 2012). The activated FLS2 triggered by flg22 recognition at PM is targeted to the intracellular compartments for degradation via an ESCRT1-dependent, but BFA-insensitive route to prevent excessive and constitutive activation of defense signaling, whereas the non-activated or newly synthesized FLS2 is shuttled between PM and cytoplasm via a BFA-sensitive route (Robatzek et al., 2006; Beck et al., 2012; Spallek et al., 2013).

images were captured by confocal laser scanning microscopy (CLSM, Leica TCS SP5 AOBS). The numbers of BFA bodies (see white arrows) were counted (I) and the fluorescence intensities of the BFA bodies and at the PM were measured, respectively, using Image J (http://rsb.info.nih.gov/ij). The GFP intensity ratios of BFA bodies to PM were measured and the statistical data were presented in (J). Bar = 50µm. a, b, c, and d indicate significant differences between the treatments by Student's *t*-test.

One may argue that the fluorescence intensity of some BFA bodies in gsnor1-3 mutant (**Figures 1E**, **2E**) could be below the detection threshold under our Confocal settings, and therefore could have an impact on the fluorescence intensity ratio of BFA bodies/PM in the mutant. If that is really the case, the average fluorescence intensity of the BFA bodies within a cell or in a given captured image (5–20 root cells/image) of gsnor1-3 root should be lower, and thus the fluorescence intensity ratio of BFA bodies/PM in the mutant should be lower than shown in **Figures 1J**, **2J**. As a result, the detection thresholds issue would not change our final conclusion that the internalization of either the transgenically expressed PIN2-GFP or the endogenous PIN2 was compromised in the gsnor1-3 mutant.

In this report, we provided genetic and pharmaceutical evidence that the NO signaling plays an inhibitory role in PIN2 internalization. However, the molecular mechanism underlying the inhibition still remains unanswered. The questions like how NO signaling inhibits PIN2 internalization and what

the agar plates without containing GSNO used as a control (D). The photos were taken 4 days after transfer. The root lengths were measured and listed in (E). a, b, c, and d indicate significant differences between the treatments by Student's *t*-test.

is the causal relationship between the NO-inhibited PIN2 internalization and polar auxin transport need to be addressed in the future. Nonetheless, our results reveal an additional layer of complex roles of NO in regulating plant growth and development through modulating internalization of auxin efflux transporter.

## MATERIALS AND METHODS

## Arabidopsis Lines and Growth Conditions

Arabidopsis Col-0 and gsnor1-3 (Feechan et al., 2005), the ProPIN2:PIN2-GFP transgenic line (Xu and Scheres, 2005) were used in this study. The ProPIN2:PIN2-GFP transgene was introgressed into the gsnor1-3 background by crossing. The homozygous gsnor1-3 line that expresses the ProPIN2:PIN2-GFP transgene was identified by the genomic PCR in combination of phenotype characterization among F3 population. These plants were grown under 16 h-light (22◦C) /8h-dark (18◦C). Seedlings were grown on 1/2 MS medium containing 1.0% sucrose (pH 5.7). Healthy six-day-old seedlings were used in this study.

## Chemical Solutions

The stock solutions of CHX (50 mM, Sigma-Aldrich), BFA (50 mM; Invitrogen) and GSNO (100 mM, Sigma-Aldrich) were prepared in DMSO. 2,4-D (10 mM, Sigma-Aldrich) was firstly dissolved in 1 M KOH and then diluted with water as described (Wang et al., 2013).

### Chemical Treatments

All chemical pretreatments except GSNO were for 30 min, and chemical treatment time is indicated in the text. For GSNO treatment, 2-day-old seedlings were transferred from GSNO-free 1/2 MS agar plates (containing 1.0% sucrose, pH 5.7) into agar plates containing different concentrations of GSNO as indicated in the text for additional 3-4 days. All seedlings were grown on MS basal salts with minimal organics (Sigma Aldrich) supplemented with 1% (w/v) Sucrose and 0.05% (w/v) MES-KOH, pH 5.6 (0.5 3 x MS), liquid medium, except otherwise specified. The final pH of the medium was 5.6–5.7.

### Polyclonal Antibody and Immunofluorescence Microscopic Analysis

Polyclonal antibody, anti-AtPIN2, was raised in rabbits as described using a synthesized peptide (Wang et al., 2013) coupled with keyhole limpet hemocyanin containing an additional N-terminal Cys (Huabio). Immunofluorescence microscopic analysis was performed as described (Wang et al., 2013). The primary PIN2 antibody was detected using Cy3-labeled anti-rabbit secondary antibodies (Sigma-Aldrich). Fluorescence images were captured using CLSM (Leica TCS SP5 AOBS). Excitation wavelengths for GFP and Cy3 were 488 nm (argon laser) and 561 nm (diode laser), respectively. The emission wavelengths were 496–532 nm for GFP and 550–570 nm for Cy3, respectively. For the purpose of direct comparisons of fluorescence intensities, laser, pinhole, and gain settings of the confocal microscope were set exactly identical when capturing the images from the seedlings of different genotypes or treatments. The intensities of fluorescence signals both at the PM and the cytoplasmic vesicles were quantified on the captured digital images using Image J (http://rsb.info.nih.gov/ ij) and the relative fluorescence intensities were presented as percentages of mock controls as described (Sauer et al., 2006; Robert et al., 2010). For the measurement of the fluorescence levels at the PM, optimal sections of the root cells were used for measurements. Using Image-J, PM regions of a captured images (over 20 cells for **Figure 1**; 5–10 cells for **Figures 2**–**4**) were selected with the rectangle tool. The mean pixel intensity readings (total intensity readings/area) for the selected PM regions were recorded and the average values were calculated subsequently. The data shown in **Figures 1**, **3**, **4** were averages from the different images captured at least from 30 individual roots. The data shown in **Figure 2** were averages from the different images captured at least from 10 individual roots. The experiments were repeated three times with similar results.

For the measurement of the fluorescence levels at the BFA bodies, the regions of the visible BFA bodies in a single cell of the captured images (over 20 cells for **Figure 1**; 5–10 cells for **Figures 2–4**) were selected with the oval tool of Image J. The mean pixel intensity readings (total intensity readings/area) for the selected regions in a single cell were recorded and the mean value of the fluorescence intensity of the BFA bodies in a single cell was calculated by dividing the number of BFA bodies in the single cell (the sum of mean values/the number of BFA bodies). The average fluorescence intensity of the BFA bodies in a captured image were calculated subsequently by dividing the number of cells in the captured image (the sum of average fluorescence intensity of the BFA bodies in a single cell/the number of cells in a captured image). The data shown in **Figures 1**, **3**, **4** were averages from different images captured from at least 30 individual roots. The data shown in **Figure 2** were averages from the different images captured at least from 10 individual roots. The experiments were repeated three times with similar results.

For calculation of the fluorescence intensity ratio of BFA/PM, the average fluorescence intensity of the BFA bodies measured in a captured image was divided by the average fluorescence intensity at the PM of the same image. The data shown in **Figures 1**, **3**, **4** were mean values of the images captured from at least 30 individual roots. The data shown in **Figure 2** were the mean values from the images captured at least from 10 individual roots. The experiments were repeated three times with similar results.

The BFA-induced internalization of PM-localized proteins was presented as the average number of fluorescence-labeled BFA bodies per cell (Robert et al., 2010). For all the quantitative data, a Student's t test (paired with two-tailed distribution) was used in statistical analysis.

### Root Elongation Assay

The 2-day-old Col-0 seedlings grown on the 1/2 MS agar plates were transferred onto the 1/2 MS agar plates containing different concentration of GSNO for additional 3–4 days. The root length was measured under dissecting microscopy using Image J (http:// rsb.info.nih.gov/ij).

## AUTHOR CONTRIBUTIONS

J-ZL and JP designed the experiments. MN, LZ, Y-FS, CW and YL performed the experiments. J-ZL and JW wrote the manuscript.

### FUNDING

This work was supported by the National Natural Science Foundation of China (31371401 and 31571423 to J-ZL; and 31370313 to JP), and Xin Miao Talent Program of Zhejiang Province (2016R404009).

### ACKNOWLEDGMENTS

The authors thank Dr. Gary Loake for providing the gsnor1-3 mutant. We also thank Mr. Zhen-Chao Li for his technical help in preparation of the figures.

### REFERENCES


development in Arabidopsis. Plant Cell 25, 499–516. doi: 10.1105/tpc.112. 108373


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling Editor declared a shared affiliation, though no other collaboration, with several of the authors CW and JP.

Copyright © 2017 Ni, Zhang, Shi, Wang, Lu, Pan and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Rice High-Affinity K<sup>+</sup> Transporter OsHKT2;4 Mediates Mg2<sup>+</sup> Homeostasis under High-Mg2<sup>+</sup> Conditions in Transgenic Arabidopsis

### Edited by:

Kai He, Lanzhou University, China

#### Reviewed by:

Kendal Hirschi, Baylor College of Medicine, United States Fouad Lemtiri-Chlieh, King Abdullah University of Science and Technology, Saudi Arabia Tamara Pecenkova, Institute of Experimental Botany (ASCR), Czechia

#### \*Correspondence:

Jiping Gao jpgao@sibs.ac.cn Wenzhi Lan lanw@nju.edu.cn

#### Specialty section:

This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science

Received: 27 July 2017 Accepted: 10 October 2017 Published: 24 October 2017

### Citation:

Zhang C, Li H, Wang J, Zhang B, Wang W, Lin H, Luan S, Gao J and Lan W (2017) The Rice High-Affinity K <sup>+</sup> Transporter OsHKT2;4 Mediates Mg2<sup>+</sup> Homeostasis under High-Mg2<sup>+</sup> Conditions in Transgenic Arabidopsis. Front. Plant Sci. 8:1823. doi: 10.3389/fpls.2017.01823 Chi Zhang<sup>1</sup> , Hejuan Li<sup>1</sup> , Jiayuan Wang<sup>1</sup> , Bin Zhang<sup>1</sup> , Wei Wang<sup>2</sup> , Hongxuan Lin<sup>2</sup> , Sheng Luan<sup>3</sup> , Jiping Gao<sup>2</sup> \* and Wenzhi Lan<sup>1</sup> \*

<sup>1</sup> State Key Laboratory for Pharmaceutical Biotechnology, NJU–NFU Joint Institute for Plant Molecular Biology, College of Life Sciences, Nanjing University, Nanjing, China, <sup>2</sup> National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China, <sup>3</sup> Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, United States

Rice (Oryza sativa; background Nipponbare) contains nine HKT (high-affinity K<sup>+</sup> transport)-like genes encoding membrane proteins belonging to the superfamily of Ktr/TRK/HKT. OsHKTs have been proposed to include four selectivity filter-pore-forming domains homologous to the bacterial K<sup>+</sup> channel KcsA, and are separated into OsHKT1s with Na+-selective activity and OsHKT2s with Na+-K<sup>+</sup> symport activity. As a member of the OsHKT2 subfamily, OsHKT2;4 renders Mg2<sup>+</sup> and Ca2<sup>+</sup> permeability for yeast cells and Xenopus laevis oocytes, besides K<sup>+</sup> and Na+. However, physiological functions related to Mg2<sup>+</sup> in planta have not yet been identified. Here we report that OsHKT2;4 from rice (O. sativa; background Nipponbare) functions as a lowaffinity Mg2<sup>+</sup> transporter to mediate Mg2<sup>+</sup> homeostasis in plants under high-Mg2<sup>+</sup> environments. Using the functional complementation assay in Mg2+-uptake deficient Salmonella typhimurium strains MM281 and electrophysiological analysis in X. laevis oocytes, we found that OsHKT2;4 could rescue the growth of MM281 in Mg2+-deficient conditions and induced the Mg2<sup>+</sup> currents in oocytes at millimolar range of Mg2+. Additionally, overexpression of OsHKT2;4 to Arabidopsis mutant lines with a knockout of AtMGT6, a gene encoding the transporter protein necessary for Mg2<sup>+</sup> adaptation in Arabidopsis, caused the Mg2<sup>+</sup> toxicity to the leaves under the high-Mg2<sup>+</sup> stress, but not under low-Mg2<sup>+</sup> environments. Moreover, this Mg2<sup>+</sup> toxicity symptom resulted from the excessive Mg2<sup>+</sup> translocation from roots to shoots, and was relieved by the increase in supplemental Ca2+. Together, our results demonstrated that OsHKT2;4 is a low-affinity Mg2<sup>+</sup> transporter responsible for Mg2<sup>+</sup> transport to aerials in plants under high-Mg2<sup>+</sup> conditions.

Keywords: Arabidopsis, HKT transporter, MGT transporter, Mg2<sup>+</sup> permeable, rice

### INTRODUCTION

fpls-08-01823 October 20, 2017 Time: 16:55 # 2

Apart from atmospheric oxygen and soil-derived water, plants require a range of minerals for their growth and development. As two major essential mineral nutrients for plant growth, K and Mg are available to plants in the ionic form (K<sup>+</sup> and Mg2+), and are transported into root cells by the plasma membrane-localized channels and transporters. Up to now, most studies are focused on identifying the active, high-affinity channels and transporters, which function in K<sup>+</sup> and Mg2<sup>+</sup> uptake from the nutrientdeficient environments (Hirsch et al., 1998; Li et al., 2001; Chérel et al., 2014; Mao et al., 2014). However, large majority of channels and transporters necessary for plants adaptation to nutrientenriched conditions remain unknown.

Due to its key role in salt tolerance, high-affinity K<sup>+</sup> transporters (HKTs) family has been widely studied and most of its members are characterized as being permeable for specific ions in heterologous expression systems (Uozumi et al., 2000; Horie et al., 2001; Mäser et al., 2002b; Garciadeblas et al., 2003; Yao et al., 2010). HKTs in plants and their K<sup>+</sup> transporter (Trk and Ktr) counterparts in fungi and bacteria form a HKT/Trk/Ktr superfamily (Rodriguez-Navarro, 2000; Corratgé-Faillie et al., 2010; Yamaguchi et al., 2013). Plant HKT transporters are divided into two subgroups based on phylogenetic analyses to date (Mäser et al., 2002a; Platten et al., 2006; Horie et al., 2009; Hauser and Horie, 2010). Group I HKT members (HKT1s) are associated with retrieval of Na<sup>+</sup> from xylem in root or sheath restricting transport and accumulation of salt in sensitive leaf tissues (Davenport et al., 2007; Munns and Tester, 2008). Grass species evolved a second class of HKT proteins, and comprehensive analysis of this group II HKTs (HKT2s) has been made in rice (Oryza sativa L.) with up to four members, OsHKT2;1, OsHKT2;2, OsHKT2;3, and OsHKT2;4 characterized for the structure, expression, and function (Ariyarathna et al., 2016). Most of HKT2s members function as Na+/K<sup>+</sup> transporters with a role in maintaining Na+/K<sup>+</sup> homeostasis in plants (Horie et al., 2007, 2011; Lan et al., 2010; Yao et al., 2010; Nieves-Cordones et al., 2016). OsHKT2;4 seems to be an exception as it exhibited permeability to a wide range of cations, including Ca2<sup>+</sup> and Mg2<sup>+</sup> when it was expressed in Xenopus laevis oocytes (Lan et al., 2010; Horie et al., 2011). However, its physiological function in rice is still unknown.

The Arabidopsis genome contains a single HKT homolog, AtHKT1;1, which functions as a Na+-selective uniporter and is not permeable to Ca2<sup>+</sup> and Mg2<sup>+</sup> (Davenport et al., 2007; Møller et al., 2009; Lan et al., 2010), suggesting that there are alternative transporters responsible for Ca2<sup>+</sup> and Mg2<sup>+</sup> transport in Arabidopsis. Ca2<sup>+</sup> and Mg2<sup>+</sup> are two of the most abundant divalent cations in living plant cells. Ca2<sup>+</sup> is utilized to strengthen cell walls and a versatile messenger in almost all physiological processes in plants (Tang and Luan, 2017). The prominent role of Mg2<sup>+</sup> is as the central atom of the chlorophyll molecule (Larkin, 2016), and it also participates in cation balance and activation of various enzymes in many fundamental processes (Shaul, 2002; Knoop et al., 2005; Bose et al., 2013). Although Ca2<sup>+</sup> and Mg2<sup>+</sup> are essential macronutrients required for plant growth, their overdose in the environment is toxic to plants (Tang et al., 2015; Oda et al., 2016). Thus, the transporters responsible for Ca2<sup>+</sup> and Mg2<sup>+</sup> homeostasis is of great importance for plant survival under low or high Ca2<sup>+</sup> and Mg2<sup>+</sup> conditions (Miedema et al., 2001; Li et al., 2008; Hermans et al., 2013; Mao et al., 2014; Oda et al., 2016). In contrast to the ambiguous research in Ca2<sup>+</sup> transport, a family of Mg2<sup>+</sup> transporters in Arabidopsis named as AtMGT (Li et al., 2001) or AtMRS2 (Gebert et al., 2009) has been studied extensively, and is found to play pivotal roles in Mg2<sup>+</sup> transport and homeostasis in Arabidopsis. One of its members, AtMGT6/MRS2-4, is a high-affinity Mg2<sup>+</sup> transporter, and loss-of-function of AtMGT6/MRS2-4 caused the severe growth retardation of Arabidopsis plants under low-Mg2<sup>+</sup> conditions (Mao et al., 2014). Interestingly, AtMGT6/MRS2-4 also confers plants adaptation to high-Mg2<sup>+</sup> conditions (Oda et al., 2016). Thus, atmgt6 plant with loss-of-function of AtMGT6/MRS2- 4 displays the deficient Mg2<sup>+</sup> transport under wide range of Mg2<sup>+</sup> concentrations, and is a promising expression system to examine whether the potential transporters possess physiological functions relevant to Mg2<sup>+</sup> in plants.

Although OsHKT2;4 was demonstrated to be permeable for Mg2<sup>+</sup> in X. laevis oocytes (Lan et al., 2010; Horie et al., 2011), this has been challenged in an independent study (Sassi et al., 2012). Here, we applied the Salmonella typhimurium MM281, a bacteria mutant lacking Mg2<sup>+</sup> transport capacity useful for identifying the Mg2<sup>+</sup> transport activities of potential transporters (Li et al., 2001; Gebert et al., 2009), to analyze the possible Mg2<sup>+</sup> transport through OsHKT2;4. Furthermore, its function on Mg2<sup>+</sup> homeostasis was also explored in X. laevis oocytes and transgenic atmgt6 Arabidopsis lines. Our results revealed that OsHKT2;4 is an effective Mg2<sup>+</sup> transporter in maintaining Mg2<sup>+</sup> homeostasis, probably through functional coordination with MGT-type transporters in planta.

### MATERIALS AND METHODS

### Plant Materials and Growth Conditions

Arabidopsis thaliana Columbia (Col-0) ecotype was used in this study. The T-DNA insertion mutant atmgt6 (SALK\_203866) was obtained from the Arabidopsis Biological Resource Center. Homozygous individuals of atmgt6 were screened by PCR using primers listed in Supplementary Table 1. For on-plate growth assays, seeds were sterilized with 75% ethanol for 2 min, washed three times, and sown on half-strength Murashige and Skoog (MS) medium containing 0.75 mM Mg2+, 1.5 mM Ca2+, 1% sucrose (Sigma) and solidified with 0.8% phytoblend (Caisson Labs). The plates were kept at 4◦C for 2 days and then were placed vertically in growth chamber under 90 µmol·m−<sup>2</sup> ·s −1 light intensity with a 16 h light/8 h dark photoperiod. Threeday-old seedlings were transferred onto media containing various ions as indicated in the figure legends. For hydroponic cultures, 7 day-old seedlings germinated in half-strength MS (1/2 MS) were transferred to one-sixth-strength (1/6 MS) hydroponic medium containing 0.25 mM Mg2<sup>+</sup> and 0.5 mM Ca2<sup>+</sup> without sucrose for another 7 days. Plants were then transferred to hydroponic 1/6 MS media containing various contents of Mg2+. Plant materials were harvested for further analyses 2 days after treatment.

### Functional Complementation of Mg2+-Transport by Salmonella typhimurium Mutant Strain MM281

The S. typhimurium mutant MM281, which lacks the Mg2<sup>+</sup> transporter-CorA, MgtA, and MgtB, is used as a system for functional complementation analysis of candidate Mg2+ transporter genes. MM281 competent cells were transformed with empty pTrc99A vector, AtMGT10-pTrc99A or OsHKT2;4 pTrc99A plasmid by electroporation. Cells were plated onto LB medium containing 10 mM Mg2<sup>+</sup> and indicated antibiotics (34 µg·mL−<sup>1</sup> chloramphenicol and 100 µg·mL−<sup>1</sup> ampicillin), and incubated at 37◦C overnight. The transformants were confirmed by PCR amplification and individual positive ones were grown in liquid LB medium containing 10 mM Mg2<sup>+</sup> and antibiotics as indicated above. Fifty micrometer IPTG was applied for the induction of protein expression. The liquid cultures were adjusted to OD<sup>600</sup> = 1.0, diluted in a 10-fold series, and spotted 3 µL onto N-minimal medium supplemented with different concentrations of MgSO<sup>4</sup> and the antibiotics. Growth of different strains was pictured after incubation at 37◦C for 2 days. The growth rate of the three strains in liquid medium was also monitored as previously described (Mao et al., 2014). After growing in liquid LB medium to OD<sup>600</sup> of 0.6–0.8, cells were harvested by centrifugation at 5000 × g for 10 min, washed twice with distilled water to remove excess Mg2+, and resuspended in distilled water. N-minimal medium was prepared with various concentrations of MgSO<sup>4</sup> (0.1, 0.5, 1, and 10 mM). Cells were then adjusted to a final OD<sup>600</sup> of 0.001–0.002. The growth of the cultures was monitored and was plotted as a function of growth time.

### Plasmid Construction and Plant Transformation

For the constructs used in functional complementation assay in MM281 strain, the OsHKT2;4 cDNA fragment was amplified using the primers OsHKT2;4-FC and OsHKT2;4-RC and ligated to the pTrc99A vector. For overexpressing OsHKT2;4 in wild type and the atmgt6 mutant, the genomic fragment (containing a 1.92 kb promoter region upstream of the ATG starting codon and 1731 bp coding region of OsHKT2;4) was amplified using primer pair OsHKT2;4-OE-F and OsHKT2;4-OE-R and cloned into pCAMBIA1300 vector. This construct was introduced into Agrobacterium tumefaciensstrain GV3101 by electroporation and was selected on 1/2 MS medium containing kanamycin. The selected positive transformant was used to transform developing floral tissues of 4-week-old atmgt6 plants using the flora dip method (Clough and Bent, 1998). For expression in X. laevis oocytes, OsHKT2;4 cDNA was cloned into the pGEMHE vector downstream from the T7 promoter using primers OsHKT2;4-FP and OsHKT2;4-RP. All primer pairs were listed in Supplementary Table 1.

### Gene Expression Analysis

Total RNA was extracted from rosette leaves using the TRizol Reagent (Invitrogen), and the first-strand cDNA was synthesized by M-MLV Reverse Transcriptase (Promega) following the manufacturer's instructions. The semi-quantitative RT-PCR analysis of gene expression using cDNA of Col-0, atmgt6, OE29, and OE24 followed by a 26 cycles of PCR amplification. AtActin2 (AT3G18780) was used as the internal reference. Primers used are listed in the Supplementary Table 1.

### Expression in Xenopus laevis Oocytes and Two-Electrode Voltage Clamp

cRNA was synthesized from 1 µg linearized DNA template using a mMessage mMachine in vitro transcription kit (Ambion) according to the manufacturer's recommendations and stored at −80◦C. Stage V to VI X. laevis oocytes were harvested, defolliculated, and cultured in ND96 solution containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 25 µg·mL−<sup>1</sup> gentamicin, pH 7.4 adjusted with 5 mM HEPES/NaOH. Approximately 50 ng of cRNA, in a total volume of 23 nL, was injected into each X. laevis oocyte. Oocytes of 2 days after injection were used for two-electrode voltage-clamp analysis. The perfusion solution was used as described previously (Lan et al., 2010) with some modifications. The perfusion solution contained (in mM) 1 K-gluconate, 1 Na-gluconate, 185 mannitol, and 10 Mes-Tris (pH 7.4). The recording pipette contained 3 M KCl. The currents were recorded by hyperpolarized pulses of a 0.2 s prepulse at −40 mV followed by voltage steps of 60 to −150 mV (in 15 mV decrements, 1.8 s duration) followed by a 1.5 s deactivation at 0 mV. The current-voltage (I-V) curves plot current values at the end of each voltage-clamp episode (t = 2 s, n = 6 for each group).

### Ion Content Measurement

Two-week-old hydroponically grown plants were exposed to solution containing different concentrations of Mg2+. After 2-day exposure, both the roots and shoots were harvested and sampled for analysis. The dry weight (DW) of the samples was measured after drying for 48 h at 60◦C. Subsequently, the samples were digested in 0.5 ml of 70% HNO<sup>3</sup> at 100◦C for 30 min on a digester (DigiBlock ED16, LabTech). Ion concentration was measured by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) (PerkinElmer NexION 300).

## RESULTS

### OsHKT2;4 Rescued the Growth of Bacterial Strain MM281 in the Mg2+-Deficient Medium

To determine whether OsHKT2;4 functions in Mg2<sup>+</sup> transport, a cDNA fragment containing the complete open reading frame with 1530 bases encoded a protein of 509 residues was cloned and expressed in S. typhimurium mutant strain MM281. MM281 is incapable of loading Mg2<sup>+</sup> into cellular compartment, as it lacks three functional Mg2<sup>+</sup> transporters CorA, MgtA, and MgtB, and its growth is retarded or arrested when the culture medium contains less than 10 mM Mg2<sup>+</sup> (Townsend et al., 1995; Li et al., 2001). Therefore, complementation of this strain has proved useful in identifying and developing information about potential

Mg2<sup>+</sup> transporters, including AtMGTs (Li et al., 2001, 2008; Hicks et al., 2003; Mao et al., 2008; Chen et al., 2009; Mao et al., 2014).

We used AtMGT10 (AT5G22830), also named as AtMRS2- 11, as the positive control in the complementation assay of MM281 due to its high affinity in Mg2<sup>+</sup> transport in MM281 system (Li et al., 2001). As shown in **Figure 1A**, the MM281 mutant strains exogenously expressing empty vector pTrc99A, AtMGT10-pTrc99A, or OsHKT2;4-pTrc99A grew normally in the medium with 10 mM Mg2+. The strains expressing OsHKT2;4 pTrc99A exhibited faster growth than those expressing the empty pTrc99A vector in the media containing low concentrations of Mg2<sup>+</sup> (1 and 2 mM), and still grew but to a less extent in the media containing 500 and 100 µM Mg2+, while the control did not grow at these conditions (**Figure 1A**), suggesting that OsHKT2;4 renders the mutant strains more tolerant to Mg2<sup>+</sup> deficiency by enhancing the Mg2<sup>+</sup> transport activity. However, OsHKT2;4 was less effective to restore the growth of mutant strains compared with AtMGT10 in the media containing insufficient Mg2+. For example, AtMGT10 rescued MM281 growth in medium containing 10 µM Mg2<sup>+</sup> (**Figure 1A**), as shown before (Li et al., 2001), while OsHKT2;4 did not (**Figure 1A**). These results indicated that although OsHKT2;4 had the Mg2<sup>+</sup> transport activity similar to AtMGT10, it might have the kinetic property with lower affinity to Mg2<sup>+</sup> in heterologous MM281 system.

To further verify the complementation of OsHKT2;4 for the growth of MM281 tested in the agar plates (**Figure 1A**), the bacteria were cultured in the liquid media containing 0.1, 0.5, 1, or 10 mM Mg2+, and their growth curves were established within 24 h after cultured. As shown in **Figure 1B**, the strain expressing AtMGT10 grew the most rapidly at these Mg2<sup>+</sup> concentrations, supporting that AtMGT10 is a high-affinity Mg2<sup>+</sup> transporter. In addition, MM281 expressing OsHKT2;4 displayed faster growth than those with empty vector pTrc99A under the conditions in which the Mg2<sup>+</sup> concentration was 1 or 10 mM. By contrast, in the presence of 0.1 or 0.5 mM Mg2+, the strains expressing OsHKT2;4 displayed the similar rate of growth to the strains expressing empty vector pTrc99A. These results were consistent with the ones observed on the agar plates, and demonstrated that OsHKT2;4 might mediate low-affinity Mg2<sup>+</sup> uptake in vivo.

### Mg2+-Dependent Currents Generated by OsHKT2;4 Expressing X. laevis Oocytes under High-Mg2<sup>+</sup> Conditions

To further assess the transporting properties of OsHKT2;4 under different Mg2<sup>+</sup> concentrations, two-electrode voltageclamp experiment using X. laevis oocytes was performed. OsHKT2;4-dependent currents were recorded from the oocytes injected with OsHKT2;4 cRNA or the oocytes injected with water perfused with different Mg2<sup>+</sup> concentrations. The oocytes injected with water produced small endogenous currents in perfusion medium with 6 mM Mg2<sup>+</sup> (**Figures 2A-a**). In contrast, OsHKT2;4-expressing oocytes generated the larger currents in the solutions containing 1.2, 6, and 20 mM Mg2<sup>+</sup> (**Figures 2A-b–d**). The current-voltage relationship displayed the currents from OsHKT2;4-expressing oocytes perfused with 6 or 20 mM Mg2<sup>+</sup> were significantly larger than those from OsHKT2;4-expressing oocytes perfused with 0.3 or 1.2 mM Mg2+. It was noteworthy that the currents from OsHKT2;4-expressing oocytes perfused with 0.3 mM Mg2<sup>+</sup> were similar to those with 1.2 mM Mg2+, implying they might not be Mg2<sup>+</sup> sensitive under low-Mg2<sup>+</sup> conditions (**Figure 2B**).

To test this possibility, we compared the amplitude and reversal potential of the currents generated from the oocytes perfused with 0, 0.3, 1.2, 6, or 20 mM Mg2+. The currents generated from the oocytes expressing OsHKT2;4 perfused with 0.3 or 1.2 mM Mg2<sup>+</sup> displayed the similar levels of the amplitudes and reversal potentials, even were similar to those without Mg2<sup>+</sup> (**Figures 2C,D**). These Mg2<sup>+</sup> insensitive currents were larger than the currents from the oocytes injected with water (**Figure 2C**), and thus they may result from other ions, such as Na<sup>+</sup> or K<sup>+</sup> currents generated by OsHKT2;4, as suggested by the previous studies (Lan et al., 2010; Horie et al., 2011). By contrast, in the presence of 6 or 20 mM Mg2+, the oocytes expressing OsHKT2;4 produced the currents with larger amplitudes and less negative reversal potentials compared with the others (**Figures 2C,D**). Thus, these results further supported the hypothesis that OsHKT2;4 exhibits permeability for Mg2<sup>+</sup> only under the conditions containing high-Mg2<sup>+</sup> concentrations.

### Overexpression of OsHKT2;4 Enhanced the Sensitivity of atmgt6 to High Mg2<sup>+</sup> But Not to Low Mg2<sup>+</sup>

We have shown previously that AtMGT6, a Mg2<sup>+</sup> deficiencyinduced Mg2<sup>+</sup> transporter, mediates directly Mg2<sup>+</sup> uptake in roots and is required for plant adaptation to low-Mg2<sup>+</sup> environment (Mao et al., 2014). An independent study reported ethyl methanesulfonate (EMS)-mutagenized AtMGT6, or named as AtMRS2-4, caused plant growth defects under both low and high-Mg2<sup>+</sup> conditions (Oda et al., 2016). Considering the critical role of AtMGT6 in Mg2<sup>+</sup> acquisition, we suggested the activity of Mg2<sup>+</sup> transport conducted by OsHKT2;4 might be covered by this transporter, and thus generated transgenic OsHKT2;4 Arabidopsis lines with the disruption of AtMGT6 to examine the potential relevance of OsHKT2;4 to Mg2<sup>+</sup> responses in planta. We used an Arabidopsis T-DNA insertion line (SALK\_203866), in which T-DNA was inserted into the third exon of AtMGT6 gene (**Figure 3A**). Transcript of AtMGT6 in the line SALK\_203866 was not detected by RT-PCR (**Figure 3C**), indicating that the T-DNA insertion line was a knockout allele, and referred to as atmgt6 line hereafter. We then expressed the coding region of OsHKT2;4 into atmgt6 line driven by its native promoter (**Figure 3B**). The transformants were screened by hygromycin, and were further analyzed for the expression levels of OsHKT2;4 by RT-PCR. We selected two of them as the representative transgenic lines due to their relatively high OsHKT2;4 expression levels, and referred to as OE29 and OE34, respectively, to perform subsequent experiments (**Figure 3C**).

We examined the growth of the Col-0, atmgt6, and transgenic lines OE29 and OE34 in Mg2+-depleted medium supplemented

with various contents of Mg2<sup>+</sup> as indicated in **Figure 3D**. The mutant atmgt6 exhibited growth defects in the medium containing 0, 0.01, 0.1, or 0.25 mM Mg2+, and had lower fresh weight and shorter roots than those of the Col-0 plants, while the growth retardation could be rescued in the Mg2+-sufficient medium (2 mM Mg2+) (**Figure 3D**), consistent with the idea that AtMGT6 confers low-Mg2<sup>+</sup> tolerance for Arabidopsis (Mao et al., 2014; Oda et al., 2016). However, OsHKT2;4 overexpression could not rescue the growth deficiency of atmgt6 in low-Mg2<sup>+</sup> conditions as expected, as transgenic lines OE29 and OE34 displayed the similar growth phenotype to atmgt6 with no significant differences on fresh weight and root length under these tested conditions (**Figures 3D–F**). These results suggested that OsHKT2;4 might not function in low-Mg2<sup>+</sup> conditions in planta.

As shown in the experiments in vitro, OsHKT2;4 exhibiting Mg2<sup>+</sup> transport activity in both heterologous MM281 system and X. laevis oocytes happened only at high external Mg2<sup>+</sup> concentrations (**Figures 1**, **2**). Therefore, we presumed that OsHKT2;4 might mediate Mg2<sup>+</sup> transport when plants were cultivated under the Mg2<sup>+</sup> abundant conditions, though it was unable to function in low-Mg2<sup>+</sup> conditions in planta (**Figures 3D–F**). To conduct assessment of the sensitivity to high-Mg2<sup>+</sup> condition of OsHKT2;4, we used 1/6 MS medium supplemented with several concentrations of Mg2<sup>+</sup> (2, 4, 6, 8, and 10 mM) for growth assays. After growing on 1/6 MS for

2 weeks, both the root length and fresh weight of transgenic lines OE29 and OE34 were comparable to those of atmgt6 under the normal condition. However, addition of 2 mM Mg2<sup>+</sup> resulted in growth arrest of OE29 and OE34 compared with atmgt6. Further increases of extra Mg2<sup>+</sup> (up to 10 mM) demonstrated a consistent dosage-dependent inhibitory manner (**Figure 4A**). Quantitative analysis of root length (**Figure 4B**) and fresh weight (**Figure 4C**) indicated that, compared with Col-0 and atmgt6, the aerial parts of OE29 and OE34 exhibited a more severe growth retardation in Mg2+-abundant conditions, while their root length were not altered. Taken together, these results demonstrated that OsHKT2;4 results in Mg toxicity on aerial tissues in high-Mg2<sup>+</sup> conditions in Arabidopsis, supporting the idea of low-affinity Mg2<sup>+</sup> uptake of OsHKT2;4.

To examine whether the high-Mg2<sup>+</sup> toxic phenotype is a consequence of the ectopic expression of OsHKT2;4 in OE lines, we conducted an RT-PCR analysis to verify the expression of OsHKT2;4 under a high-Mg2<sup>+</sup> condition supplemented with 6 mM Mg2+. As demonstrated in Supplementary Figure 5, OsHKT2;4 was mainly expressed in shoot tissues under normal growth conditions, consistent with the previous report in rice (Lan et al., 2010). Moreover, the expression of OsHKT2;4 in the shoots was not significantly induced by 6 mM Mg2+, and even decreased after 24 h<sup>0</sup> treatment with 6 mM Mg2+. However, expression of OsHKT2;4 in the roots was dramatically induced after 4 h<sup>0</sup> treatment with 6 mM Mg2+, and became even stronger after 24 h of this treatment (Supplementary Figure 5). The oppose effect on OsHKT2;4 expression roots and shoots upon high Mg2<sup>+</sup> suggested a disturbance on Mg2<sup>+</sup> balance between roots and shoots under the high-Mg2<sup>+</sup> conditions.

### Overexpression of OsHKT2;4 Affected Mg2<sup>+</sup> Homeostasis in the atmgt6 Lines

To probe the reason responsible for the increased sensitivity to high external Mg2<sup>+</sup> in the OE plants, Mg2<sup>+</sup> concentration of the atmgt6 and OsHKT2;4 overexpression lines was determined using ICP-MS. Plants were grown hydroponically for 2 weeks and then transferred to a fresh hydroponic medium containing 0, 0.25 (referred to as "Control" in **Figure 5**), and 6 mM Mg2<sup>+</sup> for another 2 days before the roots and shoots were harvested, respectively, for analysis. As shown in **Figure 5A**, Mg2<sup>+</sup> content was consistently higher in the OE lines than atmgt6 in shoot tissues when plants grown in all Mg2<sup>+</sup> regimes tested (0, 0.25, and 6 mM). In analysis of Mg2<sup>+</sup> content of root tissues among different plants, although they exhibited similar and incremental

Mg2<sup>+</sup> content in the Mg2+-deficient and normal medium, the OE lines contained ∼30% less Mg2<sup>+</sup> compared with the atmgt6 plants in 6 mM Mg2<sup>+</sup> condition (**Figure 5B**). These results indicated an altered Mg2<sup>+</sup> distribution ratio in shoot and root. We thus analyzed the Mg2<sup>+</sup> partitioning between shoots and roots in atmgt6 and OE lines, and noticed that as the Mg2<sup>+</sup> level elevated, more Mg2<sup>+</sup> sequestered in root tissues than in low-Mg2<sup>+</sup> condition. In medium containing 6 mM Mg2+, the shoots of atmgt6 accumulated ∼37%, while the OE lines accumulated over ∼50% of the total Mg2<sup>+</sup> enclosed in plants (**Figure 5C**), further confirming the critical role of OsHKT2;4 in the Mg2<sup>+</sup> allocation between shoots and roots.

### Increased Sensitivity of atmgt6 Lines Expressing OsHKT2;4 to Excess Mg2<sup>+</sup> Was Alleviated by Adding Ca2<sup>+</sup>

Due to the similar physical properties, Ca2<sup>+</sup> and Mg2<sup>+</sup> compete for the same sites of substrates (Yermiyahu et al., 1994), and the balance of Ca2<sup>+</sup> and Mg2<sup>+</sup> is an important factor for plant growth. Previously, evidence was presented that OsHKT2;4 acts as a channel for the transport of both Ca2<sup>+</sup> and Mg2<sup>+</sup> (Lan et al., 2010; Horie et al., 2011). To examine whether Ca2<sup>+</sup> affects the high Mg2+-sensitive phenotype in OsHKT2;4 overexpressing lines, we assessed the growth of Col-0, atmgt6, atmgt6 overexpressing OsHKT2;4 lines (OE29 and OE34) on Mg2+-abundant 1/6 MS medium supplemented with different concentrations of excess Ca2+.

In normal 1/6 MS medium, OE29 and OE34 exhibited the similar growth as atmgt6. As the concentration of external Mg2<sup>+</sup> increased, OE29 and OE34 started to show a more severe growth arrest than atmgt6 (**Figure 4A**). However, increasing Ca2<sup>+</sup> improved the plant growth (**Figure 6A**) of all genotypes, and the improvement was more obvious in the OE29 and OE34 lines (Supplementary Figure 2). For example, fresh weight of OE lines was ∼50% of that in atmgt6 in 1/6 MS medium with extra 8 mM Mg2<sup>+</sup> ("basal medium" in **Figure 6**), however, when 1 mM Ca2<sup>+</sup> was added to this basal medium, the fresh weight of OE lines was restored and reached to ∼80% to that in atmgt6. Moreover, when 3 mM

Col-0, atmgt6, OE29, and OE34 under the Mg2+-abundant conditions described in (A). Six independent 10-day-old seedlings of each genotype were gathered as one biological repeat for root length and fresh weight measurement. Data are represented as the mean ± SD, n = 3, in which n is the number of biological repeat. Asterisks indicate statistically significant differences compared with atmgt6 (Student's t-test, <sup>∗</sup>P < 0.05).

Ca2<sup>+</sup> were added to the basal medium, both root length and fresh weight of OE29 and OE34 were recovered to almost an identical level with that of atmgt6 (**Figures 6B,C** and Supplementary Figure 2), supporting the notion of Ca2+-Mg2<sup>+</sup> antagonism.

It has been reported that low Ca2<sup>+</sup> in the medium triggered the increase of Mg2<sup>+</sup> concentration, mimicking high-Mg2<sup>+</sup> conditions (Rios et al., 2012). To analyze further whether Ca2<sup>+</sup> deficiency is also responsible for the phenotype induced by high Mg2+, we thus tested the sensitivity among different plants to low-Ca2<sup>+</sup> conditions. In Ca2+-depleted 1/6 MS medium ("basal medium" in **Figure 7**), similar phenotype was observed in atmgt6 and OE lines, suggesting that OsHKT2;4 is not responsible for Ca2<sup>+</sup> deficiency. On the contrary, as the increasing content of extra Mg2<sup>+</sup> (2, 4, 6, and 8 mM) was added to the basal medium, differences of fresh weight between atmgt6 and OE lines started to occur (**Figure 7** and Supplementary Figure 3). These results demonstrated that high Mg2+, rather than Ca2<sup>+</sup> deficiency, is the primary factor that caused growth defects in the OE lines than atmgt6.

### DISCUSSION

Mg2<sup>+</sup> is an essential macronutrient for plant growth, development and reproductive success (Li et al., 2001; Hermans et al., 2013; Mao et al., 2014), while it could be detrimental at high concentrations (Visscher et al., 2010). Plants possess specific Mg2<sup>+</sup> transport systems that can function under a wide range of concentrations to secure intracellular Mg2<sup>+</sup> homeostasis. Despite several transporters have been shown to function in Mg2<sup>+</sup> uptake and distribution in Arabidopsis, including the AtMGT/AtMRS2-type transporters (Li et al., 2001) and Mg2+/H<sup>+</sup> antiporter AtMHX (Shaul et al., 1999), little is known about the transporters responsible for Mg2<sup>+</sup> homeostasis in rice. OsHKT2;4 has been reported to function as a non-selective transporter for diverse cations, including Mg2<sup>+</sup> and Ca2+. Our study here provided further evidence that OsHKT2;4 exhibits characteristics of low-affinity transport of Mg2+, and plays a key role in Mg2<sup>+</sup> homeostasis for plant's adaptation to high-Mg2<sup>+</sup> conditions.

Rice contains up to nine HKT genes (depending on variety), and OsHKT2;4 is the member of class II HKTs with the conserved Gly residues at the four P-loop filter positions (Mäser et al., 2002b). OsHKT2;4 is localized at the plasma membrane of rice cells, and its exogenous expression caused X. laevis oocytes to produce large currents when the extracellular Mg2<sup>+</sup> concentrations were at the range of millimolar levels (Lan et al., 2010; Horie et al., 2011). Triticum aestivum HKT2;1 (TaHKT2;1) was also found to result in robust Mg2<sup>+</sup> permeability of the oocytes, although to a lesser degree (Horie et al., 2011). OsHKT2;4-mediated currents exhibited the shifts to positive reversal potentials upon increased Mg2<sup>+</sup> concentration from 5 to 50 mM (Horie et al., 2011). The present study further analyzed the capability of Mg2+-uptake of OsHKT2;4 in three systems, the oocytes, bacteria, and Arabidopsis under the conditions containing high-Mg2<sup>+</sup> concentrations. The current amplitudes and reversal potentials in the oocytes expressing OsHKT2;4 were not changed when the extracellular Mg2<sup>+</sup> concentration was less than 1.2 mM, until its concentration reached 6 mM (**Figure 2**). Similarly, the expression of OsHKT2;4 rescued growth defects of MM281 bacteria cells that are deficient in Mg2<sup>+</sup> uptake in the presence of relatively high-Mg2<sup>+</sup> concentration, but the rescuing effect was much less than MGT10, the high-affinity Mg2<sup>+</sup> transporter (**Figure 1**). Complementary to the observations in the oocytes and bacteria, the phenotype relating to Mg2<sup>+</sup> stress in transgenic OsHKT2;4-overexpressed atmgt6 lines happened at the Mg2+-abundant (**Figure 4**), but not at Mg2+-deficient conditions (**Figure 4**). Taken together, our findings showed that OsHKT2;4 has a distinct low-affinity Mg2<sup>+</sup> transportation, and confirm Mg2<sup>+</sup> permeability of OsHKT2;4 as reported (Lan et al., 2010; Horie et al., 2011). It is worth mentioning that OsHKT2;4

was reported to be impermeable to Mg2<sup>+</sup> when it was expressed in the oocytes by an independent study (Sassi et al., 2012). Due to the genetic diversity in rice during evolution and amino acid variation of HKTs among Oryza accessions (Horie et al., 2001; Ren et al., 2005), the differences might result from the sources of OsHKT2;4 from different rice varieties. In the previous studies (Lan et al., 2010; Horie et al., 2011) and the present study, genetically tractable rice (O. sativa; background Nipponbare) was used.

Mg2<sup>+</sup> is taken up from the soil by the plant root system, which is likely to be mediated by AtMGT6/MRS2-4. AtMGT6/MRS2- 4 is located in the plasma membrane or the endoplasmic reticulum and highly expressed in the root epidermal cells, and its disruption resulted in growth retardation of Arabidopsis under the low-Mg2<sup>+</sup> condition (Mao et al., 2014; Oda et al., 2016). OsHKT2;4 might not be a key factor for roots to uptake Mg2<sup>+</sup> from the low-Mg2<sup>+</sup> environment as its overexpression did not cause the changed growth phenotype of transgenic lines (**Figure 3**), fitting the idea of its low-affinity Mg2<sup>+</sup> transportation. After satisfying the needs of the roots, the rest of the Mg2<sup>+</sup> will be transported to the shoot through the process involving AtMGT6/MRS2-4 activity (Oda et al., 2016). However, plants will display Mg2<sup>+</sup> toxicity symptom when Mg2<sup>+</sup> is over accumulated in the shoot. To deal with this toxicity, plants might restrain the Mg2<sup>+</sup> distribution in shoot or sequester the excess intracellular Mg2<sup>+</sup> into the vacuoles (Hermans et al., 2013; Tang et al., 2015). The OE lines had higher Mg2<sup>+</sup> content in shoot compared with atmgt6 under both low-Mg2<sup>+</sup> and normal conditions (**Figure 5**), indicating the expression level of OsHKT2;4 was high enough to drive transportation of Mg2<sup>+</sup> from root to shoot. However, once the expression level of OsHKT2;4 was further enhanced under high-Mg2<sup>+</sup> conditions (Supplementary Figure 5), the Mg2<sup>+</sup> transportation to shoot was also strengthened, thus leading to an increased Mg2<sup>+</sup> distribution ratio of shoot to root and enhanced sensitivities of OE lines to high-Mg2<sup>+</sup> conditions. Therefore, we suggested that OsHKT2;4 plays a key role in Mg2<sup>+</sup> homeostasis and might control the Mg2<sup>+</sup> translocation between roots and shoots under the high-Mg2<sup>+</sup> conditions.

Although roles of Ca2<sup>+</sup> and Mg2<sup>+</sup> are distinct in diverse physiological and biochemical processes, they may play an antagonistic function in plants. (Tang and Luan, 2017). For example, growth retardations induced by individual knockouts of genes in AtMRS2/AtMGT family under low

Mg2<sup>+</sup> could be ameliorated when Ca2<sup>+</sup> concentrations were concomitantly lowered (Lenz et al., 2013). Mutation of AtCAX1, which encodes a vacuolar Ca2+/H<sup>+</sup> exchanger, resulted in reduction of Ca2<sup>+</sup> in the vacuole, thus leading to more Ca2<sup>+</sup> retaining in the cytosol to counteract

with excess Mg2<sup>+</sup> (Cheng et al., 2003; Bradshaw, 2005). Consistently, our study demonstrated that addition of Ca2<sup>+</sup> to the high-Mg2<sup>+</sup> medium could partially rescue the Mg2+-induced growth defect of atmgt6 and the OsHKT2;4-overexpressed lines to a wild type level (**Figure 6**), which is also a supportive evidence for the antagonistic interaction between Ca2<sup>+</sup> and Mg2<sup>+</sup> in planta. However, Mg2<sup>+</sup> currents through OsHKT2;4 in oocytes were not inhibited and their reversal potentials were not significantly shifted in the presence of 1.8 mM Ca2<sup>+</sup> in the perfusion solution (Supplementary Figure 4), indicating that Ca2<sup>+</sup> did not inhibit Mg2<sup>+</sup> uptake in oocytes expressing OsHKT2;4. Thus, the effects of changes in Ca2<sup>+</sup> additions on Mg2<sup>+</sup> toxicity might result from the physiological antagonism between Ca2<sup>+</sup> and Mg2<sup>+</sup> in planta (Bradshaw, 2005; Tang et al., 2015), although further evidence is needed.

In our previous study, we found that OsHKT2;4 had the diverse expression pattern in rice plants, including leaves, stems and primary/lateral roots, and was highly expressed at xylem and phloem of epidermis (Lan et al., 2010). However, the homozygous lines of Tos-tagged oshkt2;4 rice lines behaved similarly to wildtype plants (Lan et al., 2010; Horie et al., 2011), and contained similar content of cations, including Mg2<sup>+</sup> and Ca2<sup>+</sup> (Lan et al., 2010). The absence of a phenotypic change in these rice lines suggested that OsHKT2;4 is functionally redundant with other transporters. Indeed, we found OsHKT2;4 rendered Arabidopsis Mg2<sup>+</sup> sensitivity when atmgt6 was knockout (**Figure 4**). Rice (O. sativa; background Nipponbare) is predicted to have nine AtMGT orthologs based on the BLAST search (Gebert et al., 2009), and Os10g0545000, the closest one to AtMGT6, is also widely expressed in rice according to the microarray gene expression data collected by Genevestigator<sup>1</sup>

<sup>1</sup> www.genevestigator.com

### REFERENCES


(Supplementary Figure 1). Elucidation of the functional relationships between MGT-type transporters and OsHKT2;4 will be a critical next step toward assessing their biological functions in rice.

### AUTHOR CONTRIBUTIONS

CZ, BZ, HL, JG, and WL designed the study. CZ, HL, BZ, and WL performed experiments. CZ, HL, JW, BZ, WW, JG, and WL analyzed and interpreted the data. CZ, HL, BZ, and WL wrote the manuscript. CZ, BZ, HL, SL, JG, and WL revised the manuscript critically. All authors read and approved the final manuscript.

## FUNDING

This research was supported by the National Natural Science Foundation of China (31271626 and 31271682).

### ACKNOWLEDGMENT

We thank Jiangsu Collaborative Innovation Center for Modern Crop Production for technical support.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2017.01823/ full#supplementary-material



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Zhang, Li, Wang, Zhang, Wang, Lin, Luan, Gao and Lan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Overexpression of Pyrabactin Resistance-Like Abscisic Acid Receptors Enhances Drought, Osmotic, and Cold Tolerance in Transgenic Poplars

Jingling Yu<sup>1</sup>† , Haiman Ge<sup>1</sup>† , Xiaokun Wang<sup>1</sup> , Renjie Tang<sup>2</sup> , Yuan Wang<sup>1</sup> , Fugeng Zhao<sup>1</sup> , Wenzhi Lan<sup>1</sup> \*, Sheng Luan1,2 \* and Lei Yang<sup>1</sup> \*

<sup>1</sup> State Key Laboratory for Pharmaceutical Biotechnology, NJU–NFU Joint Institute for Plant Molecular Biology, College of Life Sciences, Nanjing University, Nanjing, China, <sup>2</sup> Department of Plant and Microbial Biology, University of California,

#### Edited by: Kai He,

Lanzhou University, China

#### Reviewed by:

Frantisek Baluska, University of Bonn, Germany Tamara Pecenkova, Institute of Experimental Botany (ASCR), Czechia

#### \*Correspondence:

Wenzhi Lan lanw@nju.edu.cn Sheng Luan sluan@berkeley.edu Lei Yang leiyang@nju.edu.cn

†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science

Received: 22 July 2017 Accepted: 25 September 2017 Published: 13 October 2017

### Citation:

Yu J, Ge H, Wang X, Tang R, Wang Y, Zhao F, Lan W, Luan S and Yang L (2017) Overexpression of Pyrabactin Resistance-Like Abscisic Acid Receptors Enhances Drought, Osmotic, and Cold Tolerance in Transgenic Poplars. Front. Plant Sci. 8:1752. doi: 10.3389/fpls.2017.01752 Abscisic acid (ABA) has been known participate in a wider range of adaptive responses to diverse environmental abiotic stresses such as drought, osmosis, and low temperatures. ABA signaling is initiated by its receptors PYR/PYL/RCARs, a type of soluble proteins with a conserved START domain which can bind ABA and trigger the downstream pathway. Previously, we discovered that poplar (Populus trichocarpa) genome encodes 14 PYR/PYL/RCAR orthologs (PtPYRLs), and two of them, PtPYRL1 and PtPYRL5 have been functionally characterized to positively regulate drought tolerance. However, the physiological function of these ABA receptors in poplar remains uncharacterized. Here, we generated transgenic poplar plants overexpressing PtPYRL1 and PtPYRL5 and found that they exhibited more vigorous growth and produced greater biomass when exposed to drought stress. The improved drought tolerance was positively correlated with the key physiological responses dictated by the ABA signaling pathway, including increase in stomatal closure and decrease in leaf water loss. Further analyses revealed that overexpression lines showed improved capacity in scavenging reactive oxygen species and enhanced the activation of antioxidant enzymes under drought stress. Moreover, overexpression of PtPYRL1 or PtPYRL5 significantly increased the poplar resistance to osmotic and cold stresses. In summary, our results suggest that

Keywords: Populus, ABA receptor, drought, cold stress, osmotic stress, resistance

### INTRODUCTION

Berkeley, Berkeley, CA, United States

Plants, as sessile organisms, have evolved sophisticated developmental and physiological strategies to adapt to the unfavorable and changing environments such as drought, high salinity, and temperature fluctuations. Drought has particularly been considered as one of the most serious natural hazards for agriculture due to the increasing water scarcity (Liu et al., 2015). Numerous

constitutive expression of PtPYRL1 and PtPYRL5 significantly enhances the resistance to drought, osmotic and cold stresses by positively regulating ABA signaling in poplar.

**99**

studies showed that drought stress exerts many negative effects on plant growth, photosynthesis, biomass accumulation, and ecosystem carbon cycling (Liu et al., 2011; Zhou et al., 2015). Desiccation results in the production of reactive oxygen species (ROS), the production of which, in turn, serves as an important indicator to multiple abiotic stresses (Apel and Hirt, 2004; Contreras-Porcia et al., 2011). Normally, ROS are rapidly scavenged as a result of the activation of an efficient antioxidant system involved in various drought-induced signaling pathways, which are modulated by abscisic acid (ABA) (Jiang and Zhang, 2002; Contreras-Porcia et al., 2011).

The physiological functions of ABA has been extensively investigated in plants since it was identified in the 1960s as an endogenous plant hormone which regulates many essential processes, including seed germination, stomatal movement, plant development and adaptive responses, to multiple environmental stresses, such as drought, extreme temperatures, hyperosmolarity, and salinity (Finkelstein et al., 2002; Park et al., 2009). Many key components in ABA signaling pathway have been identified at the molecular level (Finkelstein et al., 2002), including ABA receptors (Shen et al., 2006; Liu et al., 2007; Pandey et al., 2009), the group A type 2 C protein phosphatases (PP2Cs) that negatively regulate ABA signaling at an early step in the pathway (Allen et al., 1999), the SnRK2 kinases that are positive regulators (Mustilli et al., 2002; Yoshida et al., 2002; Fujii et al., 2007), transcription factors (Seki et al., 2002; Himmelbach et al., 2003) and ion channels (Lee et al., 2009).

As the initial sensor of ABA signaling pathway, 14 genes designated as Pyr1 and Pyl1-Pyl13 (for PYR1-Like) have been identified in the Arabidopsis genome and encode proteins belonging to members of the cyclase subfamily of the START/Bet v I superfamily, which share a conserved hydrophobic ligandbinding pocket (Ma et al., 2009; Park et al., 2009). In presence of ABA, these proteins perceive ABA, then undergo conformation changes, and subsequently bind to clade A subfamily of PP2Cs, thus de-repressing the inactivation of the downstream SNF1 related protein kinase 2 (SnRK2) kinases. The PYR/PYL/RCAR-PP2C-SnRK2 signaling module is conserved across land plants.

Genetic evidence suggested that higher-order mutants lacking multiple ABA receptors in Arabidopsis (Pyr1;Pyl1;Pyl2;Pyl4;Pyl5;Pyl8) exhibit severe ABA insensitive phenotypes, establishing their critical physiological roles in ABA signaling (Gonzalez-Guzman et al., 2012). PYL5 over-expression in Arabidopsis led to a globally enhanced response to ABA and enhanced drought resistance (Santiago et al., 2009). Constitutive overexpression of PYL8/RCAR3 confers ABA hypersensitivity in Arabidopsis seeds (Saavedra et al., 2010). Overexpression of NtPYL4 in tobacco hairy roots resulted in a reprogramming of the cellular metabolism that is represented by a decreased alkaloid accumulation and conferred ABA sensitivity to the production of alkaloids (Lackman et al., 2011).

Tree species in the genus Populus spp., commonly known as poplars, aspens, and cottonwoods, are widespread in the northern hemisphere with nutrient-poor environments, and are increasingly important for bioenergy, wood products, and environmental services (Doty et al., 2016). Most of the poplar cultivation and distribution area are in the desolate lands on the earth where it is usually cold and dry in winter, causing the restriction of poplar forestry. Therefore, it is very critical to improve the resistance of poplars to abiotic stresses, including drought-resistance and cold-resistance. We have previously shown that PtPYRL1 and PtPYRL5 (poplar AtPYR1-like 1 and 5) physically interacted with PP2Cs, which interacted with SnRK2 kinase, suggesting they might act as the ABA receptors in mediating ABA signal transduction through phosphorylation and dephosphorylation (Yu et al., 2016). Furthermore, overexpression of PtPYRL1 or PtPYRL5 in Arabidopsis enhanced ABA sensitivity and drought-resistance. However, it is still unknown if they also play a similar function in poplars which protect against drought stress. Here, we report that the overexpression of PtPYRL1 and PtPYRL5 in poplar enhances resistance to drought, osmosis, and cold, the abiotic stresses that poplar frequently encounters. Our results also provide a potential biotechnological tool in engineering stress-resistant poplar cultivars.

### MATERIALS AND METHODS

### Plant Materials and Growth Conditions

The sterile wild-type and transgenic hybrid poplar (Populus davidiana × Populus bolleana) were amplified by micropropagation with leaf bud explants and kept under a 16 h-light/8-h-dark photoperiod (120 µmol·m−<sup>2</sup> ·s −1 ) at 21–24◦C. The plantlets were sub-cultured onto fresh 1/2 Murashige and Skoog (MS) medium supplemented with 0.1 mg·L −1 naphthaleneacetic acid (NAA) and 1% (w/v) agar. Two-week-old wild type (WT) or transgenic hybrid poplars with new roots were transferred to 7.5 cm-width pots containing nutrient soil. The plants were grown in greenhouse under a 16-h-light/8-h-dark (120 µmol·m−<sup>2</sup> ·s −1 ) at 21–24◦C.

### Poplar Transformation

To construct the pCAMBIA1301S2-PtPYRL1 (or PtPYRL5) plasmid, the entire coding region of PtPYRL1 (or PtPYRL5) was amplified by PCR with Xba I-Sal I (or BamH I-Sal I) linker primers and cloned into modified pCAMBIA1301S2 with the 2×CaMV 35S promoter via the Xba I-Sal I (or BamH I-Sal I) site (**Supplementary Figure S1**). The hybrid poplars (Populus davidiana × Populus bolleana) were transformed using Agrobacterium tumefaciens (EHA105 strain) infection method. Briefly, leave explants excised from 1- to 2-month-old were inoculated in the EHA105 culture resuspended with liquid MS for 10 min and then were plated on solid MS medium containing 0.4 mg·L −1 6-BA, 0.1 mg·L <sup>−</sup><sup>1</sup> NAA and 0.01 mg·L <sup>−</sup><sup>1</sup> TDZ. After 2 days of co-cultivation with Agrobacterium, the explants were transferred onto fresh MS medium containing 400 mg·L −1 timentin, and 10 mg·L <sup>−</sup><sup>1</sup> hygromycin for selective regeneration. When regenerated shoots reached 1 cm tall, they were excised and placed on rooting medium. Then, the rooted seedlings were transferred into soil and grown in the greenhouse. The presence of transgene was verified by PCR from genomic DNA, using primers specific for P35S and PtPYRL1-RT-R or PtPYRL5-RT-R.

### Histochemical GUS Analysis

fpls-08-01752 October 11, 2017 Time: 15:49 # 3

Detection of β-glucuronidase (GUS) activity was performed as described by Jefferson et al. (1987) with some modifications: leaf explants were incubated in GUS assay buffer (50 mM sodium phosphate, pH 7.0, 0.1% [v/v] Triton X-100, 0.5 mM ferricyanide, 0.5 mM ferrocyanide, and 2 mM 5-bromo-4-chloro-3-indolylβ-D-glucuronide) for 12 h at 37◦C. Then the plant tissues were decolorized in 75% ethanol for three times. The samples were photographed after treatment.

### Quantitative Real-time PCR Analysis

Total RNA was extracted from various samples using TRIzol reagent (Invitrogen, Carlsbad, CA, United States), which was sequentially treated with DNase I (Invitrogen) and reverse transcribed by M-MLV reverse transcriptase (Promega). In semiquantitative RT-PCR and qRT-PCR assays, the poplar elongation factor gene EF1β was used as an internal reference. qRT-PCR was performed with a CFX Connect Real-Time System (Bio-Rad). The relative expression of series indicated genes was calculated based on the comparative threshold cycle method using EF1β as a control and normalized to the WT hybrid poplars (under normal conditions). All primers used in this study were listed in Supplementary Table S1.

### Drought–Rehydration Experiments

Two-week-old WT or transgenic poplars were transplanted to each 7.5 cm-width pot containing 50 g nutrient soil for greenhouse cultivation. We chose the 2-month-old WT or transgenic poplar seedlings with the same height to perform the drought and rehydration experiments. As the control, half of WT and transgenic poplar seedlings were normally watered, and the rest of these seedlings were not watered. After 1 week, the plantlets were watered again. After 3 days, the re-watered plants were photographed.

### Water Potential Determination

Leaf water potential was measured on the same location of the blade with a WP4-T Dew point PotentiaMeter (Decagon Devices, Inc., United States). Five individual plants from WT or different transgenic poplars were measured after drought treatment for 5 days.

### Measurement of Contents of H2O2, MDA, and Proline

H2O<sup>2</sup> in fresh leaves was analyzed using the method reported by Hu et al. (2012). The absorbance was recorded at 390 nm with the spectrophotometer (Biomate 3S, Thermo). Malondialdehyde (MDA) content was quantified using the method reported by Heath and Packer (1968), which is related with the level of lipid peroxidation in the leaves. The absorbance was read at 532 and 600 nm by the spectrophotometer (Biomate 3S, Thermo) with thiobarbituric acid (1%) in 20% trichloroacetic acid as control. The Proline content in leaves was quantified using the method by Bates et al. (1973). The absorbance was measured spectrophotometrically (Biomate 3S, Thermo) at 520 nm and toluene was used as blank.

### Extraction and Assay of Antioxidant Enzymes

Fresh leaves (0.5 g/sample) were homogenized in presence of 100 mM Tris-HCl (5.0 ml, pH 7.5), 3.0 mM β-mercaptoethanol, 1.0 mM EDTA (ethylenediaminetetraacetic acid), and 1.5% polyvinylpyrrolidone-40. The mixture was better supplemented with serine and cysteine proteinase inhibitors [1.0 mM phenylmethanesulfonyl fluoride (PMSF) + 1.0 µg·mL−<sup>1</sup> aprotinin]. The homogenate was centrifuged at 10,000 × g for 15 min (4◦C) after the filtration through cheese cloth. The supernatants were collected and served as the crude enzyme for determination of SOD (EC1.15.1.1), CAT (EC1.11.1.6), and POD (EC 1.11.1.7) activities. For the determination of APX activity, leaf sample was separately grounded in a homogenizing medium supplemented with 2.0 mM ascorbic acid (AsA) to maintain the enzyme stability. SOD activity was analyzed after the photoreduction of nitroblue tetrazolium (NBT) according to the method of Giannopolitis and Ries (1977). The absorbance was recorded spectrophotometrically (Biomate 3S, Thermo) at 560 nm. One unit of SOD is the quantity of protein that hampers 50% photoreduction of NBT and the activity was expressed as enzyme unit (EU)·mg−<sup>1</sup> protein. The method of Chance and Maehly (1955) was employed to analyze CAT activity. The absorbance was read at 240 nm through a UV-Visible spectrophotometer (Biomate 3S, Thermo) and EU·mg−<sup>1</sup> protein represented the CAT activity. The method of Nakano and Asada (1981) was used for the APX activity (EC1.11.1.11) measurement. The absorbance was recorded at 290 nm each 30 s for 3 min spectrophotometrically (Biomate 3S, Thermo). The POD activity was determined by examining the absorbance of reaction buffer at 420 nm based on guaiacol oxidation (Maehly and Chance, 1954). APX activity was calculated by

the consumption rate of ASC using the ASC extinction molar coefficient (e = 2.8 mM−<sup>1</sup> ·cm−<sup>1</sup> ). APX activity was expressed with EU·mg−<sup>1</sup> protein. One unit of APX is the quantity of protein used to break down 1.0 µmol of substrate per min at 25◦C. The activity of GR (EC 1.6.4.2) and GPX (EC 1.11.1.9) was determined using GR Assay Kit (S0055, Beyotime, China) and Total GPX Assay Kit (S0058, Beyotime, China), respectively, according to the manufacturer's instructions. The absorbance was read at 340 nm with spectrophotometer (Biomate 3S, Thermo). GR activity was expressed as µmol NADPH oxidized min−<sup>1</sup> (EU·mg−<sup>1</sup> protein) (Carlberg and Mannervik, 1985). The GPX activity was calculated by measuring the reduction of NADPH to NADP<sup>+</sup> at 340 nm of absorbance.

### Analysis of Relative Water Content (RWC)

The extent of desiccation in WT and transgenic poplars aboveground part was indicated with RWC (%) following the formula RWC % = (Wde – Wdr)/(W<sup>f</sup> − Wdr) × 100, where W<sup>f</sup> is the wet weight of fully hydrated aboveground part, Wde is the dehydrated weight after desiccation for a period of time, and Wdr is the dry weight determined after 48 h of drying at 80◦C. Through this, the RWC reflects the extent of desiccation, with a fully hydrated shoot having a RWC of 100% and a fully dehydrated shoot having a RWC close to 0%. A lower RWC indicates higher desiccation.

### Stomatal Aperture Measurements

To measure stomatal aperture in response to ABA, epidermal peels of the leaves in the same location were floated on a stomatal opening medium containing 50 mM KCl, 10 mM MES-KOH (pH 6.15), and 0.1 mM CaCl<sup>2</sup> and incubated in a growth chamber under white light (150 µmol·m−<sup>2</sup> ·s −1 ) for 2 h. The epidermal strips were transferred to the opening medium with 0 or 20 µM ABA and incubated for a further 2 h before stomatal apertures were measured.

FIGURE 2 | Expression of PtPYRL1 or PtPYRL5 in wild type (WT) and transgenic hybrid poplars. Expression of PtPYRL1 (A) or PtPYRL5 (C) in 2-month-old WT and transgenic hybrid poplars (L1-13, L1-10, and L1-3) grown at normal medium. The level was detected using gene-specific primers after 26 PCR cycles. EF1β was used as endogenous control in all cDNA samples. Relative levels of PtPYRL1 transcripts (B) or PtPYRL5 transcripts (D) in three transgenic lines by qRT-PCR. Total RNA was isolated from 2-month-old leaves grown at normal medium. The relative expression level was calculated as the ratio of PtPYRL level to endogenous control EF1β level.

### Hyperosmotic Stress Treatment

fpls-08-01752 October 11, 2017 Time: 15:49 # 5

The 5 cm apical shoot segments of WT and transgenic poplars were transferred into the 1/2 solid Murashige and Skoog (MS) medium containing 0.1 mg·L <sup>−</sup><sup>1</sup> naphthaleneacetic acid and 1% (w/v) agar supplemented with 0, 200, or 300 mM mannitol. The photos were taken at the 30th day after mannitol treatment. The experiments were repeated three times.

## H2O<sup>2</sup> Staining

H2O<sup>2</sup> in leaves was visualized by 3,3-diaminobenzidine (DAB). Populus leaves were cut at the leaf base and infiltrated in 1 mg/mL DAB solution (50 mM Tris-HAC, pH 5.0) for 2–8 h. Samples were then decolorized in 95% ethanol at 80◦C for 2 h. Brown flecks indicate the accumulation of H2O<sup>2</sup> (Yang et al., 2004). The leaves were observed and photographed under stereomicroscope.

### Statistical Analysis

The experiments were repeated three times and all comparisons of average values were analyzed using one-way ANOVA test. Post hoc comparisons were performed by applying least significant difference test. Significant differences were indicated with the threshold of P < 0.05.

### RESULTS

### Molecular Characterization of the Transgenic PtPYRLs Poplar Plants

To obtain transgenic poplar lines that overexpress the ABA receptors PtPYRL1 and PtPYRL5 genes under the control of the CaMV 35S promoter were introduced into the leaf explants from

(C) After drought, WT and transgenic hybrid poplars were then re-watered for 3 days. The shoot weight after drought stress treatment (D), shoot height after re-watered for 3 days (E), water potential value (F) of WT and transgenic hybrid poplars were measured. Values are means ± standard deviation (SD) (one-way ANOVA test; <sup>∗</sup>P < 0.05 as compared to WT).

a hybrid poplar cultivar (Populus davidiana × Populus bolleana), respectively. After cultured in the medium containing timentin and hygromycin, shoots were regenerated from some transfected explants (**Supplementary Figure S2A**). Partial regenerated shoots formed roots when they were placed on rooting medium (**Supplementary Figure S2B**), and these rooted plantlets were selected to analyze 35S-PtPYRL1 or 35S-PtPYRL5 insertion by PCR analysis of genomic DNA. A reporter gene encoding GUS was co-transferred into the leaf explants via the same vector, to authenticate the transgene expression in the putative transgenic lines. The GUS staining analysis revealed that all rooted seedlings expressed GUS activity properly (**Figure 1A**), confirming the integration and expression of the transgene in the genome of the transformants (**Figure 1B**). More than 10 independent lines were obtained for each of transgenes, including the lines L1-3, L1-10, and L1-13 expressing PtPYRL1, and the lines L5-1, L5-2, and L5-3 expressing PtPYRL5 (**Figure 1B**). After the rooted seedlings were transferred into soil, the survival seedlings were selected for further analysis of the differential expression levels of PtPYRLs by RT-PCR (**Figures 2A,C**) and qRT-PCR (**Figures 2B,D**). Among of them, Lines L1-3, L1-10, and L1-13 had over eightfold increment of the relative expression level of PtPYRL1 than in WT (**Figure 2B**), and lines L5-1 and L5-3 had over threefold increment of the relative expression level of PtPYRL5 compared with WT (**Figure 2D**). Thus, two lines with high level of PtPYRL1 (L1-3 and L1-10) or of PtPYRL5 (L5-1 and L5-3) were used for subsequent physiological analysis.

### Overexpression of Poplar ABA Receptors Enhanced Drought Tolerance in Transgenic Poplar Plants

To investigate the role of PtPYRLs in drought tolerance, 2-month-old wild-type, PtPYRL1 transgenic plants (L1-3 and L1-10), and PtPYRL5 transgenic plants (L5-1 and L5-3) were subjected to water withhold for 5 days and then re-watered for 3 days. After 5 days of water deprivation, wild-type plants began to wilt, while most of transgenic plants remained fresh and alive (**Figure 3B**), similar to those grown under the watering condition (**Figure 3A**). When plants were re-watered for 3 days,

WT became wilted permanently and eventually died, whereas transgenic plants showed less damaged and most of them recovered growth (**Figure 3C**). Similarly, PtPYRL5 transgenic plants [L5-1 and L5-3 and another two PtPYRL1 transgenic lines (L1-8 and L1-5)] displayed a healthier growth compared with the WT after rewatering (**Supplementary Figures S3A–C**). Furthermore, transgenic poplars overexpressing PtPYRL1 or PtPYRL5 showed more shoot biomass (**Figure 3D** and **Supplementary Figures S3D, S4D**) and shoot height (**Figure 3E** and **Supplementary Figures S3E, S4E**) than WT. They also had higher leaf water potential 9 than WT (**Figure 3F** and **Supplementary Figures S3F, S4F**), indicating the leaves of transgenic lines retained more water during the stress.

Drought stress severely impairs the cellular lipid structure and function in tree species (Štajner et al., 2011), and thus the corresponding products proline and MDA were measured in these poplars. Though the proline and MDA contents in both WT and transgenic poplars were increased after drought stress treatment, the transgenic poplars contained higher proline level (**Figures 4A,C**) and less MDA content (**Figures 4B,D**) than WT. It is noteworthy that the changes of biomass, water potential, proline, and MDA in the transgenic PtPYRL5 lines (L5-1 and L5-3) were less significant than the transgenic PtPYRL1 lines (L1-3 and L1-10), suggesting that PtPYRL1 may be more potent than PtPYL5 in inducing the downstream protective responses. Moreover, relative water content (RWC) of detached leaves was higher from transgenic PtPYRL1 lines (L1-3 and L1-10) than from WT plants after exposed in air for 6 h (**Figure 5**). Taken together, these results indicated that overexpression of PtPYRL1 or PtPYRL5 in poplars enhanced the resistance to drought stress, probably by reducing water loss rate.

### Effects of PtPYRLs Overexpression on Antioxidant Metabolism in Poplars under Drought Stress

Lipid hydroperoxidation is considered as the biochemical indicator of cellular oxidative damage, which is induced by excessive accumulation of the reactive oxidative species (e.g., superoxide and H2O2) in plant cells (Yoshimura et al., 2004; Sochor et al., 2012). Therefore, we measured H2O<sup>2</sup> content in the leaves of WT and transgenic plants. In parallel with the increase in MDA contents indicated in **Figure 4**, cellular H2O<sup>2</sup> levels were also increased by drought stress in the WT and transgenic PtPYRL1 or PtPYRL5 plants. However, H2O<sup>2</sup> level was significantly lower in these transgenic hybrid poplars than that in the WT leaves (**Figure 6A**). Correspondingly, the activity of the key enzymes controlling ROS

scavenging, including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidases (APX), and catalase (CAT), were analyzed in these poplars under drought stress. Indeed, the activity of SOD, POD, APX, and CAT were remarkably enhanced in transgenic hybrid poplars compared with the WT (**Figures 6B–E**). Besides these antioxidant enzymes, plants have several non-enzymatic antioxidants important for redox equilibrium, such as glutathione (GSH) (Cao et al., 2009). GSH content is controlled by glutathione reductase (GR), which has an important role in maintaining the level of glutathione, and by glutathione peroxidases (GPx), which can catalyze the reduction of lipid peroxide through glutathione. Upon drought stress, transgenic hybrid poplars had higher activity of GR and GPx than WT (**Figures 6F,G**). These results indicated that overexpression of PtPYRL1 and PtPYRL5 resulted in greater stimulation of the activity of enzymes responsible for ROS and GSH metabolism in poplars upon drought stress.

### Overexpression of PtPYRL1 or PtPYRL5 Accelerated Stomatal Closure Induced by ABA

The stomatal movement is one of the most important responses in plants under drought stress, and stomatal behavior is highly controlled by ABA. ABA was thus applied to assess the potential difference in stomatal movement between the WT and the transgenic PtPYRLs poplars. Without any treatment, even though overexpressing lines have more closed stomata, the difference was not significant, as shown by ANOVA test. With 20 µM ABA treatment, a significant decrease in stomatal aperture was found for both WT and the transgenic PtPYRLs poplars (**Figure 7A**). There was a significant difference between WTmock and WT-ABA using ANOVA test. Stomatal apertures in the PtPYRL1- and PtPYRL5-overexpressing lines L1-3, L1- 10, L5-1, and L5-3 reduced to 0.394, 0.395, 0.387, and 0.392, respectively, while stomatal aperture of WT became 0.490 (**Figure 7B**), which was significantly larger than that of PtPYRL1 and PtPYRL5-overexpressing lines. These results indicate that the overexpression of PtPYRL1 and PtPYRL5 enhanced poplar stomatal closure in response to ABA, which might be the cause of the enhanced drought tolerance of transgenic plants.

### Overexpression of PtPYRL1 or PtPYRL5 Enhanced Osmotic Stress Resistance

Osmotic damage is a major consequence of drought stress in plants, and ABA signaling contributes to damage reduction (Skirycz and Inzé, 2010). Overexpression of PtPYRL1 or PtPYRL5 enhanced resistance to water deficit (**Figure 3** and **Supplementary Figures S3, S4**), which suggested a possible role of PtPYRL1 or PtPYRL5 in high osmotic stress resistance. To confirm this possibility, wild-type and transgenic shoots were cultured on 1/2 MS supplemented with 200 or 300 mM mannitol. The supplementation of mannitol inhibited the growth of WT and transgenic poplars (**Figure 8A**). At the presence of 300 mM mannitol, the WT plants did not even survive and their leaves show yellowish and withered, while young leaves of transgenic plants overexpressing PtPYRL1 or PtPYRL5 kept green and some

old leaves displayed yellowish at leaf edge (**Figures 8A,B**). In addition, unlike WT plants, all transgenic plants generated new roots successfully in 1/2 MS medium supplemented with 300 mM mannitol (**Figures 8A,B**). According to the statistical analysis shown in **Figure 8C**, transgenic plants overexpressing PtPYRL1 or PtPYRL5 had roots with an average length of around 4.5 cm, while the WT did not have visible roots. These results indicated that the transgenic poplar overexpressing PtPYRL1 or PtPYRL5 improved the resistance to hyperosmotic stress.

### Overexpression of PtPYRL1 or PtPYRL5 Reduced the Injury Induced by Low Temperature

We also subjected WT and transgenic PtPYRL1 or PtPYRL5 poplars to low temperature which is a common stress in poplars growth and found that PtPYRL1 and PtPYRL5 transgenic poplars displayed the increased resistance under chilling stress (**Figure 9**). After 4◦C treatment for 5 days, the apical young leaves of WT were damaged more severely with leaf wilting and necrosis than that of transgenic poplars (**Figure 9A**). Furthermore, the leaves of WT had a higher accumulation of hydrogen peroxide visualized by 3,3-diaminobenzidine (DAB) (**Figure 9B**). Meanwhile, the

leaves of WT contained less proline accumulation, a key antifreeze component (**Figure 9C**). These results suggested that overexpression of PtPYRL1 or PtPYRL5 alleviated the injury induced by low temperature in poplars.

### DISCUSSION

As initial factors in triggering ABA signaling, the physiological function of PYR/PYL/RCARs family is supposed to be critical in the evolution from aquatic to terrestrial plants. PYR/PYL/RCAR genes have been reported to be present as 13–14 members in the genome of Arabidopsis, rice and Populus (Ma et al., 2009; Tian et al., 2015; Yu et al., 2016). As a class of ubiquitous soluble protein, the PYR/PYL/RCARs family plays a critical role in ABA response and signal transduction in plants. Overexpression of Arabidopsis PYR/PYL/RCAR receptors is known to enhance ABA response and plant drought tolerance (Santiago et al., 2009; Saavedra et al., 2010; Pizzio et al., 2013). Our previous study showed the transgenic Arabidopsis overexpressing PtPYRL1 or PtPYRL5 were both hypersensitive to ABA and enhanced drought resistance (Yu et al., 2016). In the present study, we further provide evidence that these two PtPYRLs are very important in poplars to confer tolerance to diverse environmental

abiotic stresses, including drought, hyper-osmosis, and low temperature.

The PYR/PYL/RCAR genes have been shown to enhance drought tolerance in Arabidopsis, rice, and tomato (González-Guzmán et al., 2014; Kim et al., 2014; Zhao et al., 2016). But the evidence of functional studies in the woody plants are still lacking. In our study, hybrid poplar Populus davidiana × Populus bolleana was transformed with the PtPYRL1 or PtPYRL5 gene and WT hybrid poplar served as control. We did not observe any phenotypic changes in PtPYRL1/5 overexpression transgenic poplars under normal growth conditions. Compared with WT poplars, overexpression of PtPYRL1 or PtPYRL5 enhanced drought tolerance. The shoot weight, leaf water potential, and proline concentration were obviously increased, and MDA content was markedly reduced in transgenic poplars under drought condition, resulted in rapid recovery with higher shoot height after drought and re-watering treatment. Low water availability in the dry soil limited evaporation and made water potential in the cell drop, these changes were associated with reduction in the leaf RWC. The overexpression of PtPYRL1 or PtPYRL5 in poplars showed larger leaf water potential (9) after drought treatment, probably due to increased water retention capacity of the cells, lower water loss rate as well as more adaptive stomatal movement during the stress (Leung and Giraudat, 1998; Zhu, 2002; Souza et al., 2004; Kwak et al., 2008). As showed in **Figure 7**, exogenous ABA promoted the stomata closure, PtPYRL1 or PtPYRL5 overexpressing transgenic poplars were more sensitive to ABA. Hereafter, recovery will be described as the visual evidence of new above ground development and growth, according to previous studies (Brodribb and Cochard, 2009; Petrov et al., 2015), which defined the process of recovery as a process of reactivated physiological processes and meristematic activity. PtPYRL1 or PtPYRL5 overexpressing transgenic poplars were taller and better recovered than WT poplars after drought and re-watering treatment (**Figure 3E**). Our findings supported the earlier finding that drought causes hydraulic restriction, with the subsequent development of high tension in the xylem water column and the closure of stomata (Tyree and Sperry, 1988). Larger aboveground biomass indicated better survival in the drought stressed PtPYRL1 or PtPYRL5 overexpressing transgenic poplars, while more proline accumulation resulted in the better water status maintenance (Bajji et al., 2001) and less MDA concentration led to better protection of the cell membrane structures in the drought treated PtPYRL1 or PtPYRL5 overexpressing transgenic poplars. The results presented here indicated that PtPYRL1/5 positively regulates ABA-related response to drought stress.

Drought commonly results in oxidative stress due to the over-production and over-accumulation of ROS derived from inefficient dissipation of excessive excitation energy (Shanker et al., 2014). The over-accumulation of ROS may lead to biochemical disruption of membranes and result in mortality (Suzuki et al., 2012; Petrov et al., 2015). ROS scavenging systems of plants detoxify ROS to minimize and/or prevent oxidative damage in cells by increasing the activity of ROS scavenging enzymes such as SOD, CAT, APX, POD, GR, and GPx (Gill and Tuteja, 2010). In the present study, the content of H2O<sup>2</sup> was significantly lower in the PtPYRL1- and PtPYRL5-overexpressing poplars than in WT under drought stress (**Figure 6A**). Consistent with this phenomenon, all of the measured antioxidant enzyme activities showed notably increased in transgenic poplars after drought treatment (**Figures 6B–G**). It was proposed that the antioxidant protection is related to higher leaf water potential 9 (**Figure 3F** and **Supplementary Figures S3F, S4F**) (Menconi et al., 1995). These results demonstrated that overexpression of PtPYRL1 or PtPYRL5 increased the antioxidant enzyme activities protecting transgenic poplars against the oxidative damage.

Consistently, osmotic stress significantly suppressed wildtype poplars roots development and leaf growth compared with PtPYRL1 or PtPYRL5 overexpressing poplars (**Figure 8**). The increased root growth rate of transgenic poplars (**Figure 8C**) has to be coupled with a mechanism that alleviates the physiological growth retardation consequences of the osmotic stress. In PtPYRL1 or PtPYRL5 overexpressing poplars, roots had increased the low water availability, as their rapid root growth resulted in more water absorption. Since the osmotic stress has been suggested to increase the ABA levels in plant cells (Zeevaart and Creelman, 1998), transgenic poplars might perceive ABA more rapidly and might trigger more efficiently the downstream responses. The phenotype of transgenic poplars under osmotic stress indicated that PtPYRL1 or PtPYRL5 increased the tolerance to hyperosmotic stress.

Another common environmental stress, a low temperature, severely inhibits plant growth and development. A recent study illustrated that co-overexpression of two genes, a bZIP

transcription factor (OsbZIP46CA1) and a protein kinase (SAPK6) involved in the ABA signaling pathway, showed improved tolerance to heat and cold stresses in rice (Chang et al., 2017). In order to enhance plant adaptability to low temperature, lipids, amino acid, membrane components, and other molecules in the cell are produced to promote cell membrane fluidity and structural rearrangement (Maruyama et al., 2014; Wu et al., 2016). Balance of the cellular ROS homeostasis also contributes to the tolerance to temperature stresses (Wang et al., 2017). Our results showed that the PtPYRL1 or PtPYRL5 overexpressing poplars enhanced low temperature stress tolerance (**Figure 9**), associated with more efficient ROS scavenging (**Figure 9B**) and more proline accumulation to maintain membrane integrity (**Figure 9C**). On the other hand, it is well known that phytohormone ABA activates a cascade of downstream signaling events in response to cold exposure (Knight et al., 2004). Further studies are required to investigate how PtPYRL1 and PtPYRL5 mediates adaptive responses to cold at the molecular level. Moreover, though our data showed that both ABA receptors are over-expressed in transgenic poplar plants at the mRNA level, it would be interesting to test their protein accumulation in future studies, since it has been suggested that PYR/PYL receptors undergo regulation at protein level by 26S proteasome pathway in Arabidopsis (Bueso et al., 2014; Irigoyen et al., 2014).

Populus is a perennial woody model plant and also economically important tree. Most Populus are sensitive to environmental factors, which considerably affects their productivities. As we know, arid and semi-arid regions account for approximately 30% of the worldwide area (Sivakumar et al., 2005). Meanwhile, the climate in poplars cultivated land of China is mostly dry and frigid. Therefore, breeding high drought- and cold-tolerant poplar cultivars is very necessary for improving land use efficiency and poplar forestry development. In our study, transgenic poplar overexpressing PtPYRL1 or PtPYRL5 was found to be significantly more drought and chilling tolerant than WT plants. We hope that the transgenic poplars generated in this study can be used for cultivating in cold as well as arid and semi-arid areas.

### AUTHOR CONTRIBUTIONS

LY, RT, and SL designed the study. LY, WL, HG, and JY revised the manuscript critically. LY, JY, RT, YW, XW, and FZ performed

### REFERENCES


experiments, analyzed and interpreted the data, and wrote the manuscript.

### FUNDING

This study was supported by the Fundamental Research Funds for the Central Universities (020814380062) and the Natural Science Foundation of Jiangsu Province of China (BK2012306).

### ACKNOWLEDGMENT

We gratefully acknowledge Hongxia Zhang for poplar material and pCAMBIA1301S2 plasmid.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2017.01752/ full#supplementary-material

FIGURE S1 | The pCAMBIA1301S2-PtPYRL1 (or PtPYRL5) Vector map.

FIGURE S2 | Transformants selection. (A) Adventitious bud regenerated from selection medium. (B) Transgenic shoots formed roots on selection medium.

FIGURE S3 | Overexpression of poplar ABA receptor PtPYRL5 in poplars enhanced drought-stress resistance. (A) 2-month-old WT (non-transgenic hybrid poplars) and transgenic poplars (L5-1 and L5-3) were cultured in the greenhouse with normal watering. (B) WT and transgenic hybrid poplars were not watered for 5 days. (C) After drought, WT and transgenic hybrid poplars were then re-watered for 3 days. The shoot weight after drought stress treatment (D), shoot height after re-watered for 3 days (E), water potential value (F) of WT and transgenic hybrid poplars were measured. Values are means ± SD (one-way ANOVA test; <sup>∗</sup>P < 0.05 as compared to WT).

FIGURE S4 | Overexpression of poplar ABA receptor PtPYRL1 in poplars enhanced drought-stress resistance. (A) 2-month-old WT (non-transgenic hybrid poplars) and transgenic poplars (L1-8 and L1-5) were cultured in the greenhouse with normal watering. (B) WT and transgenic hybrid poplars were not watered for 5 days. (C) After drought, WT and transgenic hybrid poplars were then re-watered for 3 days. The shoot weight after drought stress treatment (D), shoot height after re-watered for 3 days (E), water potential value (F), proline content (G) and MDA content (H) of WT and transgenic hybrid poplars were measured. Values are means ± SD (n = 18, three independent experiments, one-way ANOVA test; <sup>∗</sup>P < 0.05).



PYL4 to regulate metabolic reprogramming in Arabidopsis and tobacco. Proc. Natl. Acad. Sci. U.S.A. 108, 5891–5896. doi: 10.1073/pnas.1103010108


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Yu, Ge, Wang, Tang, Wang, Zhao, Lan, Luan and Yang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fpls-08-01752 October 11, 2017 Time: 15:49 # 13

# Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

Chris Gehring1,2 \* and Ilona S. Turek1,3

<sup>1</sup> Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia, <sup>2</sup> Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy, <sup>3</sup> Leibniz Institute of Plant Biochemistry, Halle, Germany

The cyclic nucleotide monophosphates (cNMPs), and notably 3<sup>0</sup> ,50 -cyclic guanosine monophosphate (cGMP) and 3<sup>0</sup> ,50 -cyclic adenosine monophosphate (cAMP) are now accepted as key signaling molecules in many processes in plants including growth and differentiation, photosynthesis, and biotic and abiotic defense. At the single molecule level, we are now beginning to understand how cNMPs modify specific target molecules such as cyclic nucleotide-gated channels, while at the systems level, a recent study of the Arabidopsis cNMP interactome has identified novel target molecules with specific cNMP-binding domains. A major advance came with the discovery and characterization of a steadily increasing number of guanylate cyclases (GCs) and adenylate cyclases (ACs). Several of the GCs are receptor kinases and include the brassinosteroid receptor, the phytosulfokine receptor, the Pep receptor, the plant natriuretic peptide receptor as well as a nitric oxide sensor. We foresee that in the near future many more molecular mechanisms and biological roles of GCs and ACs and their catalytic products will be discovered and further establish cNMPs as a key component of plant responses to the environment.

### Edited by:

Gerald Alan Berkowitz, University of Connecticut, United States

### Reviewed by:

Frantisek Baluska, University of Bonn, Germany Zhi Qi, Inner Mongolia University, China

### \*Correspondence:

Chris Gehring c.a.gehring@molecular-signals.com

### Specialty section:

This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science

Received: 25 July 2017 Accepted: 19 September 2017 Published: 04 October 2017

### Citation:

Gehring C and Turek IS (2017) Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling. Front. Plant Sci. 8:1704. doi: 10.3389/fpls.2017.01704 Keywords: signaling, cGMP, cAMP, guanylate cyclase (GC) and adenylate cyclase (AC), receptor kinases, Arabidopsis thaliana

## INTRODUCTION

There is a growing awareness that cyclic nucleotide monophosphates (cNMPs) and their cyclases are universal signaling molecules across the tree of life (Schaap, 2005). They are also key to many plant responses and the novel biological roles of these molecules and their mechanisms of action are discovered at an increasing rate (Meier et al., 2009; Wong et al., 2015; Marondedze et al., 2016a). Here, we review some of the early work on the detection of cNMPs in plants and the discovery of the cyclases that catalyze formation of 3<sup>0</sup> ,50 -cyclic adenosine or guanosine monophosphate (cAMP or cGMP) and pyrophosphate from their corresponding nucleoside 5<sup>0</sup> -triphosphate. We will also review recent approaches to elucidate systems responses to cNMPs in plants (e.g., Isner et al., 2012; Marondedze et al., 2013; Alqurashi et al., 2016). A summary of cGMP-dependent processes is provided in **Figure 1**.

### A BRIEF SUMMARY OF THE ROLES OF CAMP AND CGMP IN PLANT FUNCTION

Despite the fact that cAMP and cGMP have been recognized as important second messengers in animals nearly half a century ago, the notion that the cNMPs signaling system is universal and operates in plants was a matter of debate until recently. The existence and physiological functions

(Chlo.), or the nucleus. Proton transport is modulated by ATPases, while cation transport, including Ca2<sup>+</sup> uptake, is enabled by cyclic nucleotide-gated channels (CNGCs) and calmodulin (Gonzalez-Fontes et al., 2014). Cyclic nucleotide-dependent processes also affect the phosphoproteome and other post-translational processes, including methionine oxidation. Established ligands of GC-coupled receptors are represented by pentagrams with green borders and include the PNP receptor (AtPNP-R1), the Pep receptor (AtPepR1), the brassinosteroid receptor (AtBRI-GC), and the phytosulfokine receptor (AtPSKR1). Candidate GC-coupled receptors include a Stomagen receptor, a CLAV3 receptor, an EPF2 receptor, and a lectin receptor.

of cNMPs in higher plants have been questioned for long time mainly due to the fact that the cAMP and cGMP levels in plants appeared to be low (usually in the nanomolar range based on fresh weight) compared to those found in animals and lower eukaryotes (concentrations in the nano- to micromolar range) and often below the detection limits of analytical methods available in the past. Furthermore, reported amounts of cNMPs in different plants largely disregarded spatial and temporal variations of these secondary messengers. Arguably, the first convincing experimental evidence for the occurrence of cNMPs in higher plants came from a report that demonstrated changes in cNMPs levels during cell expansion and division in tobacco (Nicotiana tabacum) (Lundeen et al., 1973). Given the relatively low concentrations of cNMPs in plants, a second controversy concerned the biological significance of the effects exerted by non-physiological micromolar concentrations of exogenously applied cNMPs analogs on plant systems. However, numerous reports of nanomolar concentrations of cNMPs stimulating plant responses have since then overcome this point (e.g., Alqurashi et al., 2016; Marondedze et al., 2016a).

A growing body of evidence from plant electrophysiology and cell and molecular biology reveals that a considerable number of physiological processes in plants are correlated with alterations in cNMPs levels. Reports from several laboratories have shown direct effects of cNMPs on cation fluxes, including K+, Na+, and Ca2+, sparking interest in these molecules as potential regulators of ion and water homeostasis. Both cAMP and cGMP cause elevation of cytosolic Ca2<sup>+</sup> concentration, involving internal and external Ca2<sup>+</sup> stores, in tobacco (Nicotiana plumbaginofolia) protoplasts (Volotovski et al., 1998) and down-regulate Na<sup>+</sup> influx and accumulation in Arabidopsis roots (Essah et al., 2003; Isner and Maathuis, 2011). Direct evidence for cAMP activation of hyperpolarization-activated Ca2<sup>+</sup> channels in plasma membrane (PM) was provided in guard and mesophyll cells of Arabidopsis thaliana (Lemtiri-Chlieh and Berkowitz, 2004) and in pollen tube of Asian pear (Pyrus pyrifolia) (Wu et al., 2011). Cyclic GMP-dependent modulation of PM H+- ATPase activity, which indirectly provides a driving force for the cellular uptake of cations, particularly K+, and hence may significantly affect nutrient uptake and solute transport and could regulate cell volume, was reported in Tradescantia multiflora stem and leaf tissue (Suwastika and Gehring, 1999). K <sup>+</sup> and Na<sup>+</sup> fluxes modulated by cGMP by the activation of non-selective ion channels were observed in Zea mays roots (Pharmawati et al., 1999). Whole-cell patch-clamp current recordings in protoplasts from Vicia faba mesophyll cells revealed dose-dependent increases in the outward K<sup>+</sup> current upon intracellular application of cAMP (Li et al., 1994), whereas cGMP was suggested to directly affect K<sup>+</sup> fluxes in Arabidopsis guard cells (Pharmawati et al., 2001) and in intact tissue (Maathuis, 2006). Furthermore, both cNMPs were demonstrated to modulate K<sup>+</sup> and Ca2<sup>+</sup> flux responses to H2O<sup>2</sup> in Arabidopsis roots (Ordoñez et al., 2014). In analogy to animal systems, many functions of plant cNMPs are primarily mediated through cyclic nucleotide-gated channels (CNGCs), many of which have already been demonstrated as critical for plant responses to both biotic and abiotic cues (Kaplan et al., 2007; Jha et al., 2016). One of the CNGCs characterized in Arabidopsis, namely AtCNGC4, that is permeable to both Na<sup>+</sup> and K+, is activated by both cAMP and cGMP and has been implicated in plant responses to biotic stress (Balague et al., 2003).

Ample evidence for the role of both cAMP and cGMP in several physiological processes, including cell cycle progression as well as cell proliferation, growth and differentiation, organelle development, seed germination, and finally, plant growth and flowering have been accumulated. Early studies revealed changes in cGMP concentration that occur during cell volume increases and division in excised pith tissues of tobacco (Lundeen et al., 1973). Stringent control of intracellular cAMP levels was observed during progression through the cell cycle of the tobacco BY-2 cells, with cAMP peaks observed in S phase and, to lesser extent, in G<sup>1</sup> phase (Ehsan et al., 1998). In contrast, reduced cAMP levels were noted at the beginning of G<sup>1</sup> phase in arrested cells (Ehsan et al., 1999). cGMP was also implicated in chloroplast development (Bowler et al., 1994a), which could be inhibited by high level of cGMP (Bowler et al., 1994b; Wu et al., 1996). Immunochemical and cytoenzymological detection

of cAMP and adenylate cyclase (AC) activity, respectively, was demonstrated in the developing tobacco chloroplasts (Witters et al., 2005), providing additional evidence for a general role of cNMPs in photosynthetic activity of higher plants, resembling well-characterized functions of these secondary messengers in lower plants. In addition to gibberellic acid (GA)-dependent elevation of cAMP concentration required for seeds germination, observed in common broomrape (Orobanche minor) (Uematsu et al., 2007) and phacelia (Phacelia tanacetifolia) seeds (Uematsu and Fukui, 2008), cGMP was identified as a positive regulator of Arabidopsis seed germination (Teng et al., 2010). In pollen, where tube grow and orientation (Prado et al., 2004) and targeting (Prado et al., 2008) are critical, the gaseous signaling molecule nitric oxide (NO) has a role too and as in other systems, cyclic nucleotides are implicated in downstream signals [for review see Domingos et al. (2015)]. In addition, cGMP was demonstrated to mediate adventitious root formation (Pagnussat et al., 2003), root gravitropism (Hu et al., 2005), and is vital for root growth and development by positive modulation of auxin-regulated signaling responses (Nan et al., 2014). Furthermore, cAMP was implicated in the regulation of symbiotic root nodule formation, with soybean (Glycine max) root nodules containing significant levels of cAMP, while low cAMP level was noted in leaves (Terakado et al., 1997), and low concentrations of this cNMP were demonstrated to induce self-incompatibility in lily (Lilium longiflorum) (Tezuka et al., 2007). Assigning guanylate cyclase (GC) activity to brassinosteroid receptor BRI1 (Kwezi et al., 2007) and phytosulfokine receptor PSKR1 (Kwezi et al., 2011) provided a link between cGMP signaling and regulation of multiple growth and developmental processes, including embryogenesis, cell proliferation and elongation, and vascular differentiation (Sauter, 2015; Wei and Li, 2016). Furthermore, cGMP has been also implicated in phytochrome-controlled induction of flowering of morning glory (Pharbitis nil) (Szmidt-Jaworska et al., 2008) and in programmed-cell death (PCD) (Clarke et al., 2000), again highlighting impact of cNMPs on nearly every aspect of plant life cycle.

It is evident that cNMPs are becoming increasingly recognized as modulators of plant metabolism and a recurrent theme that has emerged since the early descriptions of these fundamental secondary messengers is the importance of their interplay with plant hormones. For instance, GA treatment of cereal aleurone layers led to a transient increase in cGMP levels and the subsequent induction of α-amylase production that in turn causes the conversion of starch to sugar (Penson et al., 1996), whereas auxin and kinetin, which cause stomatal opening, also signal via this cNMP (Cousson and Vavasseur, 1998; Pharmawati et al., 2001). Elevated concentrations of cAMP coincide with the early stages of the response to phytoalexins and evoke production of an antifungal isocoumarin, 6-methoxymellein, in cultured carrot (Daucus carota) cells (Kurosaki et al., 1987; Kurosaki and Nishi, 1993). Furthermore, NO elicits many processes in plants [for review see Domingos et al. (2015)] and often does so together with cyclic nucleotides and notably cGMP, which in analogy to mammalian systems, has been strongly implicated in NO signaling in plants (Pfeiffer et al., 1994; Durner et al., 1998; Dubovskaya et al., 2011). Examples include calcium-dependent development of cell polarity in Ceratopteris richardii (Salmi et al., 2007), NO increases in mitochondrial respiration in Arabidopsis callus (Wang et al., 2010), and NO- and redox signaling in the phloem upon localized leaf wounding (Gaupels et al., 2016). Finally, cGMP mediates plant natriuretic peptides (PNPs) signal (Morse et al., 2004), pinpointing a general function of cNMPs in regulation of ion and solute homeostasis in plants.

Importantly, cNMPs are involved in perception of extracellular abiotic and biotic stimuli and subsequent amplification and transduction of the signals to corresponding responses. Numerous observations have provided incontrovertible evidence that cNMPs function in plant abiotic stress responses and this is often accomplished by regulation of the ion fluxes. Addition of membrane permeable cNMPs during growth assays of A. thaliana roots affected voltage-independent channels and reduced levels of Na<sup>+</sup> accumulation, thus improving plant salinity tolerance (Maathuis and Sanders, 2001). A rapid (<1 min) time-dependent increases in cellular cGMP levels also occur after the salt and osmotic stress treatments (Donaldson et al., 2004) and CNGC19 and CNGC20 were speculated to presumably be involved in these early responses (Kugler et al., 2009). Elevated cGMP was also noted in late (>2 h) response to ozone (Pasqualini et al., 2009), whereas increased cGMP level upon a 30 min-long heat stress was associated with CNGC16 (Tunc-Ozdemir et al., 2013). These increases point to physiological roles of cNMPs in abiotic stress responses. It is noteworthy that proteomic study also suggested that cAMP functions in responses to environmental threats, such as temperature, as well as in light signaling and regulation of photosynthesis (Thomas et al., 2013). Early studies revealed considerable fluctuations of cGMP levels occurring in response to light (Brown et al., 1989; Bowler et al., 1994b), and a direct effect of cGMP concentrations on the phytochrome signal transduction was elucidated by investigating aurea mutant of tomato that is deficient in phytochrome A, a photoreceptor responsible for regulating many morphogenesis responses, including flowering, seed germination, and diurnal rhythms; cGMP not only triggered the production of photoprotective compounds anthocyanins, but in combination with Ca2<sup>+</sup> induced development of fully mature chloroplasts containing all the photosynthetic machinery (Bowler et al., 1994a). In line with these observations, recently reported interactors of cNMPs include several enzymes involved in Calvin cycle (Donaldson et al., 2016), implying specific roles of cNMPs in photosynthesis.

An impact of cNMPs on gas exchange can also be assumed based on early observations describing effect of cAMP analogs in stomata opening in V. faba (Curvetto et al., 1994), while cGMP analog was reported to induce stomata opening in a number of plants, including Arabidopsis (Pharmawati et al., 1998, 2001); this occurs downstream of abscisic acid (ABA)-induced changes in H2O<sup>2</sup> and NO (Dubovskaya et al., 2011). Consistent with the notion of cNMPs involvement in photorespiration, several photorespiratory enzymes were observed among proteins identified as cAMP or cGMP interactors (Donaldson et al., 2016). Furthermore, differential effects of cGMP and 8-nitrocGMP on stomata opening (Joudoi et al., 2013) were reported, whereas, similarly to cGMP, cyclic adenosine 5<sup>0</sup> -diphosphoribose

(cADPR) was shown to positively function in ABA and methyl jasmonate-induced stomatal closure in Arabidopsis (Hossain et al., 2014). Since stomata can be considered a frontline of plant interaction with the environment, their closure can be induced by adverse conditions, including not only drought and salt, but also pathogen attack, to reduce water loss and cell dehydration. Thus, function of cNMPs in plant responses to biotic stress appears plausible.

During the past two decades, the role of endogenous cAMP and cGMP in plant defense responses gained more recognition. cGMP was also reported to cause transcript increases of the defense-related genes encoding pathogenesis-related 1 and phenylalanine ammonia lyase proteins in tobacco (Durner et al., 1998) and to participate in signal transduction pathways activated by a group of danger-associated molecular patterns (DAMPs), such as AtPeps (Qi et al., 2010). NO has also a central role in defense against pathogens and the salicylic acid pathogen defense response pathways do not just include NO but also cGMP (Klessig et al., 2000) and this holds true for NO-triggered PCD (Clarke et al., 2000). In accordance with this finding, challenging A. thaliana with avirulent strain of Pseudomonas syringae results in cGMP accumulation (Meier et al., 2009). More recently it has been demonstrated that infection of A. thaliana with avirulent pathogens (and not avirulent strains) can cause a biphasic increase of cGMP downstream of NO. However, constitutive cGMP accumulation can compromise systemic acquired resistance (Hussain et al., 2016) and this would suggest that "swamping" the cell with a signaling molecule can cause disfunction. Accumulation of cGMP was also demonstrated in extrafascicular phloem, a defensive structure against herbivorous animals, in leaf wounded pumpkin (Cucurbita maxima) (Gaupels et al., 2016). Similarly, pathogen attack and mechanical wounding elevated cGMP content in Hippeastrum hybrid (Swiezawska et al., 2015). On the other hand, cAMP, apart from being implicated in activation of phytoalexin synthesis in sweet potato (Ipomoea batatas) (Oguni et al., 1976) and in early signaling events in the apoplastic oxidative burst (Bindschedler et al., 2001), was also reported to be elevated at the infection site initiation in pathogen-related cytosolic Ca2<sup>+</sup> signaling (Ma et al., 2009). Moreover, production of elevated concentrations of cAMP by the administration of elicitors has been demonstrated in French bean (Phaseolus vulgaris) (Bolwell, 1992), carrot (D. carota) (Kurosaki and Nishi, 1993), alfalfa (Medicago sativa) exposed to Verticillium alboatrum glycoprotein (Cooke et al., 1994), Mexican cypress (Cupressus lusitanica) cell culture treated with yeast oligosaccharides (Zhao et al., 2004), and Arabidopsis treated with Verticillium dahliae toxins, resulting in improved disease resistance of the plants (Jiang et al., 2005). It is noteworthy that many signaling cascades generated in response to biotic stresses not only employ cNMPs, but also critically depend on CNGCs. For instance, AtCNGC4, activated by cAMP and cGMP, is induced as a result of pathogen infection and certain pathogenrelated signals (Balague et al., 2003), whereas CNGC11 and CNGC12 activate multiple pathogen resistance responses and act as positive mediators of plant resistance against avirulent isolate of oomycete Hyaloperonospora parasitica (Yoshioka et al., 2006). Interestingly enough, recently several other CNGCs, including CNGC2 and CNGC4, have also been associated with plant responses to biotic stresses (Ali et al., 2007; Chin et al., 2013). In Arabidopsis, AtCNGC2 has recently been demonstrated to induce apoplastic Ca2<sup>+</sup> influx in response to jasmonic acid (JA) and thus linking JA-induced cAMP increases to Ca2+-dependent downstream signaling (Lu et al., 2016). The rapid (<5 min) JAdependent cytosolic cAMP increases and concomitant increase in cytosolic Ca2<sup>+</sup> are indeed consistent with cAMP being an AtCNGC2 ligand (Lu et al., 2016). It is noteworthy, that another recent report suggests that AtCNGC2 delivers Ca2<sup>+</sup> from veins into leaf cells thereby preventing apoplastic Ca2<sup>+</sup> accumulation but does not seem to have a direct influence on the hypersensitive response (Wang et al., 2017).

Furthermore, wounding stress was shown to lead to a rapid approximately five-fold increase in concentration of the noncanonical 2<sup>0</sup> ,30 -isomers of cAMP and cGMP in A. thaliana leaves (Van Damme et al., 2014). These reports, taken together, strongly argue that both the host–pathogen recognition and the downstream processes are transduced by complex cNMP signatures and often include regulation of CNGCs. The role of cNMPs in both abiotic and biotic stress responses does not come as a surprise considering the fact that in biotic interactions pathogens modulate plant host ion and water homeostasis, inflicting abiotic stress conditions, to the detriment of the host (Gottig et al., 2008).

Since the low concentrations of cNMPs in higher plants definitely constituted a major problem in cNMP research in this system, it can be noteworthy to emphasize the impact of technological progress in the development of methods for extraction, purification, detection, and quantitation of cNMPs, with a very low limit of detection, on the advancement in investigation of plant cNMPs signaling witnessed over last few decades. Initial methods of cNMPs separation and detection, such as radio-immune assays, characterized by a femtomole dynamic range, suffered from lack of accuracy due to coelution of interfering plant metabolites, including derivatives of cNMPs and hence significantly compromising the data. This problem was largely overcome due to the use of anti-cAMP or anti-cGMP antibodies in an immunopurification step, allowing more powerful sample clean-up. In the 1980s, chromatographic analyses in plant cNMPs analysis, including paper and thin-layer chromatographies, were replaced by high-performance liquid chromatography (LC) by virtue of its capability to separate the 3<sup>0</sup> ,50 -cyclic nucleotides and their 2<sup>0</sup> ,30 -isomers (Brown, 1983). Nevertheless, unambiguous demonstration of the identity of the putative cNMPs obtained in cell extracts and as the product of incubations with AC and guanylyl cyclase (GC) was accomplished by the use of physical techniques, mass spectrometry (MS) in particular. The fast atom bombardment (FAB) collision-induced dissociation (CID) tandem MS (MS/MS) not only allowed the unequivocal identification of cAMP and cGMP, but also demonstrated natural occurrence of cytidine, inosine, uridine, and 2<sup>0</sup> -deoxythymidine 3<sup>0</sup> ,50 -cyclic monophosphate (cCMP, cIMP, cUMP, and cdTMP, respectively) in pea (Pisum sativum) (Newton et al., 1991) in the early 1990s. Due to its greater sensitivity, LC–electrospray ionization (ESI)– MS/MS set-up, which decreased detection limits to femtomole

level, gradually replaced FAB–MS/MS and is successfully used in cNMP detection and quantification studies until now, providing an excellent platform for large-scale investigation of cNMPs interactors and system-level analyses of proteins that are differentially expressed and/or post-translationally modified in a cNMP-dependent manner. Further advancement of separation techniques enabled analysis of 2<sup>0</sup> ,30 -cNMPs in plants (Pabst et al., 2010) and facilitated studies on physiological relevance of both the canonical and non-canonical cNMPs in plants over the last decade (Van Damme et al., 2014).

The analytical chemistry methods developed for determination of cNMPs concentrations with fine enough spatial and temporal resolution that provides confidence in the biological significance of the results, although powerful, are labor intensive and costly in terms of infrastructure, require high levels of expertise and large amount of tissue. The enzyme immunoassays (EIAs), tailored for animal cells, used to determine contents of cAMP or cGMP in analyzed plant samples suffered greatly from lack of specificity and selectivity, often leading to inconsistent results (Newton and Smith, 2004). Newer enzyme-linked immunosorbent assays (ELISAs) are more specific for cNMPs but may under-report amounts of cNMPs present in plant cells (Meier et al., 2010), whereas a recently improved EIA method for cAMP detection in plant tissue was promised to be 10 times more sensitive than previously used methods (Lomovatskaya et al., 2011). The advent of biosensorbased techniques in cell biology, and in particular reporter systems used to record cNMP levels non-destructively in real time and with relatively high spatial and temporal resolutions, was a significant step forward in plant cNMPs research. However, in plant research, we are still quite distance away from the development and application of high-resolution sensing, e.g., with cytosolic Förster resonance energy transfer-based cGMP biosensors (Gotz et al., 2014) that are currently applied in medial research. Another obstacle that is intrinsic to plant work is the very small cytosolic compartment of cells where by far the biggest volume is taken up by the vacuole which is almost certainly not the place where cNMP-dependent signaling has a key role. Still, owing to the ease of use, the currently available plant reporters have significantly contributed to establishing the physiological functions of cNMPs in plants and these reporters also offer an affordable alternative to the advanced techniques of analytical chemistry.

In brief, firstly, the imaging of cAMP cellular distribution and quantification was reported in living pollen tubes (Moutinho et al., 2001) and it was achieved by microinjecting a cAMP fluorosensor. The pollen tube was chosen to investigate the role of cAMP not least because it is a large single cell that lends itself to microinjecting a fluorescent sensor dye into its cytoplasm. Secondly, reliable quantification of cytoplasmic cGMP concentrations in plant cells transiently and stably transformed with δ-FlincG in response to various external stimuli has also been reported (Isner and Maathuis, 2011). The reported δ-FlincG dissociation constant for cGMP was approximately 200 nM and was reported to result in a dynamic range spanning from approximately 20 nM to 2 µM. Thirdly, the development of luciferase-based promoter reporter systems. The principle here is that the promoter of a cGMP-inducible gene [e.g., OLIGOPEPTIDE TRANSPORTER X (OPTX)] is fused to a luciferase reporter gene (Wheeler et al., 2013) and luciferase activity is a proxy for cGMP and can be calibrated.

In summary, the development and application of sophisticated methods in the fields of immunocytochemistry, electrophysiology, and separation technologies combined with MS have assisted in the in-depth characterization of components of the plant cNMP-dependent signalosome.

### THE DISCOVERY OF THE FIRST GCS IN HIGHER PLANTS

Despite the fact that by the year 2000 the presence of cNMPs in higher plants was established beyond reasonable doubt and numerous cAMP- and cGMP-dependent processes have been reported, the cyclases responsible for the formation of cNMPs have remained elusive. While basic local alignment search tool (BLAST) searches with annotated GCs from lower eukaryotes and animals did return candidate GC in cyanobacteria perhaps surprisingly, when ACs and GCs from lower or higher eukaryotes were used to query the newly available A. thaliana genome no plausible plant candidates were returned (Ludidi and Gehring, 2003). This suggested that plant cyclases had lost ancestral cyclases domains and evolved new ones, or more likely, that plant cyclases had evolved in such a way that only the key catalytic residues remained conserved and that those few key amino acid (aa) residues where beyond the detection limit of BLAST searches. In order to test the second hypothesis, we firstly determined the key aa residues with annotated roles in the catalytic process (Liu et al., 1997; Tucker et al., 1998). These have in part been determined based on the crystal structure of the rat type II AC C2 catalytic domain that was in turn used to homology model a mammalian AC C1–C2 domain pair, a homodimeric AC of Dictyostelium discoideum, a heterodimeric soluble GC (sGC), and a homodimeric membrane GC. These authors also docked Mg2+-complexed adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to the active site to inspect stereo-chemical constraints of the resulting conformation (Liu et al., 1997). The authors report that their "models are consistent with the activities of seven mutants" in the catalytic site where an aspartic acid (D) and glutamine (Q) of type I ACs can coordinate the Mg2<sup>+</sup> ion. They also show that mutating D310 residue to serine (S) and (D) 310 to alanine (A) decreases the reduced Vmax of the reaction as well as altering [Mg2+] dependence. Furthermore, it was proposed that the purine moieties bind in hydrophobic pockets and that specificity is due to a lysine (K) and D in AC, and a glutamic acid (E), an arginine (R), and a cysteine (C) in GCs. It was also predicted that an asparagine (N)–R pair would stabilize the transition state. With these structural parameters in mind, we then inspected alignments of known catalytic centers of different types of nucleotide cyclases (McCue et al., 2000) before aligning catalytic centers of annotated, and in some cases, experimentally confirmed nucleotide cyclases in lower and higher eukaryotes (Ludidi and Gehring, 2003). From this alignment, we extracted a 14 aa-long core catalytic center search motif (**Figure 2**) and

when used to query the Arabidopsis proteome, it returned seven candidate GCs, including AtCG1 (At5g05930) and AtWAKL10 (At1g79680).

A recombinant AtCG1 was first to be tested experimentally and shown to be capable to generate cGMP for GTP in vitro in the presence of Mg2+. AtGC1 is a dual-domain 274 aa-long sGC with the catalytic center on the very N-terminus, but contrary to sGCs in animals, AtGC1 is not sensitive to NO and therefore represented a new type of sGC. The second domain has been predicted to be a peptidase family C39-like domain (IPR005074) typical for antibiotic bacteriocin-processing endopeptidases from bacteria where the cleavage is mediated by its ATP-binding cassette (ABC) transporter domain as part of the secretion process and this is supported by fold studies suggesting that AtGC1 is a member of a novel eukaryotic C proteinase family that might function as a protease (Ginalski and Zemojtel, 2004). While preliminary studies have confirmed the protease activity, the interrelationship between the GC activity and the protease activity as well as the biological function(s) has remained elusive.

We also tested AtWAKL1 that is a receptor type wall associated kinase-like molecule that can also generate cGMP in vitro as well as having kinase activity (Meier et al., 2010).

Furthermore, a co-expression and stimulus-specific expression analysis of AtWAKL10 has suggested that it is consistently co-expressed with extensively characterized pathogen defenserelated genes and, much like these genes, is induced early and pronouncedly in responses to both a range of pathogens (including Botrytis cinerea and P. syringae) and their elicitors (Meier et al., 2010).

### EXPANDING THE SEARCH AND DISCOVERY OF NOVEL CYCLASE IN HIGHER PLANTS

How many mononucleotide cyclases (MNCs; ACs and GCs) are there in the proteomes of higher plants? This question has remained unsolved, but several lines of evidence suggest that the number may be considerably higher than previously suspected and may in fact be >100. Firstly, we noted that Chlamydomonas reinhardtii, a unicellular green alga, contains >100 MNCs annotated on the basis of sequence homology with predicted and/or experimentally confirmed ACs and GCs. These MNCs come in >20 different domain combinations and configurations with 13 different partners including Heme NO/OXygen (H-NOX), periplasmic-binding protein, GAF-like and protein kinase-like domain, ATPase domain of HSP90, RNIlike, ribonuclease-H, duplicated hybrid motif, CheY-like, family AG protein-coupled receptors, homodimeric domain of SIG, SAM/pointed domain, and C proteinase (Meier et al., 2007). Still, while the dependence on cNMPs of biological processes such as sexual signaling by gametes (Pasquale and Goodenough, 1987) and nitrate assimilation (de Montaigu et al., 2010) have been described, the cyclases at the source of the signaling pathway have remained elusive.

Secondly, given the number and diverse nature of cNMPdependent processes such as stomatal guard cell movement (Curvetto et al., 1994; Pharmawati et al., 1998; Joudoi et al., 2013), responses to light and temperature (Brown et al., 1989; Bowler et al., 1994b; Thomas et al., 2013), responses to pathogens (Bindschedler et al., 2001; Meier et al., 2009; Gaupels et al., 2016; Hussain et al., 2016) and Pep peptides (Qi et al., 2010), responses to NO and ozone (Clarke et al., 2000; Pasqualini et al., 2009), cation transport (Hoshi, 1995; Pharmawati et al., 1999; Maathuis, 2006; Ordoñez et al., 2014), the modulation of CNGCs (Ali et al., 2007; Zelman et al., 2012; Chin et al., 2013; Tunc-Ozdemir et al., 2013), and the specific cGMP-dependent protein phosphorylation (Marondedze et al., 2016a) and methionine oxidation (Marondedze et al., 2013), it seems highly unlikely that only a very small number of ACs and CGs is responsible for all of these complex reactions. It appears more likely that higher plants, much like Chlamydomonas, harbor a large number of diverse MNCs that are capable to deliver distinct spatial, temporal, and stimulus-specific transient signals.

Thirdly, Chlamydomonas MNCs do not seem to be closely related to proteins in A. thaliana. The lowest e-value of a Chlamydomonas MNC protein (or protein fragment) compared against Arabidopsis proteins is 0.009 and this protein is a putative ethylene-responsive DEAD box RNA helicase (At5G63120). Another three Chlamydomonas MNC proteins have BLAST e-values < 0.05 but >0.01. This makes the evolution of MNC from Chlamydomonas to Arabidopsis at best a matter of speculation only (Meier et al., 2007).

Given these considerations, we hypothesize that higher plants are likely to contain a large number of MNCs and that the most promising way forward was to use catalytic center motifs as a means to identify them. Modifications of the motif would be based on rational considerations and in some cases tested by sitedirected mutagenesis of experimentally proven cyclases (Kwezi et al., 2007). This led to a derived more relaxed search motif ([R]X{5,20}[RKS][YFW][GCTH][VIL][FV]X{3}[VIL]X{4}[KR] X {1,2}[D]) that occurs in 27 A. thaliana proteins of which AtBRI1 (At4g39400), the receptor for the brassinosteroids, a class of plant hormones, was the first to be tested (Kwezi et al., 2007). AtBRI1 is a leucine-rich repeat receptor-like kinase (LRR-RLK) from which we have cloned and expressed a 114 aa-long recombinant protein (AtBRI1-GC) that harbors the GC domain. This domain can convert GTP to cGMP in vitro, a finding that suggested that AtBRI1 may belong to a novel class of GCs that contains both a cytosolic kinase and GC domain and we have subsequently shown that the GC activity of AtBRI1 enables brassinosteroid signaling by rapidly phosphorylating the downstream brassinosteroid signaling kinase 1 (BSK1) (Wheeler et al., 2017). Incidentally, AtBRI1 and other plant LRR-RLKs have a domain organization that is not dissimilar to that of animal atrial natriuretic peptide (ANP) receptors, NPR1 and NPR2 (Singh et al., 1988; Chinkers et al., 1989). Since ANPs bind specifically to PMs and elicit physiological responses (e.g., by stomatal opening) (Gehring et al., 1996) and plants also contain highly biologically active compounds that turned out to be functional homologs of ANPs, that also cause cGMP transients [for review see Gehring and Irving (2003) and Gehring and Irving (2013)], it was not entirely surprising when a recent report found and described the PNP receptor and showed that it contains a cytosolic GC domain that is activated by the ligand PNP (Turek and Gehring, 2016).

A second candidate tested in this group is AtPepR1 (At1g73080). It is a receptor for a family of peptide signaling molecules (AtPeps) that act as DAMPs in pathogen defense signaling cascades in plants (Qi et al., 2010). We showed that this LRR-RLK also has GC activity and that AtPep-dependent expression of biotic stress defense genes (PDF1.2, MPK3, and WRKY33) is linked to Ca2<sup>+</sup> signaling pathway that in turn are associated with AtPeps and Pep receptors.

Interestingly, these 27 Arabidopsis proteins are highly significantly (p-value < 1e−<sup>5</sup> ) enriched for the gene ontology (GO) categories of "phosphorus metabolic processes," "protein metabolic process," "cellular macromolecular metabolic process," and "biopolymer metabolic process." Furthermore, several proteins are annotated as LRR-RLKs and they include AtBRI1 (brassinosteroid insensitive 1; At4g39400), which is the receptor for brassinosteroids. What appears quite unusual about the domain architecture of this trans-membrane receptor kinase is that the GC domain is encapsulated in the cytoplasmic kinase domain, begging question of the evolutionary and functional relationship of these two domains. We have subsequently shown

that AtBRI1 has GC activity (Kwezi et al., 2007) and that cGMP enables brassinosteroid signaling by rapidly phosphorylating the downstream BSK1 (Wheeler et al., 2017).

Also included in the GC candidate list are three receptor kinases of the ERECTA family: At2g26330 ERECTA (ER), At5g62230 ERECTA-like1 (ERL1), and At5g07180 (ERL2) [for review see Ho et al. (2016), Ikematsu et al. (2017), and Lin et al. (2017)]. Arabidopsis erecta (er) mutants show altered organ shape and the ecotype Landsberg (Ler) is typified by compact inflorescences with flowers clustering at the top and also have round leaves with short petioles and siliques (Bowman, 1993). Furthermore, they include photomorphogenesis, phytohormone biosynthesis and signal transduction, and flower organ identity mutants. More recently, many of theses mutants have been identified as LRR-RLKs (Torii et al., 1996). At2g26330 (ERECTA) is a quantitative trait locus for transpirational efficiency due, in part, to its influence on epidermal and mesophyll development including stomatal density and porosity of leaves, and it has been shown to be required for callose deposition upon infection (Sanchez-Rodriguez et al., 2009) and implicated in the resistance to the soil borne bacterium Ralstonia solanacearum, the necrotrophic fungus Plectosphaerella cucumerina, and oomycetes from Pythium irregulare (Godiard et al., 2003; Llorente et al., 2005; Adie et al., 2007) as well as in thermotolerance (Shen et al., 2015). The second candidate, At5g62230 (ERL1), also encodes an RLK that, together with ER and ERL2, has a key role in the fate of protodermal cells to divide proliferatively and produce pavement cells or, alternatively, to undergo asymmetric divisions that will eventually form stomatal complexes. The third candidate, the RLK At5g07180 (ERL2), determines whether protodermal cells divide proliferatively to produce pavement cells or divide asymmetrically to generate stomatal complexes as well as for maintaining stomatal stem cell activity in order to prevent terminal differentiation into the guard mother cell (Staff et al., 2012). In silico identification of the candidate GC catalytic centers in these proteins may suggest that cGMP acts as a messenger in the developmental programming of stomatal formation. However, the biochemical proof of GC activity of these RLKs still awaits confirmation.

Furthermore, CLAVATA1 (CLV1; At4g20270) is another annotated LRR-RLK. It is expressed in the shoot apical meristem where stem cells are at their undifferentiated stage by inhibiting WUSCHEL (WUS) and FANTASTIC FOUR (FAF). WUS and FAF2 expression is inhibited by the CLV3 peptide in wild-type plants and impaired with Ca2<sup>+</sup> channel blockers and GC inhibitors. This inhibition was also observed in the DEFENSE NO DEATH 1 (dnd1) and 2 (dnd2) mutants that lack the CNGC 2 and 4, respectively (Chou et al., 2016). This may suggest that when CLV3 binds to the CLV1 receptor in the meristem, an increase in cGMP results and that may in turn activate CNGC2 channels and consequently increase cytoplasmic Ca2+. Elevated Ca2<sup>+</sup> concentrations have been reported to cause altered expression of WUS and FAF2 and eventually change the fate of the meristem cell (Isner and Maathuis, 2016). Perception of CLV3 peptide by CLV1 activates mitogen-activated protein kinases (MPK) MPK3 and MPK6 signaling (Betsuyaku et al., 2011; Lee et al., 2011), that in turn is involved in mediating multiple developmental and stress responses, including responses to biotic cues. Interestingly enough, involvement of CLV1 in signaling and/or perception of CLV3/endosperm surrounding region (CLE)-like effector proteins secreted by plant-parasitic beet cyst nematodes was revealed since infection with Heterodera schachtii is reduced on clv1 mutant and CLV1 expression was induced in the nematode feeding sites (Replogle et al., 2013). More recent study investigated the function of both nematode A- and B-type CLE peptides in the regulation of cell proliferation at site of feeding, linking the function of CLE receptors, including CLV1 to the modulation of the vascular stem cell pathways (Guo et al., 2017).

In order to identify additional candidate GCs we have further modified the search motif ([R]X{5,40}[RKS][YFW] [GCTH][VIL][FV]X{3}[VIL]X{4}[KR]X{2,3}[D]) and retrieved 107 candidates and subjected them to GO search. Of the significantly enriched terms, we chose these in the category of "trans-membrane receptor protein tyrosine kinase signaling pathway" [p-value: 2.7e−10; false discovery rate (FDR): 2.1e−09] and identified four candidates: At2g02220, phytosulfokine receptor 1 (AtPSKR1); At3g13065, strubbelig-receptor family 4 protein (SRF4); At5g58150, an uncharacterized LRR-RLK; and At5g65710, the HAESA-Like 2, HSL2. Of those, AtPSKR1 is perhaps the best studied GC. Not only was it shown that the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 has serine/threonine kinase activity (approximate K<sup>m</sup> of 7.5 M and Vmax of 1800 nmol min−<sup>1</sup> mg−<sup>1</sup> protein), but also that this domain has a GC activity in vitro. It was further demonstrated that over-expression of the full-length AtPSKR1 receptor in A. thaliana leaf protoplasts caused a >20-fold increase in endogenous cGMP levels and, importantly, that the addition of the active sulfonated ligand, PSK, induces rapid increases in cGMP levels in protoplasts (Kwezi et al., 2011). This is entirely consistent with the idea that the cytosolic GC domain of the receptor is activated by the ligand and enables to downstream signal via cGMP and suggests that the receptor GCs where the catalytic GC center is embedded in functional kinases are in fact dual-functioning – moonlighting – enzymes (Irving et al., 2012). The arising question of the functional relationship of the two domains was also addressed and it was demonstrated firstly, that Ca2<sup>+</sup> is the switch between the kinase and the cyclase activity in these moonlighting enzymes (Muleya et al., 2014) and secondly, that specific auto-phosphorylation and dimerization are essential mechanisms for ligand-mediated catalysis and signaling (Muleya et al., 2016). The remaining three candidates: SRF4, the un-annotated LRR-RLK, and HSL2 remain biochemically uncharacterized. SRF4 is likely to operate as a "positive regulator" of leaf size (Eyüboglu et al., 2007) but it is also transcriptionally activated by, e.g., ABA, drought, and the plant pathogen Golovinomyces cichoracearum. HSL2 participates in abscission and in particular floral abscission (Patharkar and Walker, 2015) and is induced by, e.g., ABA and the root-knot nematode Meloidogyne incognita.

### SOLUBLE GCS AND NO SENSING

Nitric oxide is a gaseous reactive oxygen species (ROS) that has evolved as a signaling hormone in many physiological processes in animals and plants, and in animal systems NO-dependent sGCs have long been known and are well characterized (Knowles et al., 1989; Denninger and Marletta, 1999). In plants, NO has been demonstrated to operate as regulator of development functioning as a signaling molecule at each step of the life cycle. NO has also been implicated in biotic and abiotic stress signaling [for review see Domingos et al. (2015)]. However, despite the multitude of plant responses to NO, many of them concomitant with cGMP increases, there have until recently been no sensor molecules discovered that can link NO to the GC activation.

In animal systems, NO binds to the heme group localized at a domain termed H-NOX, and this binding activates the GC catalytic center leading to the generation of cGMP from GTP (Palmer et al., 1987; Bellamy et al., 2002). Mutational and structural studies have confirmed the histidine (H) residue as a proximal ligand for the docking to the iron core of the heme porphyrin moiety (Wedel et al., 1994; Zhao et al., 1998) and the binding results in a 5-coordinate complex (Stone et al., 1995) that becomes a nitrosyl complex when bound to NO (Stone and Marletta, 1994). It can then sever the proximal H–Fe bond and lead to the subsequent displacement of the heme moiety (Dai et al., 2012). Given the chemistry of the structurally highly conserved H-NOX reaction center (Boon et al., 2005), we have again built a consensus motif search term (HX{12}PX{14,16}YXSXR) and retrieved four candidate molecules presumably capable of NO binding (At1g62580, At4g01160, At5g19160, and At5g57690). One of the candidate molecules (AtNOGC1, At1g62580) also contains the key residues required for GC catalytic activity ([RKS]X[GCTHS]X{9,10}[KR]) and is annotated as a flavin monooxygenase (Mulaudzi et al., 2011). Electrochemistry revealed that the recombinant binds NO and has higher affinity for NO than for O<sup>2</sup> and that AtNOGC1 can generate cGMP from GTP in vitro in a NO- and timedependent manner. More recently, it was also demonstrated that in NO-dependent stomatal closing nitration of cGMP (8-nitrocGMP) is key and that in the Atnogc1 mutant neither cGMP nor the cell permeant 8-bromo-cGMP induce stomatal closure (Joudoi et al., 2013) making AtNOGC1 an essential component in this signaling pathway.

### FINDING ACS IN HIGHER PLANTS

Establishing the presence and functions of cAMP and ACs in plants and higher plants in particular has been a long and at times rather controversial process (Gehring, 2010). Until a few years ago a Z. mays protein that participates in pollen tube growth and reorientation has remained the only experimentally confirmed AC in plants (Moutinho et al., 2001). In order to systematically identify candidate ACs in A. thaliana, we decided to undertake searches with modified GC motifs (**Figure 2**) since the aa in position 3 confers substrate specificity (Tucker et al., 1998; Roelofs et al., 2001; Wong and Gehring, 2013a). In GCs the residues in position 3 are [C, T, G, or H] whereas in ACs they are [D] or [E] (**Figure 2**). We then queried the Arabidopsis proteome using this substituted motif ([RKS][YFW][DE][VIL]X(8,9)[KR]X(1,3)[DE]) and retrieved 341 candidates proteins (Al-Younis et al., 2015). We then narrowed the search by including an N-terminal [R] 5–20 aa upstream of position 1 since [R] is essential for pyrophosphatebinding (Liu et al., 1997). This extended AC motif identifies 14 candidates. Testing of AC activity is somewhat more convenient than testing for GC activity since one can make use of an Escherichia coli cyaA mutant SP850 strain deficient in the AC (cyaA) gene (Shah and Peterkofsky, 1991) for rescue-based selection. Several of the candidates have since been tested and shown to be capable to rescue the cyaA mutant. One of them is the A. thaliana K <sup>+</sup>-uptake permease 7 (AtKUP7, At5g09400). In AtKUP7, an AC catalytic center containing cytosolic fragment has also been shown with tandem MS methods to generate cAMP from ATP (Al-Younis et al., 2015). The biological role of this molecule remains to be elucidated since it might act as a cAMP-dependent K+-flux sensor and is still under investigation.

It is noteworthy that in Paramecium, cAMP formation has been reported to be linked to K<sup>+</sup> conductance, and this conductance is an intrinsic property of the AC (Weber et al., 2004). This multi-domain AC therefore functions as both an AC and a K<sup>+</sup> channel in which a S4 voltage-sensor is part of the N-terminus and a K<sup>+</sup> pore-loop occupies the C-terminus on the cytoplasmic side (Weber et al., 2004). AtKUP7 also has such dual domain architecture and may function as both K<sup>+</sup> transporter and an AC; however, AtKUP7 is likely to be a protoncoupled carrier rather than a K channel. The future will tell if cAMP production is dependent on K<sup>+</sup> fluxes and/or if cAMP can modulate K<sup>+</sup> fluxes.

### PREDICTING NOVEL ACS AND GCS WITH THE SUPPORT OF STRUCTURAL MODELING

Molecular methods for the study of signal transduction have steadily developed together with technical advances (Irving and Gehring, 2013) and this is also reflected in the discovery and characterization of cNMPs and their cyclases. These advances include computational methods for large-scale sequence analyses of the ever-increasing number of fully sequenced (plant) genomes in the public domain and the growing number of published structures of MNCs, ACs, and GCs. These two resources have indeed allowed a computational approach to the identification of candidate MNCs in higher plants (Wong and Gehring, 2013b). A method to achieve accurate predictions of Arabidopsis candidate MNCs has been detailed and includes pattern searches with catalytic center motifs of ACs or GCs. It is also recommended that these centers of candidate MNCs are compared with orthologs of closely as well as more distantly related species since conservation, particularly in the key residues, to add confidence to the predication.

An example are the systemically mobile peptidic plant hormones (the PNPs) that elicit many physiological responses

at nanomolar concentrations (Ludidi et al., 2002; Maryani et al., 2003; Gottig et al., 2008; Gehring and Irving, 2013; Turek et al., 2014). The A. thaliana PNP-A (At2g18660) is a ligand of AtPNP-R1 (At1g33612) receptor (Turek and Gehring, 2016) that we have shown to be a ligand-activated receptor kinase with GC activity. The ortholog in tomato is a protein (K4D0Y5\_SOLLC) that also contains the key residues essential for catalysis. And given that a Verticillium natriuretic peptide-like molecule is the ligand for the tomato Verticillium wilt immune receptor (C4NAS0| C4NAS0\_SOLLC Verticillium wilt disease resistance protein), it does not come as a surprise that this receptor also comes with a GC motif and may conceivably be a GC as well (de Jonge et al., 2012).

Furthermore, it has also proven useful to subject candidate MNCs to structural modeling against structure templates of annotated MNCs and in addition, docking of the substrate, ATP or GTP, to ascertain if the catalytic center forms a cavity with the key residues for ATP of GTP interaction and the metal-binding residue [an example is shown in Wong and Gehring (2013a)]. Incidentally, such an approach can also be used to model mutations and predict if candidates are conceivably functional as cyclases (Wong et al., 2015).

### IN SEARCH OF NOVEL BIOLOGICAL FUNCTIONS OF CNMPS AND MNCS

Systems-based approaches to biology have also contributed and continue to contribute to or understanding of the breadth and complexity of cNMP-dependent cellular processes. The cGMP-dependent signals and their role in phosphorylation was examined using a phosphoproteomics approach and it was shown that upon treatment of A. thaliana roots with membranepermeable cGMP a number of plant hormone-dependent microsomal proteins changed their phosphorylation status thus linking plant hormone responses to hormone-dependent cGMP transients and cGMP-dependent downstream phosphorylation (Isner et al., 2012). More recently a quantitative cGMP-dependent phosphoproteome of A. thaliana suspension-cultured cells that were metabolically labeled with <sup>15</sup>N has revealed highly specific responses and points to hitherto unknown cGMP-dependent phosphorylation and dephosphorylation events (Marondedze et al., 2016a). A pathway analysis of the identified proteins revealed that seven of them are part of the spliceosome (Matera and Wang, 2014) and belong to four different spliceosome complexes. They include the pre-mRNA processing protein (At1g44910) and splicing factor PW1 (At1g60200) that belong to the U1 complex; the WD40 repeat protein (At2g32700) is part of the U4/U6 complex; the periodic tryptophan protein 2 (At1g15440, AtPWP2) belongs to the U5 complex; the UBP1 associated protein 2A (At3g56860), the RNA-binding protein 47C (At1g47500), and the R/S-rich splicing factor that are common spliceosome components. There is firm evidence that the alternative splicing and hence the spliceosome have an important role in biotic and abiotic stresses responses and that mutations in splicing factors and spliceosomal proteins are predicted to modulate the circadian clock and the highly complex plant defense responses (e.g., Staiger and Brown, 2013) and that the phosphorylation status of spliceosome components can affect splicing (Feng et al., 2008; Lipp et al., 2015) including in responses to stimuli. This points to a direct or indirect role of cGMP in splicing-mediated stress responses.

A recent study has used an affinity purification approach to identify cyclic nucleotide-binding proteins in A. thaliana (Donaldson et al., 2016). Of the 15 novel candidate cNMPbinding proteins (**Table 1**), several have key functions in the Calvin cycle and photorespiration pathway, and 8 of the 15 were shown to contain cyclic nucleotide-binding domains. What is particularly interesting is the fact that the identified proteins are post-translationally modified by NO, co-expressed, and annotated to function in responses to H2O<sup>2</sup> and defense response (Donaldson et al., 2016). The activity of one of cGMP interacting proteins, glycolate oxidase 1 (At3g14420) is a photorespiratory enzyme that produces H2O<sup>2</sup> in response to Pseudomonas, and was shown to be repressed by the combination of cGMP and NO treatment. This gave rise to the hypothesis that these cNMPbinding proteins (co-)operate as points of cross-talk between cyclic nucleotides, NO, and ROS signaling during defense responses (Donaldson et al., 2016).

TABLE 1 | List of candidate cNMP interacting proteins.


The GO analysis was performed in: http://bioinfo.cau.edu.cn/agriGO/. 1, GO:0009409: response to cold (FDA 6.3e−08); 2, GO:0009266: response to temperature stimulus (FDA 3.1e−07); 3, GO:0009628: response to abiotic stimulus (FDA 6.6e−06); 4, GO:0009526: plastid envelope (FDA 8.5e−12); and 5, GO:0009507: chloroplast (FDA 1.9e−05).

### OUT-LOOK

The discovery of an increasing number of plant MNCs does suggest that cAMP and cGMP have critical and complex roles in plant development, physiology, and responses to the environment. This is also supported by the diversity of the domain organizations of the MNCs themselves, which include NO sensors, receptor kinases, and ion channels. Not only do these "moonlighting" MNCs have to be characterized at the single molecule level, it will also be necessary to use genetic tools to elucidate their biological functions and in particular the role the cNMPs play in the downstream responses. There are already indications that the cNMPs act together with other messengers such as Ca2+. We can also expect to obtain further information on cNMP-dependent effects through investigations at the systems level, in particular extensive studies on cAMP- and cGMPspecific phosphorylation events, and the effect of the nucleotides on the metabolome. The recent report on Arabidopsis RNAbinding proteome has revealed that >20 RNA-binding proteins are spliceosome components (Marondedze et al., 2016b) and it will therefore be interesting to see if stress responses affect the RNA-binding proteome particularly since cGMP has been shown to cause specific cGMP-dependent phosphorylation of spliceosome components (Marondedze et al., 2016a).

In addition, there is increasing evidence that not only cAMP and cGMP function as messengers or modulators but also noncanonical nucleotides such as cCMP or cIMP. These molecules have long been known to be present in plants (Newton et al., 1999), and it has recently been demonstrated that particularly

### REFERENCES


cIMP can induce significant ROS production. Incidentally, when homology models of experimentally confirmed plant GCs were probed with substrates other than GTP, it appears conceivable that they might function (Marondedze et al., 2017). This is of course not answering the question if there are cyclases that are specific for cCMP or cIMP.

A possibly the most intriguing unanswered question is where the plant 3<sup>0</sup> ,50 -cyclic-nucleotide phosphodiesterases (PDEs) in higher plants are. Again, much like in the case of the cyclases, a BLAST search with an annotated C. reinhardtii PDE (A8HNW2\_CHLRE) does not return any plausible candidate with reasonable similarity. However, recently a liverwort (Marchantia polymorpha) protein has been reported that has both AC and PDE domain and both activities (Kasahara et al., 2016). Incidentally, the molecule does contain an AC catalytic center motif ([RKS]X[CTGH]X{8,12}[KR]X{1,3}[DE]) but has no ortholog in Arabidopsis or other higher plants for that matter.

### AUTHOR CONTRIBUTIONS

Both authors have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### FUNDING

The cost for the publication of this article was covered by King Abdullah University of Science and Technology.

in suspension cultured French bean cells involve cAMP and Ca2+. New Phytol. 151, 185–194. doi: 10.1046/j.1469-8137.2001.00170.x




immunoassay method. Plant Cell Rep. 30, 125–132. doi: 10.1007/s00299-010- 0950-5




**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Gehring and Turek. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# ALA6, a P4-type ATPase, Is Involved in Heat Stress Responses in Arabidopsis thaliana

Yue Niu\*, Dong Qian, Baiyun Liu, Jianchao Ma, Dongshi Wan, Xinyu Wang, Wenliang He and Yun Xiang\*

MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China

Maintaining lipid membrane integrity is an essential aspect of plant tolerance to high temperature. P4-type ATPases are responsible for flipping and stabilizing asymmetric phospholipids in membrane systems, though their functions in stress tolerance are not entirely clear. Aminophospholipid ATPase6 (ALA6) is a member of the P4-type ATPase family, which has 12 members in Arabidopsis thaliana. Here, we show that a loss-of-function mutant of ALA6 (ala6) exhibits clear sensitivity to heat stress, including both basal and acquired thermotolerance treatments. Overexpression of ALA6 improves seedling resistance to heat stress, while mutated ALA6 transgenic plants, in which the conserved functional site of the ALA family has a point mutation, are still susceptible to heat stress like ala6 loss-of-function mutant. In addition, ala6 displays higher ionleakage during heat treatment, suggesting that the lipid flippase activity of ALA6 plays a vital role in heat stress responses. Transcriptome analysis reveals differences in gene expression between ala6 and wild-type plants with or without heat stress. The differentially expressed genes are involved primarily in the physiological processes of stress response, cellular compartment maintenance, macromolecule stability and energy production. Our results suggest that ALA6 is crucial for the stability of membrane when plants suffer from high temperature stress.

Keywords: Arabidopsis P4-type ATPase, Aminophospholipid ATPase6 (ALA6), ALA6 T-DNA insertion mutant (ala6), lipid flippase, membrane, heat stress response

### INTRODUCTION

The phospholipid bilayer of cell membranes is asymmetrical, as the abundance of various lipid species on one side differs from that on the other. Within the membrane leaflets, the positions of the phospholipids are not fixed. Phospholipids perform multiple intramolecular motions, including rotation and lateral diffusion, and can also flip-flop between the two leaflets (Pomorski and Menon, 2016). The ambient temperature has a direct effect on the rate and frequency of these movements, eventually altering membrane fluidity. Thus, maintenance of the thermodynamic balance of the

#### Edited by:

Yi Ma, University of Connecticut, United States

#### Reviewed by:

Shengcheng Han, Beijing Normal University, China Joshua Blakeslee, The Ohio State University, United States

#### \*Correspondence:

Yue Niu niuy@lzu.edu.cn Yun Xiang xiangy@lzu.edu.cn

#### Specialty section:

This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science

Received: 03 May 2017 Accepted: 21 September 2017 Published: 04 October 2017

#### Citation:

Niu Y, Qian D, Liu B, Ma J, Wan D, Wang X, He W and Xiang Y (2017) ALA6, a P4-type ATPase, Is Involved in Heat Stress Responses in Arabidopsis thaliana. Front. Plant Sci. 8:1732. doi: 10.3389/fpls.2017.01732

**Abbreviations:** AGP, Arabinogalactan protein; AOC, Allene oxide cyclase; BBX, B-box domain protein; Chl, Chlorophyll; DEG, Differentially expressed gene; EL, Electrolyte leakage; ERF, Ethylene response factor; FPKM, Fragments per kilobase of transcript per million fragments mapped; GUS, β-Glucuronidase; HSP, Heat shock protein; JA, Jasmonic acid; MGDG, Monogalactosyldiacylglycerol; PC, Phosphatidylcholine; PCR, Polymerase chain reaction; PE, Phosphatidylethanolamine; PS, Phosphatidylserine; PSII, Photosystem II; SLAH, Slow anion channel-associated homolog; UBQ, Ubiquitin; WRKY, Transcription factor with conserved amino acid sequence WRKYGQK.

membrane is vital for cell function, especially during abrupt temperature changes (Murata and Los, 1997; Vigh et al., 1998, 2007; Horváth et al., 2012).

Studies have revealed that lipid asymmetry via flipflopping across membrane leaflets is involved in numerous functions of the membrane system, including formation and maintenance of cell shape; vesicle budding; membrane trafficking, impermeability, and rigidity; extra- and intracellular signaling; fertilization; apoptosis; and membrane-coupling protein regulation (Puts and Holthuis, 2009; Andersen et al., 2016). Generally, the chemical traits of phospholipids dictate that transbilayer exchange of polar lipids should occur slowly and rarely. In fact, lipid translocation across the two leaflets is greatly assisted by flippases/floppases and/or scramblases. The membrane proteins that carry out these lipid translocation processes are classified into two categories based on whether the lipid transportation across the bilayer is driven by ATP (Pomorski and Menon, 2006, 2016). P-type ATPases are found in all kingdoms of life and possess a conserved aspartic acid residue within the P-type motif DKTGT that mediates reversible phosphorylation and conformational changes during substrate transport (Pedersen and Carafoli, 1987). Based on substrate specificity and sequence characteristics, P-type ATPases are divided into five subclasses, P1- through P5-type ATPases (Palmgren and Axelsen, 1998). P4-ATPases are flippases, which utilize the energy from ATP hydrolysis to transport specific phospholipids from the exoplasmic to the cytoplasmic face of the membrane against their concentration gradient (Coleman et al., 2009, 2013; Zhou and Graham, 2009; Lopez-Marques et al., 2014). These proteins are unique in that they flip "giant" phospholipids across membranes and are found only in eukaryotic cells (Paulusma and Elferink, 2010; Van Der Mark et al., 2013; Andersen et al., 2016). The first five P4-ATPases (Neo1p, Drs2p, Dnf1p, Dnf2p, and Dnf3p) were found in yeast, and at least 14 such proteins (ATP8A1 to ATP11C) occur in mammals (Paulusma and Elferink, 2010; Sebastian et al., 2012; Van Der Mark et al., 2013; Andersen et al., 2016). Most P4-ATPases have unique lipid specificities and subcellular localization. For example, Drs2p/ATP8A1 carries out PS and PE translocation in the late secretory pathway, while ATP8B2 and ATP10A were found to mediate PC asymmetry specifically (Ding et al., 2000; Alder-Baerens et al., 2006; Paterson et al., 2006; Xu et al., 2009; Zhou and Graham, 2009). Flippases with a relatively wide range of phospholipid specificities also exist, such as Dnf1p and Dnf2p, which can transport PC, lysophospholipids and synthetic alkylphospholipids in plasma membranes (Pomorski et al., 2003; Riekhof and Voelker, 2006; Baldridge et al., 2013).

Unlike those in yeast and mammals, the P4-ATPase in the plant Arabidopsis thaliana was identified much later and its function remains unclear. There are12 P4-ATPase proteins, ALA1 through ALA12 (Aminophospholipid ATPase subfamily) in Arabidopsis (Axelsen and Palmgren, 1998). Recent reports indicate that ALA2 internalizes PS in the endosomal system, whereas ALA3, localized in the Golgi apparatus, carries out flipping of a broad range of lipids, including PS, PE and PC (Poulsen et al., 2008; López-Marqués et al., 2010). ALA10 is located in the plasma membrane and internalizes various phospholipids, including lysoPC (Poulsen et al., 2015). With respect to biological function, ALA1 may play an important role in chilling tolerance (Gomès et al., 2000), while ALA2 may function with ALA1 in antiviral defense (Guo et al., 2017). ALA3 is involved in secretory processes of the Golgi apparatus at the root tip to regulate root growth (Poulsen et al., 2008). ALA6 and ALA7 are crucial for pollen fitness (McDowell et al., 2015). In addition, ALA10 has been found to function in leaf and root development, as well as in stomatal control (Poulsen et al., 2015; Botella et al., 2016). Some reports have also indicated that the physiological functions of several plant P4-ATPases can be affected by changes in temperature (Gomès et al., 2000; McDowell et al., 2013, 2015; Botella et al., 2016). Nevertheless, the ways in which these flippases respond to some types of temperature stresses remain unclear.

In the present study, our results suggest that seedlings of a lossof-function mutant of ALA6 (ala6) grow normally at standard temperatures, while they wither, turn yellow and eventually die under basal and acquired thermotolerance treatments. Overexpression of ALA6 can improve plant tolerance of high temperature, and ALA6 point-mutated transgenic plants lacking a conserved aspartic acid residue are still sensitive to heat stress. In addition, the results of a transcriptome analysis and observation of increased ion-leakage and Chl b/a ratios in ala6 plants during heat treatment further suggest that ALA6 may protect plant cells from heat stress via the maintenance of membrane stability and integrity.

### MATERIALS AND METHODS

### T-DNA Insertion Mutants

The Arabidopsis thaliana plants used here were in the Col-0 background. The T-DNA insertion mutant used in this experiment was ala6 (SALK\_150173), obtained from the Arabidopsis Biological Resource Center (Ohio State University<sup>1</sup> ) with homozygous progeny determined via PCR screening (Alonso et al., 2003). The T-DNA insertion site is shown in **Figure 2A**, and all PCR primer sequences are given in Supplementary Table S1.

### Plant Growth Conditions

Seeds were surface-sterilized in 20% bleach for 12–15 min, rinsed five times with sterile water, and sown on <sup>1</sup>/<sup>2</sup> MS medium containing <sup>1</sup>/<sup>2</sup> Murashige and Skoog salts (MS; PhytoTech, Lenexa, KS, United States), 1% (w/v) sucrose, and 0.8% (w/v) agar. Plates were incubated at 4◦C for 3 days in the dark and then transferred to a growth chamber at 21 ± 2 ◦C with long days (16-h light/8-h dark cycles). The growth chamber was illuminated with white light at ∼110 µmol m−<sup>2</sup> s −1 .

### Gene Cloning and Transgenic Line Creation

Full-length cDNAs of ALA6 and ALA6 with a point mutation in the N-terminal aspartic acid at position 426 were amplified

<sup>1</sup>http://abrc.osu.edu/

via PCR. These cDNA fragments were cloned into the pBIB binary vectors driven by the ALA6 gene promoter. Fourweek-old wild-type (WT) Col-0 and ala6 mutant plants were transformed with A. tumefaciens (strain GV3101) using the floral dip method as described by Clough and Bent (1998). Homozygous transgenic plants were isolated on Basta. Four transgenic Arabidopsis lines were generated: complementary and overexpressing lines containing ALA6-autologous-promoter-ALA6, a tissue localization line containing the GUS gene driven by the ALA6-autologous-promoter, and a line expressing a point mutation in ALA6 driven by ALA6 gene promoter. The point mutation was an A-to-C conversion at base 1277 relative to the start codon of the full-length ALA6 cDNA (Supplementary Figure S1). All PCR primer sequences and vectors are shown in Supplementary Table S1.

### Basal and Acquired Thermotolerance Treatments

Thermotolerance assays were performed on 14-day-old seedlings as previously described (Larkindale and Vierling, 2008; Mittler et al., 2012). The basal heat treatment consisted of incubation at 43.5◦C for 45, 60, or 90 min. The acquired thermotolerance treatment consisted of incubation at 37◦C for 1.5 h followed by recovery at 22◦C for 2 h and then by heat shock at 43.5◦C for 1, 1.5, or 2 h. Plants were then moved to a growth chamber for 30, 36, or 72 h prior to sample preparation, measurement or photography. All assays were repeated at least in triplicate.

### Determination of Electrolyte Leakage

Fourteen-day-old seedlings were subjected to thermotolerance treatments and allowed to recover for 36 h. EL was determined as described by Sairam and Srivastava (2002). The sample (0.5 g) was placed in a 15-ml tube with 10 ml deionized water and incubated at 25◦C for 2 h, and the conductivity in the solution was measured using a conductometer (R1). Samples were then heated for 15 min in a boiling water bath, and conductivity was measured again after the samples cooled to 25◦C (R2). EL was calculated as the ratio of the initial conductivity to the conductivity after heating in boiling water (EL (%) = R1 R2 × 100%). All assays were repeated at least in triplicate.

### Measurement of Chlorophyll Contents

Samples were collected from 50 to 100 mg leaves for chlorophyll extraction. Chlorophyll contents were measured using 95% ethanol as described previously (Woo et al., 2001). Leaf samples of 50–100 mg were placed in a test tube and pulverized in liquid nitrogen. The freeze-dried powder was incubated with 1 ml 95% ethanol in the dark at room temperature for 2–3 h. After dilution with solvent to 1.5 ml, the mixture was centrifuged at 13400 × g for 5 min. One milliliter of the supernatant was diluted 2- to 10-fold with 95% ethanol (OD value = 0.1–0.6), and the chlorophyll level was determined by a spectrophotometer at 665 and 649 nm. All steps above were performed at room temperature (25◦C). The assay was repeated at least in triplicate. Chlorophyll contents were calculated using the following formulae: Chl a = 13.7 × OD<sup>665</sup> − 5.76 × OD649, Chl b = 25.8 × OD<sup>665</sup> − 7.6 × OD649, and Chl a + b = 6.10 × OD<sup>665</sup> + 20.04 × OD649. Chl a is the chlorophyll a content, Chl b is the chlorophyll b content, Chl a+b is the total chlorophyll content, and OD is the absorbance.

### Semi-Quantitative RT-PCR

Fourteen-day-old WT and ala6 seedlings grown on MS agar plates were collected, placed in tubes, and stored in liquid nitrogen. Total RNA was isolated using the MiniBEST Plant RNA Extraction Kit (Takara Biotechnology Co., Ltd., China). First-strand cDNA synthesis reaction was performed as Takara manual using Reverse Transcriptase M-MLV (RNase H-) (Takara Biotechnology Co., Ltd., China). PCR was carried out via a TP350 thermal cycler (Takara Bio Inc., Otsu, Japan) with the following program: 3 min at 94◦C (1 cycle); 30 s at 94◦C, 30 s at 53∼60◦C and 30 s∼1.5 min at 72◦C (28/32 cycles); and 5 min at 72◦C (1 cycle). Each PCR reaction was replicated for three times. ACTIN2 was selected as a reference gene (see Supplementary Table S1 for primer sequences).

### Quantitative PCR

Fourteen-day-old WT, ala6 and COM seedlings grown on MS agar plates were treated at 43.5◦C for 45 min and recovered for 30 h, then collected in liquid nitrogen. Total RNA was reversetranscribed into cDNA by the methods above. qPCR was carried out using a Stratagene MX3005P Real-Time System (Agilent, United States) with SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd., China) as manufacturer's instructions. Program for the reaction was as follows: 95◦C for 30 s, 40 cycles of 95◦C for 5 s, 60◦C for 30 s. Melt curves (0.5◦C increments in a 60–95◦C range) were performed for each gene to assess the sample for non-specific targets and primer dimers. UBQ11 was used as an internal control to normalize the expression of the target gene. A list of the primers used in these experiments is found in Supplementary Table S1. The 11Ct method was used to analyze relative transcript abundance (Rieu and Powers, 2009). The results were based on three independent experiments.

### GUS Staining

To identify the tissue-specific localization of ALA6, independent plants from each GUS-transgenic line were selected and various tissues were stained (Jefferson et al., 1987). Samples were incubated in a solution of 2 mM 5-bromo-4-chloro-3-indolylβ-D-glucuronide (X-gluc), 3mM K3(Fe(CN)6), 3M K4(Fe(CN)6), 0.2% Triton-X-100, and 50 mM KH2PO4/K2HPO4(pH 7.2) at 37◦C in the dark for overnight. Samples were then rinsed in 95% ethanol to remove chlorophyll. The samples were observed and photographed via anatomic microscope (SMZ-168, Motic Inc.).

### Transcriptome Analysis

Fourteen-day-old WT and ala6 seedlings grown on MS agar plates were incubated at 43.5◦C for 45 min and allowed to recover for 30 h. Gene expression analysis was performed by the Novogene Corporation, Beijing, China. RNA libraries were sequenced on a HiSeq 2500 instrument. TopHat version 2.0.12<sup>2</sup>

<sup>2</sup>http://tophat.cbcb.umd.edu/

was used for mapping against the Arabidopsis genome<sup>3</sup> . HTSeq v0.6.1 was used to determine the read numbers for each gene. Based on the length of each gene and the corresponding number of reads, the FPKM value of each gene was calculated. Three independent experiments were conducted, and only genes having consistent expression changes in the three microarray assays were reported. DEG analysis was performed using the DESeq R package (1.18.0) with an adjusted P-value (<0.05). In addition, Gene Ontology (GO) enrichment analysis of DEGs was carried out using the GOseq R package, in which results were corrected for gene length bias. GO terms with corrected P-values less than 0.05 were considered to be significantly enriched DEGs.

### Statistical Analysis

fpls-08-01732 October 3, 2017 Time: 16:43 # 4

Each experiment was repeated at least three times. Data were analyzed using one-way ANOVA with Turkey's multiplecomparison test under a 0.05 confidence coefficient.

### RESULTS

### Tissue Expression Pattern of ALA6

To analyze the tissue specificity of ALA6, its promoter region was fused to a GUS reporter gene and stably expressed in Arabidopsis plants. After staining, GUS signals were found throughout the young seedling except in the root tip, with particularly strong expression at the junction between the root and hypocotyl (**Figure 1A(a)**). In the leaf, the signal penetrated the veins and was especially strong in the leaf margin (**Figure 1A(b)**). In the flower and silique, staining was strong in the stigma, anther and base and tip of the silique; the signal was also particularly intense in pollen (**Figure 1A(c,d)**). Gene expressed in different tissues via semi RT-PCR analysis also showed that ALA6 was expressed in reproductive organs more than that in vegetative tissues (**Figure 1B**). The expression of ALA6 in numerous tissues suggests that ALA6 may be involved in multiple physiological activities in the plant.

### Loss of Function of ALA6 Confers Hypersensitivity to Heat Stress in Arabidopsis Seedlings

To determine the function of ALA6, a T-DNA insertion mutant was obtained from the SALK collection (SALK\_150173). The T-DNA was inserted into the first exon of ALA6 (**Figure 2A**). The homozygous F2 progeny of ala6 plants were screened (**Figure 2B**) and tested to determine ALA6 expression levels, and it was found that the expression of ALA6 in this T-DNA insertion line was completely suppressed (**Figure 2C**).

As ALA6 has been predicted to function in response to high temperature stress, two different experiments were performed to determine the heat-sensitive phenotype of ala6 at a lethal temperature of 43.5◦C (**Figure 3B**). As shown in **Figure 3A**, the first treatment consisted of incubation at high temperature for different lengths of time (45, 60, or 90 min) followed by recovery

Niu et al. ALA6 Participates in Heat-Stress Responses

for 3 days. Compared to WT, ala6 was extremely sensitive to heat stress. In the 45-min treatment, some ala6 seedlings began to wilt, with leaves shrinking, curling, and yellowing. Seedlings also grew more slowly or died (**Figure 3A(b)**). After longer heat-treatments, the lethal effect of the ala6 mutation was more obvious (**Figure 3A(c,d)**).

The second heat treatment consisted of exposure to a constant, non-lethal temperature (37◦C) to allow acclimation, followed by incubation at an optimum temperature (22◦C) prior to exposure to a lethal temperature (43.5◦C) (**Figure 3B**). As shown in **Figure 3A(g,h)**, ala6 remained sensitive to high temperature. Compared with the basal heat-treatment, both WT and ala6 plants showed improved tolerance to high temperature after a brief period of acclimation. With preliminary heat acclimation, most WT seedlings survived, and approximately 50% of ala6 seedlings turned yellow after 90 min at 43.5◦C (**Figures 3A(g),C**). Without previous heat acclimation, only a few WT seedlings showed any viability, and all ala6 seedlings were dead after 90 min at 43.5◦C (**Figures 3A(d),C**). These results indicated that plants exposed to non-lethal temperatures in advance of abrupt exposure to high temperature showed improved responses to heat stress. In addition, it appeared that ala6 was inherently heat-sensitive, showing susceptibility to heat treatment even after warm acclimation. ALA6 might play a crucial role in plant heat-tolerance.

Considering the conserved function of the ALA family in phospholipid transport, the membrane status of ala6 plants under hyperthermia was investigated. WT and mutant seedlings

<sup>3</sup>www.arabidopsis.org

were grown on MS plates for 14 days and incubated at 43.5◦C for 45 min, followed by a 36-h recovery under optimum conditions. After this treatment, the growth of all seedlings was affected. Seedlings were collected, and EL was quantified; the results were shown in **Figure 3D**. Heat treatment significantly increased the rate of EL in both WT and mutant plants. Leakage rates increase by 48.9 and 68.9% due to heat treatments in WT and ala6 seedlings, respectively. Notably, the membrane status of ala6 cells was altered more than that of WT cells, implying that ALA6 might protect plant cells from heat stress by regulating plasma membrane permeability and stability.

Changes in the contents of photosynthetic pigments (chlorophylls) and in Chl b/a ratios are useful indicators of stress and tolerance in plants (Zhang et al., 2008). We found that chlorophyll levels decreased during heat stress treatments in both WT and ala6 mutant plants, indicating possible heatinduced damage to the photosynthetic activity of the chloroplasts (**Figure 3E**). In addition, the chl b/a ratio increased dramatically in ala6 mutants after basal heat treatment, reaching a level 36.4% higher than in ala6 mutants without treatment, whereas the ratio in WT increased only 28.2% (**Figure 3F**). These results indicated that a lack of ALA6 might increase the injurious impact of heat stress on membranous organelles.

### The Intrinsic Function of ALA6 Is Critical for Thermotolerance

To confirm the function of ala6 under high temperature, an exogenous ALA6 gene (normal or deficient in conserved gene function) was expressed in ala6 and WT plants. The complementation lines resulting from the former transformation were subjected to heat treatment, as shown in **Figure 4**, and it was found that the expression of ALA6 could rescue the heatlethal phenotype of the loss-of-function mutant. The growth of the complementation lines resembled that of WT plants, even after heat treatment. In addition, the ALA6 overexpression lines exhibited greater vitality than WT plants (**Figures 4A,C**), showing that ALA6 was involved in intracellular responses to heat stress, and the heat-sensitive phenotype of ala6 was due to the loss of function of ALA6.

P-type ATPases, including the ALA family in Arabidopsis, contain a motif that carries out phosphorylation during phospholipid transport; this motif contains an aspartic acid residue (Pedersen and Carafoli, 1987). To determine the relationship between the phospholipid transport activity of ALA6 and the heat stress response, transgenic lines containing a point mutation in the phosphorylation motif were generated. This mutation converted an A to a C in the codon encoding the conserved aspartic acid residue of ALA6. As expected, the transgenic lines showed a heat-hypersensitive phenotype like that of ala6 under high-temperature treatment (**Figure 5A**). This result further demonstrated that the native phospholipid transport activity of ALA6 was implicated in heat-tolerance, and the specific phenotype of ala6 was associated with a defect in its enzymatic activity. The levels of ALA6 transcript in these transgenic lines were measured and were significantly higher than those of the WT (**Figures 4B**, **5B**). This observation was also supported by the survival rate shown in **Figures 4C**, **5C**.

### Microarray Analysis Reveals ALA6-Related Genes

To investigate the underlying mechanism of heat sensitivity in ala6, we analyzed the RNA-seq data of WT and ala6 Arabidopsis samples. A total of 426 DEGs were found to be related to ALA6 (**Figure 6A**). Of these, 286 genes showed down-regulated and 140 showed up-regulated expression in ala6. All the transcripts listed here exhibited twofold or larger changes in abundance. These DEGs were enriched in GO terms containing genes responsive to endogenous stimuli, chemical or organic substances, and biotic or abiotic stresses (shown in Supplementary Table S2). The top 30 DEGs that were annotated as stress response elements include various transcription factors,

but the heat shock time was 1.5 h; h received acclimation at 37◦C prior to heat treatment for 2 h. (B) Schematic diagram for heat treatment corresponding to (A); from top to bottom, linear graphs represent basal thermotolerance (no acclimation) and acquired thermotolerance (acclimation). Red lines indicate the temperatures and blue lines indicate different treatment times. Arrows illustrate the times of the heat shock at 43.5◦C. (C) The percentage of seedlings, including dead, half-dead and alive. The six columns correspond to b, c, d, f, g, and h in (A) are calculated from three independent experiments (average results ±SE). Columns with different letters have significantly different values (P < 0.05). (D) Electrolyte leakage (%) of 14-day-old seedlings treated at 43.5◦C for 45 min and allowed to recover for 36 h. (E) Chlorophyll contents (mg/g) of seedlings as in (D). WT\_H, wild type treated with heat stress; ala6\_H, ala6 treated with heat stress; Chla, chlorophyll a; Chlb, chlorophyll b; Chlt, total chlorophyll. (F) Chlb/Chla ratio of (E). Results are calculated from three independent experiments (average results ±SE). Columns with different letters have significantly different values (P < 0.05).

membrane components and transporters (**Table 1**). These results indicated that ALA6 might be involved in multiple cellular responses.

To identify genes related to ALA6 under heat stress, the RNA-seq data of Arabidopsis WT and ala6 samples with and without heat treatment were analyzed as described in the Section

"Materials and Methods." Quantitative PCR analyzing the expression of several DEGs in WT, ala6, ALA6 complementary transgenic lines with or without heat stress also confirmed the results from RNA-seq data above (**Figure 7**). **Figure 6A** showed the DEGs for ala6 vs. WT (a), WT with heat treatment vs. WT (b), ala6 with heat treatment vs. ala6 (c) and ala6 with heat treatment vs. WT with heat treatment (d). It was believed that genes related to ALA6 under heat treatment should be found in b but not in c (**Figure 6A**). A total of 1058 DEGs were identified and implicated in heat stress responses related to ALA6. The top

30 DEGs in **Table 2** are involved in transcription factor activity, organelle components, and signal transduction and substrate transport. Of these genes, two transcription factors [BBX14 (AT1G68520) and ERF104 (AT5G61600)] had previously been implicated in stress response. And most of genes we screened were involved in photosynthesis-related activities (AT1G16720, AT4G12800, AT1G52870, AT2G30570, and AT1G06430) and transmembrane transport activity (AT5G49730, AT1G64720, AT5G46110, AT4G25570, and AT3G13062).

Differentially expressed genes from the basal heat treatment were subjected to GO enrichment analysis (**Figure 6B**). The genes associated with ALA6 under heat stress included 112 genes categorized as "response to endogenous stimulus" and 194 categorized as "response to stress." Other categories of enriched genes include "response to oxygen-containing compound" (141 genes), "defense response" (100 genes), "response to acid chemical" (92 genes), and "aromatic compound biosynthetic process" (160 genes). These results indicated that ALA6 might have versatile effects on cellular responses triggered by heat stress.

### DISCUSSION

### ALA6 Is Involved in Heat-Induced Cellular Responses via Maintenance of Membrane Stability

Temperature is a major environmental cue that has a rapid impact on cellular homeostasis, including both membrane stability and protein activity, and also has significant effects on plant development and growth. Because land plants are sessile, they are exposed to daily and seasonal temperature fluctuations (Saidi et al., 2011). To survive, plants evolved a series of mechanisms to respond to the ambient environment, including membrane-related stress signaling mechanisms for intact cell (Vigh et al., 2005, 2007). Stress-triggered changes in the lipid species of membranes could influence both the physical properties of the membrane and the distribution

and activity of membrane proteins (Vigh et al., 2005). P4 type ATPases have recently been found to alter membrane lipid composition by transporting specific lipids, and some members of this protein family have been found to play crucial roles in stress responses in yeast and mammals (Axelsen and Palmgren, 2001; Sebastian et al., 2012). Among the 12 members of the P4-type ATPase family in Arabidopsis, ALA1 was first reported to be implicated in cold tolerance (Gomès et al., 2000). In addition, pollen tube development in an ala6/7 double mutant is described to be vulnerable under hot-day/cold-night temperature stress (McDowell et al., 2015). ALA3 has been demonstrated to participate in secretory processes of the Golgi apparatus, regulating root growth and reproductive development as well as tolerance to temperature stress (Poulsen et al., 2008; McDowell et al., 2013). Though ALA10 is responsible for root and leaf development independent of temperature, it can improve MGDG synthesis at low temperature (Poulsen et al., 2015; Botella et al., 2016). However, the functions of other members of this subclass have not been determined. In fact, the members of the ALA family that exhibit lipid substrate specificity have been shown to be involved in many physiological processes in a temperaturedependent manner. Considering the relatively close phylogenetic relationship among ALA family members 3–10 (Poulsen et al., 2015), we chose to investigate ALA6 and found that this protein is essential for responses to high temperature stress. As shown in **Figure 2A**, though a short-term acclimation can alleviate heat injury to seedlings, two heat treatments lead to the death of ala6 seedlings, whereas WT seedlings survive. In addition, the partially sterile siliques of ala6 plants under heat stress (data not shown) are comparable to the description given by McDowell et al. (2015). Since plants also suffer other stresses while acquiring thermotolerance via heat acclimation (Larkindale and Vierling, 2008; Mittler et al., 2012), this study shows that ALA6 can be implicated directly in heat stressrelated intracellular responses rather than in other stresses. To determine whether the enzyme activity of phospholipid translocation is involved in the heat stress response, ALA6 with a point-mutation at a conserved functional site was analyzed and was confirmed not to rescue the heat-sensitive phenotype of an ala6 knockout mutant (**Figure 5A**). Taken together with the greater EL and higher Chl b/a ratio of


Gene\_id: Gene identity; FPKM: Fragments Per Kilobase of transcript per Million fragments mapped; padj: P-value by adjustment.

ala6 mutants (Demidchik et al., 2014; Bi et al., 2016), this result implies that ALA6 might serve as a membrane stabilizer via altering membrane permeability, especially during heat stress.

### ALA6 Can Induce Changes in the Expression of Heat Stress-Related Genes

Under high temperature, changes in lipid composition affect membrane dynamics and initiate the corresponding signal transduction pathways (Vigh et al., 1998). It has been reported that membrane fluidity can modulate the transcription of heatinducible genes (Carratù et al., 1996; Horváth et al., 1998). To identify the downstream signaling triggered by ALA6, we analyzed gene expression in WT and ala6 plants with and without heat shock. DEGs between WT and ala6 Arabidopsis were shown to be involved in many functions. Among the 286 down-regulated genes are numerous genes related to stress and hormone responses, including heat, cold, salt, drought, oxidative, and pathogen stress, as well as ethylene, abscisic acid, JA and salicylic acid in various other processes. These responses are involved in the control of gene expression by transcription factors, such as the F-box protein family, the B-box zinc finger protein family, WRKYs and ERFs (**Table 1**). For instance, the expression of BBX14 (AT1G68520), a B-boxtype zinc finger protein, was significantly lower in ala6 than in WT Arabidopsis. Although the function of BBX14 is still unknown, expression of its homolog, BBX18, has been confirmed to be induced by heat shock and to affect seed germination and seedling survival negatively by suppressing the expression of heat-responsive genes (Wang et al., 2013). Other members of this family are also involved in responses to various biotic or abiotic stresses (Gangappa and Botto, 2014), likely explaining why ala6 plants are sensitive to heat stress. In addition, it was found that 140 genes with up-regulated expression in ala6 plant are responsible for various biological processes such as phosphorylation, substrate transport and synthesis, and lipid metabolism, but most of these genes have an integral membrane component (**Table 1** and Supplementary Table S2). Of these genes, only two fell within the top 30 DEGs with increased expression; these were SLAH3 (AT5G24030) and AGP17 (AT2G23130). The products of these genes both localize to the plasma membrane. SLAH3 is an anion channels involved in mediating ion homeostasis, while AGP17 is a glycosylphosphatidylinositol (GPI)-anchored protein involved in extracellular signal perception and interaction with proteins in lipid rafts (Gaspar et al., 2004; Zhang et al., 2016). The results imply that lack of ALA6 might not only lead to the alteration of membrane permeability

and integrity but also to corresponding changes in membrane proteins.

Interestingly, the expression of many chloroplast-related genes in the top 30 DEGs are shown to be up-regulated in ala6, whose functions depend greatly on membrane stability (**Table 2**; Boudière et al., 2014). A HSP81-3 (HSP90.3, AT5G56010), which functions as a molecular chaperone, and AOC2 (AT3G25770), which has AOC activity, are among the most interesting genes we identified. The major functions of HSPs include preventing proteins from misfolding and disaggregation and protecting membranes that are exposed to high temperature (Banti et al., 2010). Proteins such as HSP20, HSP70, HSP90 and HSP101 have been shown to be components of the heat shock response and assist in thermotolerance by adjusting protein aggregation (Nieto-Sotelo et al., 2002; Cazalé et al., 2009; Fragkostefanakis et al., 2015; Yu et al., 2016). Thus, the decreased expression of HSP genes in an ALA6 loss-of-function mutant may also demonstrate that ALA6 plays an important role in heat-inducible signaling. Moreover, AOC2 catalyzes the biosynthesis of JA, for which a role in basal thermotolerance has been illustrated recently (Clarke et al., 2009; Stenzel et al., 2012). In conclusion, when plants are exposed to high temperature, it is speculated that ALA6 may have an effect on the membrane permeability and stability, and this alteration can induce changes in membrane proteins and eventually trigger heat-associated gene expression. However, the physiological functions and underlying mechanisms of these altered genes in ala6 mutants require further study.

### CONCLUSION

ALA6, a P4-type ATPase of Arabidopsis, possesses the conserved structure and function of this family and serves as a phospholipid flippase to maintain membrane stability and fluidity under normal or adverse conditions. The seedlings of loss-of-function mutant ala6 were found to wither, turn yellow and eventually die with exposure to basal thermotolerance or acquired thermotolerance treatments. Overexpression of ALA6 can rescue this phenotype, while transgenic plants containing a pointmutation in a conserved region of ALA6 show no response to heat stress. This suggests that ALA6 is

TABLE 2 | Genes related to ALA6 under basal thermotolerance.


Gene\_id: Gene identity; FPKM: Fragments Per Kilobase of transcript per Million fragments mapped; padj: P-value by adjustment.

significant for response to high temperature stress. Moreover, ala6 seedlings exhibit higher ion-leakage and lower chlorophyll content after heat treatment, indicating that ALA6 can protect membranes from the disordered state that results from heat stress. This protective effect involves the maintenance of membrane permeability, stability and integrity. In addition, transcriptome analysis shows that ALA6 affects the expression of heat-inducible genes. Taken together, these evidences show that ALA6 plays an essential role in cellular responses to high temperature. In addition, characterization of ALA6 activity and its roles should further illuminate the mechanisms of the involvement of P4-ATPase in stress responses.

### ACCESSION NUMBER

fpls-08-01732 October 3, 2017 Time: 16:43 # 12

RNA sequencing data is available at the National Center for Biotechnology Information (NCBI) data repository (accession PRJNA390831).

### REFERENCES


### AUTHOR CONTRIBUTIONS

YN and YX designed research; YN, DQ, BL, JM, DW, XW, and WH performed research; YN, DQ, BL, JM analyzed data; and YN, DQ, and YX wrote the paper.

### FUNDING

This work was supported by the National Natural Science Foundation of China (31400220) and the Fundamental Research Funds for the Central Universities (lzujbky-2014-92, lzujbky-2017-156, lzujbky-2017-k14).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.01732/ full#supplementary-material


Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 114, 1377–1382. doi: 10.1073/pnas. 1614204114


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Niu, Qian, Liu, Ma, Wan, Wang, He and Xiang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fpls-08-01732 October 3, 2017 Time: 16:43 # 13

# Transcriptome and Differential Expression Profiling Analysis of the Mechanism of Ca2<sup>+</sup> Regulation in Peanut (Arachis hypogaea) Pod Development

Sha Yang1†, Lin Li 2†, Jialei Zhang<sup>1</sup> , Yun Geng<sup>1</sup> , Feng Guo<sup>1</sup> , Jianguo Wang<sup>2</sup> , Jingjing Meng<sup>1</sup> , Na Sui <sup>3</sup> , Shubo Wan<sup>4</sup> \* and Xinguo Li <sup>1</sup> \*

#### Edited by:

*Gerald Alan Berkowitz, University of Connecticut, United States*

### Reviewed by:

*Changai Wu, Shandong Agricultural University, China Kun Wang, Michigan State University, United States Daowen Wang, Institute of Genetics and Developmental Biology (CAS), China*

#### \*Correspondence:

*Shubo Wan wansb@saas.ac.cn Xinguo Li xinguol@163.com*

*† These authors have contributed equally to this work as co-first authors.*

#### Specialty section:

*This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science*

Received: *02 June 2017* Accepted: *04 September 2017* Published: *28 September 2017*

#### Citation:

*Yang S, Li L, Zhang J, Geng Y, Guo F, Wang J, Meng J, Sui N, Wan S and Li X (2017) Transcriptome and Differential Expression Profiling Analysis of the Mechanism of Ca*2<sup>+</sup> *Regulation in Peanut (Arachis hypogaea) Pod Development. Front. Plant Sci. 8:1609. doi: 10.3389/fpls.2017.01609* *<sup>1</sup> Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan, China, <sup>2</sup> College of Agronomy, Hunan Agricultural University, Changsha, China, <sup>3</sup> College of Life Sciences, Shandong Normal University, Jinan, China, <sup>4</sup> Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Shandong Academy of Agricultural Sciences, Jinan, China*

Calcium not only serves as a necessary nutrient for plant growth but also acts as a ubiquitous central hub in a large number of signaling pathways. Free Ca2<sup>+</sup> deficiency in the soil may cause early embryo abortion, which eventually led to abnormal development of peanut pod during the harvest season. To understand the mechanisms of Ca2<sup>+</sup> regulation in pod development, transcriptome analysis of peanut gynophores and pods was performed by comparing the treatments between free Ca2<sup>+</sup> sufficiency and free Ca2<sup>+</sup> deficiency using Illumina HiSeqTM 2000. 9,903,082,800 nt bases are generated totally. After assembly, the average length of 102,819 unigenes is 999 nt, N50 is 1,782 nt. RNA-seq based gene expression profilings showed a large number of genes at the transcriptional level changed significantly between the aerial pegs and underground swelling pods under free Ca2<sup>+</sup> sufficienct or deficiency treatments, respectively. Genes encoding key members of Ca2<sup>+</sup> signaling transduction pathway, enzymes for hormone metabolism, cell division and growth, transcriptional factor as well as embryo development were highlighted. This information provides useful information for our further study. The results of digital gene expression (DGE) indicated that exogenous calcium might contribute to the development of peanut pod through its signal transduction pathway, meanwhile, promote the normal transition of the gynophores to the reproductive development.

Keywords: calcium, peanut, pod development, RNA-Seq, differential expression analysis

## INTRODUCTION

Peanut (Arachis hypogaea L.) is an economic crop that contributes 20% to the global oil production and 11% of the protein supply per year (Chen et al., 2014). Aerial flowering and underground development of fruit are special genetic characteristics of peanuts, and the ecological environment of dark, moist and mechanical stimulation is essential conditions for pod development. So, the ovule-carrying pegs (gynophores) must elongate to bury the fertilized ovule into the soil in order

**140**

to survive and reproduce (Arya et al., 2016). When gynophores are buried into the soil at 2–8 cm deep, the ovule swells to provide room for the embryo to grow. During this complex period, the peanut can directly absorb moisture, calcium, and other inorganic salts from the soil to maintain its reproductive development (Beringer and Taha, 1976). Calcium-deficient soil may result in the termination of pods expansion and eventually lead to embryo abortion. By contrast, peanuts will produce filled pods when sufficiently supplied with calcium (Jain et al., 2011). Therefore, the development of peanut pod is extremely sensitive to calcium, and free-Ca2<sup>+</sup> deficiency in the field will greatly reduce the yield of peanut.

Ca2+, a universal second messenger, plays an important role in plant growth and development, including cell division and apoptosis, polarity formation, differentiation and senescence (Hepler, 2005; Zhang et al., 2017). In our previous study, we reported that free-Ca2<sup>+</sup> deficiency results in peanut dwarfism, and Ca2+/CaM signal transduction pathway was involved in the turnover of PSII reaction center components to alleviate the damage of photoinhibition to PSII (Yang et al., 2013, 2015). However, little was known about the regulation of calcium on the development of peanut pods over the last century. With the development of molecular biological techniques, the transcriptome and proteomics benefited the peanut genomics research, and was applied in the seed development. Recently, several studies have compared genes and proteins between the aerial and underground peanut pods (Zhu et al., 2013). Chen et al. (2013) speculated that categories in the photosynthetic pathway were altered greatly in aerial gynophores, while the growth regulators such as IAA, ABA and kinetin may be the key factors contributing to the swelling of the subterranean pods. Other studies have explored changes in the gene expression at the different developmental stages of the gynophores. Xia et al. (2013) assembled 13 million short sequences into 72,527 unigenes to study peanut gynophores in three developmental stages. The results showed that numerous enzymes involved in plant hormone biosynthesis and signaling pathway, as well as light signaling, changed significantly when the ovary began to enlarge. These candidate genes and proteins have been identified to understand the regulation mechanisms that control the development of peanut pods and provide valuable resource to illustrate the specific mechanism of calcium signaling on pod development.

Digital gene expression (DGE) profiling, by comparing reads with the reference genome, can comprehensively and rapidly detect the specific gene expression of a particular species and distinguish the differences in the expression of poorly expressed genes (Glazinska et al., 2017). However, only reference transcripts from wild peanuts, not those of the cultivated peanut, have been published. Therefore, transcriptome profilings obtained from a mixture of all samples tested in our study will supply the data for the reference genome. Transcriptome sequencing can be used to sequence all mRNAs transcribed by eukaryotes, specific tissues, or cells in a particular state, and also analyze the structure of the genes and new transcripts produced (Sathiyanathan et al., 2017). This method has been widely applied in Arabidopsis and other model plants, e.g., rice and soybean, and has gradually become a useful tool in biological research.

In this study, RNA-Seq and digital gene profiling were combined to investigate and compare differentially expressed genes (DEGs) between aerial gynophores and underground pods grown under free-Ca2+-sufficient or deficient Our objectives were to: (1) compare the DEGs of aerial gynophores between free Ca2<sup>+</sup> sufficiency and free Ca2<sup>+</sup> deficiency treatments, (2) identify candidate genes related to pod swelling affected by calcium, (3) find out the main regulatory pathways of calcium to promote pod development.

### MATERIALS AND METHODS

### Plant Materials and Treatments

The Arachis hypogaea "XiangHua 2008," a peanut cultivar from the Hunan Agriculture University, was used as material. The red soil overburden including exchangeable calcium 0.74 cmol(1/2 Ca2+)/kg in Gengyuan practice base, Hunan Province of China, was used for the free Ca2<sup>+</sup> deficient treatment. The soil fertilized with 100 kg plaster (CaO) per 667 m<sup>2</sup> was used as free Ca2<sup>+</sup> sufficiency treatment. CaO was applied before sowing.

### The Method of Sampling

In peanut production, 15 days after pegging (DAP) was confirmed to be the most sensitive period for the free Ca2<sup>+</sup> content in the soil. In this study, gynophores under free Ca2+ deficient treatment 1 (GD1), free Ca2+-deficient treatment 2 (GD2), free Ca2+-sufficient treatment 1 (GS1), free Ca2+ sufficient treatment 2 (GS2), as well as pods under free Ca2+ deficient treatment 1 (PD1), free Ca2+-deficient treatment 2 (PD2), free Ca2+-sufficient treatment 1 (PS1), and free Ca2+ sufficient treatment 2 (PS2), were harvested, frozen in liquid nitrogen and then stored at −70◦C. The 8 samples were used to detect global changes in gene expression. Two biological replicates were used in this study. The mixed samples were used in transcriptome sequencing.

### RNA Extraction and Sequencing

Total RNA was isolated from the harvested materials using the total RNA extraction Kit (TaKaRa, Inc., Dalian, China) according to the manufacturer's instructions. Agilent 2100 and NanoDrop were used to detect RNA quality and purity. Only high-quality RNA samples were chosen for RNA-seq analyses. Beads with Oligo (dT) were used to enrich for mRNA and the RNA was fragmented into short fragments. First-strand cDNA was synthesized using random hexamer primers and buffer, while dNTPs, RNaseH and DNA polymerase I were added to synthesize the second strand (Wang et al., 2009). After purification, the short fragments were then connected using sequencing adapters. Finally, the library was sequenced from directions on Illumina HiSeqTM 2000 System (Illumina, San Diego, CA). Data analysis and base calling were achieved by applying the Illumina instrument software. Whole dataset has been deposited in the NCBI Sequence Read Archive with accession number SRX1795063.

### Digital Expression Profile Analysis

Differential expression profiling was conducted among the gynophores and pods under free- Ca2+-deficient or sufficient treatments. Prior to further analysis, raw data should be filtered to decrease data noise and obtain clean data (Cock et al., 2010). Gene expression level was quantified by a software package RNASeq by Expectation Maximization (RSEM). RSEM computes maximum likelihood abundance estimates using the Expectation-Maximization (EM) algorithm for its statistical model, including the modeling of paired-end (PE) and variablelength reads, fragment length distributions, and quality scores, to determine which transcripts are isoforms of the same gene (Li and Dewey, 2011). Fragments per kilobase of exon per million fragments mapped (FPKM) method was used to calculate the expression level as follows: FPKM = [106C/(NL/10<sup>3</sup> )], where FPKM is the expression of gene A, C is the number of fragments uniquely aligned to gene A, N is the total number of fragments uniquely aligned to all genes, and L is the number of bases on gene A (Ning et al., 2013). The FPKM method can eliminate the influence of different gene lengths and sequencing discrepancy on the calculation of gene expression. The intensity values of each sample were further transformed on log2-scale and used to analyze differential expression. The probe sets with P < 0.01 in at least one of the comparisons were considered as DEGs for further analysis. The WEGO software was used to obtain gene ontology (GO) annotations for the DEGs, while pathway analyses were used to analyze KEGG and BLASTX (E < 0.00001) against the NCBI Nr database (Ye et al., 2006).

The contents of endogenous hormone indole acetic acid (IAA) and gibberellin (GA3) were determined by enzyme linked immunosorbent assay (ELISA), which was performed as a commercial service in China agricultural university.

### Quantitative Real Time PCR (qRT-PCR) Analysis

Gynophores and pod samples under the same treatments as those in RNA-seq were analyzed using qRT-PCR. cDNAs were reverse transcribed using the PrimeScriptTM first-strand cDNA synthesis kit (K1622 Thermo scientific). The PCR was amplified using SYBR Premix Ex TaqTM following the manufacturer's instructions (TaKaRa, Inc., Dalian, China) with the qRT-PCR amplification instrument (ABI 7500, USA). The target gene primers (**Table S3**) were designed to detect the sample mRNA. Tua5-F and Tua5- R were used as controls to normalize the expression data (Yang et al., 2013). The relative gene expression was calculated using the 2 <sup>−</sup>11CT method as described by Livak and Schmittgen (2001). For log2-transformed FPKM values, the maximum expression level of each selected gene was considered to be 100, and the expression levels of the other genes were transformed accordingly (Zhang et al., 2016).

### RESULTS

### Transcriptome Sequencing and Data Analysis

Ca2<sup>+</sup> deficiency in soil induces early embryo abortion in peanuts, and this process produces empty pods. This phenomenon was TABLE 1 | The effects of calcium treatment on peanut phenotype.


*a, b means significant difference compared with CK and calcium-treated plants (NC12) using Student's t-test at P < 0.05.*

verified by the full pod number and dry weight of pods (**Table 1**). In our study, we attempted to reveal the molecular mechanism of the effect of free calcium on the development of peanut pods using a RNA-seq approach. A total of 99,030,828 clean reads with an average length of 90 bp, which corresponded to approximately 8.91 gigabase pairs (Gb) of raw data, were obtained. An overview of the sequencing and assembly is outlined in **Table S1**. 141,819 contigs with an average length of 391 bp were produced. Among these contigs, the length of 81,881 (57.73%) from 100 to 200 bp, 18,847 (13.28%) from 200 to 300 bp, 9256 (6.52%) from 300 to 400 bp, 5633 (3.97%) from 400 to 500 bp, and 26,202 (18.47%) were more than 500 bp in length. The results of the assembly showed 102,819 unigenes with a total length of 102.72 Mb and an average of 999 bp. Of these, the length of 46,924 (45.63%) were from 100 to 500 bp, 17,974 (17.48%) from 500 to 1,000 bp, 12,891 (12.53%) from 1,000 to 1,500 bp, 10,011 (9.73%) from 1,500 to 2,000 bp, and 15,019 (14.60%) were more than 2,000 bp (**Figure S1**). The length of assembled sequences is a criterion for successful assembly. We calculated the length distribution of contigs and unigenes, and the results showed that the Illumina sequencing solution was reproducible and reliable (**Figure S1**).

### Unigene Function Annotation

Functional annotation of unigenes gives protein functional annotation, COG functional annotation and Gene Ontology (GO) functional annotation. Unigenes were annotated with the databases of NR, NT, Swiss-Prot, KEGG, COG, and GO. Then the number of annotated unigenes was counted with each database (**Figure 1**). Analysis of species distribution showed that 42.2% of the unigenes were homologous with the sequence of Glycine max, and 13.8, 13.1 and 11.5% of the unigenes with the sequence of Cicer arietinum, Phaseolus vulgaris, and Medicago truncatula, respectively (**Figure 2**).

We use Blast2GO program with NR annotation to obtain the GO annotation of the unigenes. GO has three ontologies: molecular function, cellular component and biological process. Every GO-term belongs to a type of ontology (Conesa et al., 2005). **Figure S2** showed that the sequences were categorized into 55 functional groups according to the sequence homologies. We mapped the unigenes to the COG database to further predict the possible functions and statistics. In the 25 COG categories, general function prediction (7,524, 32.03%) represented the largest group, followed by transcription (4,202, 17.89%), and replication, recombination, and repair (4,128, 17.57%) (**Figure S3**).

A total of 14,985 sequences, containing 18,215 potential EST-SSRs, were detected with software MicroSAtellite (MISA). The quantity statistics of SSR classification showed that trinucleotide (40.86%) was the most abundant type of repeat motif, followed by di-nucleotide (29.94%), mononucleotide (21.61%), hexa-nucleotide (2.67%), quad-nucleotide (2.53%), and pentanucleotide (2.38%) repeat units (**Table S2**). Within the cSSRs detected, AG/CT represented the dominant type, followed by AAG/CTT, ATC/ATG, and AAT/ATT (**Figure S4**).

### Digital Gene Expression (DGE) Profiling

Eight DGE libraries were created from four independent biological samples using gynophores and pods with free

Ca2+-deficient or -sufficient treatments as described in the method section. The clean reads (in millions) obtained from the different libraries were as follows: GD1, 14.91; GD2, 15.59; GS1, 14.54; GS2, 14.34; PD1, 16.02; PD2, 16.67; PS1, 14.19; and PS2, 12.84. The saturation analysis showed that when the number of reads reached a certain amount, the growth curve of detected genes tended to flatten in all libraries, indicating that the sequencing was saturated for gene identification (**Figure S5**).

GO annotation of the DEGs from two pairwise comparisons (GD/GS and PD/PS) was used to classify genes into different sub-categories belonging to the three main GO categories. In the biological process category, we identified three abundant sub-categories across both comparisons, namely, metabolic process, cellular process, and single organism process. Cell, cell part, and membrane were the main sub-categories identified in the cellular component category, while catalytic activity and binding dominated the molecular function category (**Figure 3**).

KEGG enrichment analysis allowed the mapping of DEGs to different pathways. The top 20 pathways in each pairwise comparison (mentioned above) are shown in **Figure S6**. DEGs were mainly enriched in photosynthesis, phagosome, glycolysis/gluconeogenesis, isoflavonoid biosynthesis, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, carbon metabolism, and ABC transporters in GD-VD-GS. Meanwhile, circadian rhythm-plant,

plant hormone signal transduction, phagosome, isoflavonoid biosynthesis, glycolysis/gluconeogenesis, fatty acid metabolism, and biosynthesis of unsaturated fatty acids were mainly enriched in PD-VD-PS.

### Analysis and qRT-PCR Validation of DGE Results

FPKM method was used to calculate the gene expression level. The reference transcripts of a progenitor of cultivated peanut (Arachis ipaensis) and our transcriptome data were used to generate an integrated reference library. For result list of each control-case pair, we draw scatter plots of all expressed genes, different color presents up-regulated, down-regulated or nonregulated genes. The total number of DEGs for each comparison was observed, and a histogram was drawn to show the number of significantly up-regulated or down-regulated genes in each control–case pair (**Figure 4**).

In order to confirm the transcriptome sequencing results, 10 DEGs in GS, GD, PS, and PD were selected randomly for qRT-PCR to analyze the expression levels. The selected genes participate in calcium signal transduction and phytohormone biosynthesis pathway, material biosynthesis, transcription factor and transporter. The consistency of trends between qRT-PCR and RNA-seq data indicated the credibility of the RNA-seq data (**Figure 5**).

### Comparative Analysis of Genes between GD and GS

More than 20 unigenes annotated as transcription factors including NAC, WRKY, bHLH, and ethylene-responsive transcription factors were regulated in GS, while M1/AGAMOUS/DEFICIENS/SRF (MADS) transcription factor family were down-regulated in GS compared with GD. Contrary to the DEGs in pods, no significant difference in calcium-related genes between GD and GS was found, while the expression of some calcium-binding proteins (CBPs), and calcium-binding transcription factors were up-regulated in GS (**Table S4**).

The expression pattern of lipoxygenases (LOX) genes showed higher expression level in GS and the expression levels of 6 LOX genes were more than 2 times higher in GS than that in GD. Genes related to hormone response were screened out by GO analysis. Several DEGs involved in biosynthesis process and signaling pathway of gibberellin (GA), auxin, ABA, and brassinosteroid were up-regulated in GS at this stage. e. Numerous transporter-related genes were screened from DEGs based on GO analysis. Several energy- and substance-related transporter genes, including sugar transporter, carbohydrate transmembrane transporter, lipid transporter, protein transmembrane transporter, inositol transporter, zinc transporter, sulfate transporter, and nucleobase transporter were all up-regulated in GS. Meanwhile MFS transporter, ABC transporter and myo-inositol transporter had both up-regulated and down-regulated. Only the peptide/histidine transporter was down-regulated in GS (**Table S4**).

### Differentially Expressed Genes between PD and PS

We identified the DEGs between PD and PS considering that calcium plays an important role in the development of

peanut pods. Unigenes encoding proteins involved in calcium signaling transduction pathways such as calcium-dependent protein kinases (CDPKs), calcium-binding protein (CBP) and calmodulin (CaM)-binding protein were up-regulated under free Ca2+-sufficient conditions. In particular, the expression of Ca2+ related protein mitogen-activated protein kinase kinase kinase (MAPKKK) was also higher in PS than that in PD (**Table S5**).

Two categories of plant hormone-related genes (auxin and GA) showed significantly different expression between PD and PS. The auxin-related genes including 7 auxin response factors (ARFs) and 1 indole-3-acetic acid-amido synthetase were upregulated in PS. Two selected DEGs, namely, GA 20-oxidase and GA receptor GID1, were up-regulated, while GA 2-oxidase, which is one of the key enzymes that catalyzes the inactivation of biological GA, was down-regulated in PS. Meanwhile, the ent-kaurenoic acid oxidase (KAO), an important enzyme in GA biosynthesis (Yamaguchi and Kamiya, 2000; Paparelli et al., 2013), was up-regulated under free Ca2+-sufficient conditions. These results were in agreement with the high contents of IAA and GA<sup>3</sup> in PS (**Figure 6**). Thus, our results indicated that the upregulated calcium-related genes increased GA level, which may play an important role in the early embryo development.

Genes encoding storage protein oleosin and some synthetases, such as tetrahydrocannabinolic acid synthase, long chain acyl-CoA synthetase, and callose synthase, exhibited higher level (5–8-folds) under free-Ca2+-sufficient conditions. In addition, eight lipoxygenase were also up-regulated in PS. Previous studies indicated that lipoxygenase is the first key enzyme in the synthesis pathway of jasmonic acid, which plays an important role in the germination, growth, and stress resistance of plant seeds (Rahimi et al., 2016). GIGANTEA, a gene involved in circadian clock and phytochrome signaling (Cha et al., 2017), was down-regulated in PS (**Table S5**). This phenomenon may be due to the fact that this light response gene cannot play a regulatory role in the subterranean areas, because darkness is also a prerequisite for the normal development of peanut pods.

### DISCUSSION

Early embryonic development is an important period for the formation of peanut yield and quality. Accumulating evidence illustrated that this complex process is regulated by light, endogenous hormones and environment stimuli. Ca2<sup>+</sup> deficiency in this process leads to early embryo abortion in peanut, resulting in unfilled pod and reduced peanut yield. This phenomenon is a long-term concern in the acidic red-yellow soil in southern China (Zhang et al., 2007). AhCYP707A4A has been identified to regulate embryo abortion induced by Ca2<sup>+</sup> deficiency using SSH cDNA libraries associated with library lift (SSHaLL) (Chen et al., 2015). However, SSHaLL is a less sensitive method that cannot detect the gene expression of whole species. Thus, this process provides limited information, and the underlying mechanism remains unclear.

De novo transcriptome sequencing facilitates the complete and rapid acquisition of almost all transcripts of a particular organ or tissue of a species based on high-throughput technologies. In this study, we characterized and compared gene expression profiles in gynophores and pods under free Ca2+-sufficient or free Ca2+-deficient treatment to identify candidate genes

related to Ca2<sup>+</sup> regulation on pod development. Our project was sequenced on the platform of Illumina Hiseq 2000. A total of 9,903,082,800 nt bases were generated. For function annotation analysis, we obtained 61,614, 62,460, 40,867, 38,079, 23,492, and 26,666 unigenes annotated to the NR, NT, Swiss-Prot, KEGG, COG, GO database, respectively (**Figure 1**). The total number of SSR was 18,215 (**Figure S4**). These unigenes and the reference transcripts of Arachis ipaensis can serve as reference sequences to determine the regulation pathway of calcium on peanut pod development.

In recent years, with widespread application of sequencing technology, transcriptome and proteomics provide views to explore the mechanism of the development of aerial peg and swelling of underground pod (Chen et al., 2013). The mechanisms of calcium on the aerial and underground part of peanut have not been reported yet. In our study, MADSbox family was significantly up-regulated in gynophores under calcium-deficient condition (**Table S4**). MADS box transcription factors are mainly known as key regulators of seed and flower development in Arabidopsis. Overexpression of OsMADS45 induces early flowering and premature senility of gynophores (Wang et al., 2013; Yu et al., 2017). Hence, calcium deficiency probably prevented the swelling of the gynophores to form a pod and resulted in abortion. LOXs, which are key genes that play important roles in the production of growth regulators and the mobilization of stored lipids during seed germination, exhibited differential transcription levels in GS and GD (Gao et al., 2011). The up-regulated expression of LOXs in GS is likely to provide sufficient nutrient preparation for pod development. Combined with the high expression of several energy- and substance-related transporter genes, which play critical roles in nutrient uptake and signal transduction (Leem et al., 2016), exogenous calcium probably promotes nutrient storage in the aerial gynophores, ascertaining the early development of pod.

Members of the Ca2<sup>+</sup> signaling pathway, including CaMbinding protein, CDPK, and MAPKKK, were found to be highly expressed in PS (**Table S5**). CaM has no inherent catalytic activity, and requires the modulation of its downstream binding proteins to function (Leba et al., 2012; Poovaiah et al., 2013). Thus, CaM has an effect on transmitting the calcium signals to downstream proteins and regulating plant growth and development. CDPKs, which are calcium sensors, are also implicated in the growth of plants, such as pollen development and gravitropism, and their response to environment stresses, such as pathogens (Ormancey et al., 2017). The MAPK cascade consists of interlinked MAPK, MAPKK, and MAPKKK and such cascades play important roles in signal transduction of plant hormone, biotic stress and abiotic stress (Wang et al., 2017). By contrast, in aerial gynophores, no significant difference was found in the Ca2<sup>+</sup> sensors between GD and GS, and only some CBPs and calcium-binding transcription factors were up-regulated in GS. It is probably interesting to investigate that calcium signal transduction pathway may participate in regulating the development of peanut pod.

Several studies have indicated that plant hormones, such as auxin, kinetin, ABA, and ethylene, can temporally and spatially coordinate the elongation of gynophore and swelling of pod (Jacobs, 1951; Ziv and Zamski, 1975; Shlamovitz et al., 1995). In this study, several enzymes in the synthesis pathway of GA were screened out to determine significant DEGs in pods between Ca2+-sufficient and Ca2+-deficient conditions,

which were not found in gynophores. Low expression of GA2 oxidase and high expression of ent-kaurenoic acid oxidase under sufficient exogenous calcium eventually led to high contents of GA (**Figure 6**). GA is reported to be required to enhance seed germination, and the increase in the synthesis of active GA contributes to the promotion of seed germination (Song et al., 2011). Therefore, calcium may promote peanut seed germination and development through GA biosynthesis pathway. This novel discovery has not been reported in previous studies. Auxin plays a critical role in plant organ development. Genes encoding auxin response factors (ARF) and indole-3-acetic acid-amido synthetase exhibited increased changes in the PS (**Table S5**). ARF is a transcription factor that activates or represses the expression of auxin response genes. In Arabidopsis, ARF5 can promote the development of cotyledon by recruiting DRN expression, and to a great extent, ABA function in seed germination depends on the TIR1/AFB-AUX/IAA-ARF–mediated auxin signaling pathway (Liu et al., 2013). Therefore, the mechanism by which auxinrelated genes identified in our study respond to calcium signaling during peanut seed development needs further research. The change in these hormones under sufficient calcium condition is beneficial for the normal development of peanut pod. Thus, the results elucidate a crucial mechanistic role of calcium on pod development. On the one hand, calcium enhanced the storage of aerial nutrients. On the other hand, it activated calcium signaling pathways and hormone-related genes in embryonic development (**Figure 7**). All these elements ensure the normal development of pods.

## CONCLUSION

In this study, we performed a comparative transcriptome and differential expression analysis of peanut gynophores and pods under free Ca2<sup>+</sup> sufficient and deficient conditions, respectively. Transcription factors including WRKY, NAC, bHLH, and MADS-box transcription factors were found up-regulated in GS, while MADS transcription factor family were down-regulated in GS compared to GD. Genes involved in plant hormone biosynthesis and transporters were found to be differentially expressed in the GS and GD. Different from the aerial part of peanut, genes involved in calcium signaling transduction pathways were identified up-regulated in pods when free Ca2<sup>+</sup> was sufficient. In addition, hormone and embryo-related genes also showed differentially expressed between PD and PS. To our best knowledge, we firstly provided mechanism of exogenous calcium on the aerial and underground parts of peanut at the early stage of seed development.

## AUTHOR CONTRIBUTIONS

SW and XL designed the experiment and drafted the manuscript. SY and LL carried out most of the experiment, analyzed the transcriptome and digital gene expression data, wrote the methods part of the manuscript. JZ, FG, and JM performed experiments, JW, NS, and YG grew the plants and prepared the samples. All authors read and approved the final manuscript.

### ACKNOWLEDGMENTS

This work was supported by Natural Science Foundation of Shandong Province (ZR2015YL077, BS2015SW020), Youth Scientific Research Foundation of Shandong Academy of Agricultural Sciences (2015YQN02, 2014QNM38, 2015YQN12), National Natural Science Foundation of China (31571581, 31571605), the Supporting Plan of National Science and Technology of China (2014BAD11B04), Major Projects of Science and Technology Innovation in Shandong Academy of Agricultural Sciences (2014CXZ06-6).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017. 01609/full#supplementary-material

Figure S1 | The length distribution of contigs and unigenes.

Figure S2 | GO classification analysis of unigenes in All-unigene. GO functions is showed in X-axis. The right Y-axis shows the number of genes which have the GO function, and the left Y-axis shows the percentage.

Figure S3 | Clusters of orthologous groups (COG) classification of Unigenes in All-Unigene.

### REFERENCES


Figure S4 | Quantity statistics of SSR classification. The X-axis is the repeat times of repeat units. The Y-axis is the number of SSRs.

Figure S5 | Sequence saturation analysis of eight samples. The X-axis shows the number of clean reads, units is 100 k, the Y-axis shows the number of genes, units is %.

Figure S6 | Scatter plot of top 20 KEGG pathways of two pairwise comparisons (GD/GS, PD/PS).

Table S1 | Summary for the transcriptome sequencing and assembly.

Table S2 | Summary of EST-SSR searching results.

Table S3 | Gene-specific primers used in quantitative real-time PCR.


at an early development stage causing early flowering in rice. Bot. Stud. 54:12. doi: 10.1186/1999-3110-54-12


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Yang, Li, Zhang, Geng, Guo, Wang, Meng, Sui, Wan and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# ERECTA Regulates Cell Elongation by Activating Auxin Biosynthesis in Arabidopsis thaliana

Xiaoya Qu, Zhong Zhao\* and Zhaoxia Tian\*

School of Life Sciences, University of Science and Technology of China, Hefei, China

The ERECTA family genes, ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERECTA-LIKE2 (ERL2), encode leucine-rich repeat receptor-like kinases in Arabidopsis thaliana. Knocking out these three genes can cause severe phenotypes, which indicates that they play significant roles in plant growth and development. However, the molecular mechanism within remains unclear. Here we show that the short hypocotyl phenotypes of er erl1 erl2 mutants are mainly due to the defects of cell elongation rather than the cell division. In contrast, in the ERECTA overexpression transgenic plants, the hypocotyl length is increased with elongated cells. Moreover, we show that the er erl1 erl2 triple mutant contains a low level of auxin, and the expression levels of the key auxin biosynthesis genes are significantly reduced. Consistent with this observation, increasing exogenous or endogenous auxin levels could partially rescue the cell elongation defects of the er erl1 erl2 triple mutant. Therefore, our results provide a molecular basis for auxin mediated ERECTA control of the hypocotyl length in Arabidopsis thaliana.

Lanzhou University, China

### Reviewed by:

Edited by: Kai He,

Anja Thoe Fuglsang, University of Copenhagen, Denmark Yuxin Hu, Institute of Botany (CAS), China

\*Correspondence:

Zhong Zhao zhzhao@ustc.edu.cn Zhaoxia Tian zxtian@ustc.edu.cn

### Specialty section:

This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science

Received: 27 July 2017 Accepted: 14 September 2017 Published: 27 September 2017

### Citation:

Qu X, Zhao Z and Tian Z (2017) ERECTA Regulates Cell Elongation by Activating Auxin Biosynthesis in Arabidopsis thaliana. Front. Plant Sci. 8:1688. doi: 10.3389/fpls.2017.01688 Keywords: Arabidopsis, ERECTA, auxin, hypocotyl length, cell elongation

## INTRODUCTION

Plant cell size is one of the most important features in plant morphology, which is controlled strictly by the inheritance and influenced by the external environment. The cell size directly correlated with cell division and cell elongation. Schleiden and Schwann (1838-1839) established the "cell theory," which regard that the cell division and cell elongation activities determine the growth and development of living organism. Cell elongation is very important for the process of plant differentiation and morphogenesis of plant organs. The growth of hypocotyls in Arabidopsis is the elongation and division of hypocotyl cells in essence. In particular, the cell elongation growth plays a decisive role in hypocotyl length (Stuart et al., 1977; Fenwick et al., 1999). The hypocotyl is an important structure connecting root, shoot tip and leaves, and also an important channel for transporting water, nutrients and signaling molecules in Arabidopsis. At the same time, hypocotyls are very sensitive to endogenous signal molecules and external environment signal (Gray et al., 1998; Nozue et al., 2007; de Lucas et al., 2008; Christie et al., 2011; Sun et al., 2012). The morphological structure of Arabidopsis thaliana hypocotyl is simple, consisting of more than 20 cells in longitudinal direction, which is below cotyledons to the above of radicle. Because most of these cells are formed in the embryonic stage, only a few are produced by cell division after germination, suggests that the hypocotyl growth is mainly caused by the cell elongation (Gendreau et al., 1997). Therefore, the hypocotyl of Arabidopsis has become an important model system for studying plant cell elongation due to its simple structure and important physiological functions.

Plant hormones are important regulatory signal molecules of hypocotyl elongation, and auxin is one of the most important types. For a single cell, the main physiological function of auxin is to regulate cell elongation, and the effect of auxin on acceleration of cell elongation is very rapid, and the lag time from treatment to effectiveness is only about 10 min. Previous studies have found that mutants with excessive synthesis of auxin in light exhibit a longer hypocotyl length, while mutants with deficient synthesis of auxin have shorter hypocotyl length (Romano et al., 1991; Jensen et al., 1998; Zhao et al., 2001; Cheng et al., 2006). This indicates that an appropriate concentration of auxin is essential for the growth and development of hypocotyls. It has now been shown that the effect of auxin on hypocotyl length is achieved by stimulating the degradation of IAA3, which in turn causes the ARF transcription factor to be released (Vernoux et al., 2011; Oh et al., 2014). ARF transcription factors, especially ARF6 and ARF8, combine the signals from endogenous development and exogenous environments to control the elongation of hypocotyl cells by activating the expression of cell elongation factors such as PRE, SAUR family genes (Bai et al., 2012; Oh et al., 2014; Challa et al., 2016).

The intercellular communication is very important for the formation of normal tissues, organs and organisms of plants. The cell surface receptor protein kinase located on the cytoplasmic membrane plays a vital role in the initiation of cell signal transmission. The ERECTA (ER) gene in Arabidopsis encodes a leucine-rich repeat receptor-like protein kinase, whose protein structure consists of a hydrophobic signal peptide in the extracellular domain, a leucine repeat sequence for protein interaction, a single transmembrane domain, and intracellular serine/threonine kinase domain (Torii et al., 1996). ERECTA is widely expressed in plant tissues and plays an important role in plant development and responses to external environmental signals (Yokoyama et al., 1998; Shpak, 2013; Jorda et al., 2016; Gao et al., 2017). ERECTA knockout mutants show obvious dysplasia phenotypes, for instance, stem node, siliques and petiole become short and inflorescences are densely clustered. Although Landsberg deficit in ERECTA gene is widely used as an Arabidopsis ecotype, there is no in-depth study on how ERECTA gene mutations have led to such a markedly intense phenotype. In addition, ERECTA, as a multi-functional major gene in the plant, has two homologous genes ERECTA LIKE 1 (ERL1) and ERECTA LIKE 2 (ERL2), both of which have partial functional redundancy with ERECTA. The triple mutant er erl1 erl2 intensifies ERECTA knockout mutant phenotype, and the plant is extremely dwarfed (Shpak et al., 2004). Previous studies have been shown that ERECTA, ERL1 and ERL2 play roles in the organ morphology and size determination in the reproductive stage of anthers and eggs by altering cell cycle progression (Shpak et al., 2004; Pillitteri et al., 2007). The mechanism of ERECTA gene family in cell-to-cell communication has been intensively studied in the stomatal development. ERECTA can recognize small peptide of the EPF/EPFL family, by forming a complex with TMM, affects stomatal development and differentiation by triggering MAPK cascade (Meng et al., 2012; Jewaria et al., 2013; Lin et al., 2017). However, the role of ERECTA family in the regulation hypocotyls remains unclear. Previous studies have shown that overexpression of YUCCA5, a member of YUCCA family of flavin monooxygenase in Arabidopsis auxin synthesis, inhibits mutant phenotype of er-103 (one of the ERECTA mutants) (Woodward et al., 2005). In addition, ERECTA family regulates the transport of auxin by regulating the expression of PIN1 at the initial stage of leaf primordia (Chen et al., 2013). However, the genetic interaction between the ERECTA family and auxin signaling in control of the hypocotyl development is still poorly understood.

In this study we found that, the hypocotyl lengths of single mutant er-105 and triple mutant er erl1 erl2 were significantly shorter than that of the wild type, whereas hypocotyls of ERoverexpressed plants was longer. We further showed that this change is due to a change in the length of a single cell in the hypocotyl rather than a change in the number of cells. In addition, we found that DR5::GFP signal of auxin reporter in the triple mutant er erl1 erl2 was significantly reduced than that of wild type. Moreover, the transcriptional levels of auxin early response gene and the major auxin synthetase genes were significantly repressed in the er erl1 erl2 triple mutant, suggesting that the auxin synthesis in the er erl1 erl2 triple mutant is sabotaged. Consistent with the observation, the exogenous auxin and endogenous auxin were able to rescue the shortened hypocotyl and cell length phenotype of the er erll erl2 triple mutant, indicating that these defects are due to the lack of auxin synthesis. Therefore we concluded that the ERECTA gene family controls the cell elongation in the hypocotyl by positively regulating the auxin biosynthesis in Arabidopsis thaliana.

### MATERIALS AND METHODS

### Plant Materials and Growth Conditions

All of the Arabidopsis lines used in this study were in the Col-0 background. The ER family mutants and overexpression plants have been described previously (Torii et al., 1996; Shpak et al., 2004; Shen et al., 2015). The seeds for er-105, er-/ erl1+/- erl2-/- mutant were kindly provided by Prof. Masao Tasaka (Nara Institute of Science and Technology). The seeds for 35S::ERECTA were kindly provided by Prof. Zuhua He (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). The seeds for DR5::GFP-NLS in wild type were kindly provided by Prof. Yuling Jiao (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). Other Arabidopsis seeds were obtained from self-preserved, genetic hybridization or agrobacterium-mediated transformation. All seeds were sterilized with 70% ethanol and 0.5% Tween for 10 min, followed by washing two times with 95% ethanol and air drying. After sterilization, seeds were sown onto 1/2 Murashige and Skoog (MS) medium containing 1% sucrose and 0.8% agar, then incubated at 4◦C in darkness for 2 days, finally grown vertically at 21◦C under long-day condition (16 h of light and 8 h of darkness).

### Plasmid Construction

For construction of pER::3 × VENUS-NLS, a 1.4-kb upstream sequence of ERECTA before ATG was used as a promoter. For the pERL1::3 × VENUS-NLS, a 4.1-kb upstream sequence of ERL1 before ATG was used as a promoter. For the pERL2::3 × VENUS-NLS, a 3.6-kb upstream sequence of ERL2 before ATG was used as a promoter. For construction of pER::ER-GFP, pERL1::ERL1- GFP and pERL2::ERL2-GFP, the genomic sequence of these three genes were amplified and used the same promoter we described above. The pER::ER-GFP construct was then transformed into the er-105 mutant to rescue its defects, while other constructs were transformed into the Col-0. For pER::iaaM, the 1.4-kb promoter of ERECTA was used to drive iaaM sequences that were obtained from Prof. Yunde Zhao (UC San Diego). This construct was then transformed into the er-/- erl1+/- erl2-/- mutant. The primer sequences used in the plasmid construction are listed in Supplementary Table S1.

### Gene Expression Analysis

fpls-08-01688 September 26, 2017 Time: 16:47 # 3

Whole seedlings were dissected and immediately transferred to liquid nitrogen. The Tripure Isolation Reagent (Roche) was used to isolate total RNA from the plant samples. The PrimeScriptTM RT Reagent Kit (TaKaRa) was used for cDNA synthesis. Quantitative PCR was performed with the Thermo PIKO REAL96 Real-Time PCR system using the GoTaq <sup>R</sup> qPCR Master Mix (Promega) with the following PCR conditions: 95◦C for 5 min and 40 cycles of 95◦C for 10 s, 57◦C for 30 s and 72◦C for 30 s, followed by 72◦C for 10 min and 20◦C for 10 s. TUBULIN was used to normalize the mRNA levels. Primers used for qRT-PCR are listed in Supplementary Table S1.

### Propidium Iodide Staining

Propidium Iodide (PI) powder (Sigma) was dissolved with PBS solution to 5 mg/ml as a stock solution, which was stored in dark. The working solution was diluted to 5 µg/ml. The whole seedling was put into the solution for staining 10 min, then washed with water three times and then observed under the confocal microscope (OLYMPUS LSM1200).

### Measurement

For the length of hypocotyls in the seedling stages, pictures were taken under the microscope; The Image J software was used for the measurement and statistical analysis. The cells in longitudinal direction from the top to the base of the hypocotyls epidermis were used for the measurement of each cell length, and 25 hypocotyls were used for biological repetitions, and the Image J software was used for the statistical analysis. The total cells number in the hypocotyls was counted directly under the microscope.

### Exogenous Chemical Substance Treatment

IAA and yucasin powder (Sigma) were dissolved in the DMSO and diluted to 50 µM for IAA and 5 mM for yucasin as the stock solutions. The stock solutions were diluted 1000x and added to the corresponding 1/2MS media. The control group was added with the same amount of DMSO in the media. The plants were grown vertically at 21◦C under long-day condition (16 h of light and 8 h of darkness).

## Statistical Analysis

Where appropriate, statistical analyses were performed with analysis of variance (ANOVA) test. Otherwise, comparisons between two groups were conducted using Student's t-test. The p-value level was set to 5%.

## RESULTS

### The Hypocotyl Length Is Shortened in the ERECTA Family Mutants

One of the most striking defects in the ERECTA family mutants (erf) mutants is the dwarf phenotype (Shpak et al., 2004). The er-105 single mutant showed reduced plant height comparing to that of wild type, whereas the plant height was increased in the ERECTA overexpressing plants (Supplementary Figure S1A) (Shen et al., 2015). In the er erl1 erl2 triple mutant, we observed even severe phenotypes in the reduction of the plant height (Supplementary Figure S1A), suggesting that ERECTA, ERL1 and ERL2 were functionally redundant in control of the plant height. To further support these observations, we measured the plant height of the 35S::ERECTA, er-105 and er erl1 erl2 plants. We observed a slight increase of the plant height in the 35S::ERECTA plants, however, a significant decrease in the er-105 and er erl1 erl2 mutants (Supplementary Figure S1B).

Consistent with the observation of plant height, in the 6-day-old seedling, we observed that the length of hypocotyl in the 35S::ERECTA plants was significantly increased comparing to that of wild type seedlings (**Figures 1A,B**). Conversely, the

hypocotyl lengths in the er-105 and er er1 erl2 mutants were dramatically reduced (**Figures 1A,B**). We conclude that the ERECTA gene family are involved in the regulation of hypocotyl length in Arabidopsis.

### Expression Patterns of ERECTA Gene Family in the Hypocotyl

Given the fact that disturbing the functions of ERECTA gene family causes the severe defects in the hypocotyl length, we assumed that ERECTA gene family are expressed in the hypocotyl cells. To test whether the ERECTA family genes are transcribed in the hypocotyl cells, we first performed the promoter activation analysis. To this end, we constructed pER::3 × VENUS-NLS, pERL1::3 × VENUS-NLS, pERL2::3 × VENUS-NLS transgenic plants. We observed evenly distributed fluorescence signals in the whole hypocotyl of these three transgenic plants (**Figures 2A–C**), suggesting that ERECTA, ERL1 and ERL2 genes are expressed in the whole hypocotyl of Arabidopsis.

Because ERECTA family genes encode leucine-rich repeat receptor-like kinases, we then test whether the ERECTA, ERL1 and ERL2 proteins are localized in the hypocotyl cells. We transformed the pER::ER-GFP construct into the er-105 mutant and observed a fully rescue of the mutant defects. By the confocal microscopy, we observed that ERECTA proteins were strongly accumulated in the hypocotyls (**Figure 2D**).

To check the protein localization of two homologous genes of the ERECTA, the pERL1::ERL1-GFP, pERL2::ERL2- GFP transgenic plants were also analyzed under the confocal microscope. Consistent with the observation of ERECTA proteins, we found that the both of proteins, ERL1 and ERL2, were also localized in hypocotyls (**Figures 2E,F**).

### The ERECTA Gene Family Controls the Cell Elongation of Hypocotyls

The organ size and shape of multicellular organism is directly related to the cells division and the size of cells. Therefore,

the individual cell length and the total number of cells could determine the hypocotyl length in Arabidopsis. Previous studies have shown that ERECTA family genes may regulate cell number in some organs, including stomata, ovule and stamen (Shpak et al., 2004; Woodward et al., 2005; Pillitteri et al., 2007; Hord et al., 2008). However, whether the ERECTA family genes control the cells division and cell elongation in the hypocotyl is still unclear. To test this, we performed Propidium Iodide (PI) staining on the hypocotyls of the wild-type plants, 35S::ERECTA plants, er-105 single mutant plants, and er erl1 erl2 triple mutant plants (**Figures 3A–D**). By the observation under the confocal microscope, we found that the individual cell size of hypocotyl in the 35S::ERECTA overexpression plants was large than that of wild type plants (**Figure 3B**). On the contrary, we observed size largely reduced cells in the hypocotyls of the er-105 single mutant and er erl1 erl2 triple mutant plants comparing to that of wild type plants (**Figures 3C,D**). By the quantitative analysis of the average cells length in the middle column of the hypocotyl epidermis, we found that the average cells length in the 35S::ERECTA overexpression plants was significantly increased, whereas it was remarkably decreased in the er-105 and er erl1 erl2 mutants (**Figure 3E**).

To test whether cells division is involved in the ERECTA family genes controlled hypocotyl elongation, we directly counted the total number of cells in the middle column of epidermis, and found that there were no significant difference in all the genotypes we test including the wild type, 35S::ERECTA, er-105 and er erl1 erl2 plants (**Figure 3F**). We concluded that the ERECTA gene family regulates the hypocotyl length by controlling the cell elongation rather than the cell division.

### Auxin Biosynthesis Is Down Regulated in the erf Mutants

Given the fact that the cell elongation was significantly reduced in the erf mutants, we then asked by what means does the ERECTA gene family regulate individual cell length in the hypocotyl? Previous studies have shown that the auxin plays an important role in regulation of cell elongation (Rayle and Cleland, 1992; Galinha et al., 2007). The endogenous auxin increased mutants exhibit hypocotyls-elongated phenotypes, whereas auxin deficient mutants have the reduced size of cells (Romano et al., 1991; Zhao et al., 2001; Cheng et al., 2006). To examine whether the auxin is involved in the ERECTA gene family mediated controls of cell elongation, two different auxin sensors, DR5::GFP and DII-VENUS, were applied to detect the endogenous auxin levels in the er erl1 erl2 mutants. The DR5::GFP is a positive auxin sensor by which GFP transcriptions are activated by auxin (Ulmasov et al., 1997; Benkova et al., 2003). Under the confocal microscope, we observed evenly distributed fluorescence signals of DR5::GFP in the entire hypocotyl (**Figure 4A**), suggesting that the auxin was distributed in all the hypocotyl of Arabidopsis. However, we can barely detect the fluorescence signals in the er erl1 erl2 mutant hypocotyl (**Figure 4B**), suggesting that the endogenous auxin levels were dramatically reduced in the triple mutant hypocotyl. To confirm this observation, the transgenic plants

that harbored the negative auxin sensor DII-VENUS by which fluorescence proteins were degradated by auxin (Brunoud et al., 2012) were further analyzed. Consistent with the observation of DR5::GFP, there were only extremely weak signals can be detected in the wild type hypocotyl (**Figure 4C**), but stronger DII-VENUS signals in the er erl1 erl2 triple mutants (**Figure 4D**). At the molecular level, we tested the transcriptional levels of auxin early response gene IAA19, which is commonly used to monitor the endogenous levels of auxin in Arabidopsis. We observed that the expression levels of IAA19 in the 35S::ERECTA transgenic plants were up regulated, whereas significantly down regulated in the er-105 and er erl1 erl2 triple mutants (**Figure 4E**). These data suggest that the ERECTA gene family is essential to maintain the endogenous auxin levels in the hypocotyl.

To shed light on the molecular mechanism underlying the ability of the ERECTA gene family controlling auxin

(E),

<sup>∗</sup>P < 0.05.

levels in the hypocotyl, we examined the expression levels of the YUCCA family genes, which encodes the major auxin biosynthesis genes (Cheng et al., 2006). We observed that the transcriptional levels of YUC2, YUC3 and YUC5 were significantly increased in the ERECTA overexpression plants compared with wild type (**Figure 4F**). Conversely, these three key auxin biosynthesis genes were markedly decreased in the er-105 and er erl1 erl2 triple mutants; especially the expression levels of YUC3 were drastically reduced in the er erl1 erl2 triple mutants (**Figure 4F**). We draw the conclusion that the ERECTA gene family positively regulates auxin biosynthesis via activating the expressions of YUC2, YUC3 and YUC5 genes.

### Exogenous Auxin Rescues the Cells Elongation Defects in erf Mutants

Given the fact that the erf mutants contain a very low amount of auxin and the key auxin biosynthesis genes are under the positive control of ERECTA gene family, we hypothesized exogenous increase of auxin might rescue the defects of cell elongation and short hypocotyl phenotypes of the erf mutants. While,

previous studies have shown that the high concentrations of auxin might inhibit the elongation of hypocotyls in wild-type Arabidopsis (Collett et al., 2000; Rashotte et al., 2003), which was also found in our experiments (data not shown). To exclude the possibility the high auxin inhibits the cell elongation in our test, we carefully select a very low concentration of IAA (50 nM), and performed our analysis. By applying this low concentration of IAA to the wild type seedlings, we did not observe any significant differences in term of the hypocotyl length comparing with the mock treatments (**Figures 5A,B**). However, this low concentration of IAA almost fully rescued the hypocotylshortened phenotypes of the er-105 single mutants and er erl1 erl2 triple mutants (**Figure 5A**). By quantifying the hypocotyl length, we observed significant increases of the hypocotyl length in the er-105 and er erl1 erl2 mutants upon the auxin treatments, which showed no differences to that of wild type seedlings (**Figure 5B**), suggesting that the hypocotyl-shortened defects in the erf mutants are due to the lacking of auxin, and the increase of exogenous auxin completely restores the mutant phenotypes. To further confirm this observation, we examine the cell elongation in the erf mutants after the auxin treatments. In the wild type hypocotyl, the 50 nM auxin treatments did not change the cell length comparing with the mock treatments (**Figures 6A,B**). However, in the er-105 and er erl1 erl2 mutants, the same amount of auxin effectively promoted the cell length to that of mock treatments (**Figure 6A**), and the cell length was significantly increased to the levels that comparable with the wild type seedlings (**Figure 6B**). To test the possibility whether the cell division was also involved in the rescue of erf mutants defects during the low auxin treatments, we directly counted the total number of cells in the middle column of epidermis with or without the low auxin treatments. We observed no significant difference among all the genotypes after the auxin treatments (Supplementary Figure S2). Therefore, we conclude that the hypocotyl-shortened phenotypes in the erf mutants are due to the cell elongation defects, which can be fully rescued by the treatments of exogenous auxin.

### The ERECTA Gene Family Mediated Endogenous Auxin Biosynthesis Controls the Cell Elongation in Hypocotyls

Given the fact that the ERECTA gene family positively regulates auxin biosynthesis in the hypocotyl by activating the expressions

of several YUCCA family genes, and the overexpression of ERECTA causes the elevated expressions of YUCs (**Figure 4F**), we then tested this interaction genetically. We treated the 35S::ERECTA seedlings with the chemical yucasin, which have been shown to reduce the exogenous auxin levels by inhibiting the expression of YUCCA genes (Nishimura et al., 2014). We carefully select a low concentration of yucasin (5 µM) to perform the analysis, which showed no effect on the hypocotyl length in the wild type seedlings (Supplementary Figures S3A,B). While in the 35S::ERECTA seedlings, the hypocotyl length was significantly decreased (Supplementary Figures S3A,B). Likely, the hypocotyl cell length of the wild type plants did not respond the low yucasin treatments. However, the cell length of the 35S::ERECTA seedlings with increased YUCCA expressions, reduced dramatically upon the low yucasin treatments (Supplementary Figure S4), demonstrating that the ERECTA gene family controls the cell elongation in hypocotyls by positively regulating the auxin biosynthesis.

The bacterial auxin biosynthesis gene iaaM, which was firstly identified in the T-DNA of agrobacterium, is commonly used in plants to increase the endogenous auxin levels (Comai and Kosuge, 1982). To further investigate the mechanism by which the ERECTA gene family mediated auxin synthesis controls the cell elongation in hypocotyls, we expressed the iaaM gene under the control of the ERECTA promoter, and transformed the pER:iaaM construct into the er−/− erl1+/− erl2−/− mutants. By analyzing the expression levels of the iaaM and the IAA19 genes in eight independent transgenic lines, we observed a correlation between these two genes, suggesting that the ectopically expressed iaaM increased the endogenous auxin levels in the er erl1 erl2 triple mutant background (Supplementary Figure S5A). We measured the hypocotyl length of eight independent transgenic lines and selected three independent T1 transgenic lines with high expression of iaaM for the further analysis. (Supplementary Figure S5B), and observed that hypocotyls and individual cell lengths of pER:iaaM transgenic plants were higher than those of er erl1 erl2 triple mutants (**Figures 7A–D**). Thus we conclude that the ERECTA gene family mediated endogenous auxin biosynthesis controls the cell elongation in hypocotyls.

background (L2, L4 and L6). (B–D) PI staining of hypocotyls in the wild type (B), the er erl1 erl2 mutant (C) and three independent pER:iaaM transgenic lines in the er erl1 erl2 mutant background (D). The yellow line represents the length of a single cell. Scale bar in (A), 1 mm; scale bars in (B–D), 50 µm.

### DISCUSSION

The ERECTA family genes, which encode leucine-rich repeat receptor-like kinases, have been shown to regulate multiple developmental processes including stomatal formation, inflorescence architecture, and ovule development (Shpak et al., 2004, 2005; Lee et al., 2012). Woodward et al have previously shown that a dominant mutant, super1-D, which causes the overexpression of one of the YUCCA family genes YUC5, is epistatic to er mutants (Woodward et al., 2005). However, little is known about the genetic interaction between the ERECTA and auxin signaling in control of the hypocotyl development. In this study, we analyzed the endogenous auxin levels in the erf mutants by using two different auxin sensors, DR5::GFP and DII-VENUS. Our results show that the endogenous auxin levels were dramatically reduced in the hypocotyl of the er erl1 erl2 triple mutant (**Figures 2A–D**). To further support this observation, we examined the expression levels of IAA19 in the hypocotyl. We found that the IAA19 transcripts are significantly reduced in the er-105 and er erl1 erl2 mutants, and increased in the 35S::ERECTA transgenic plants (**Figure 4E**). To further investigate the mechanism by which the ERECTA family genes regulate auxin levels in the hypocotyl, we examined the key auxin biosynthesis genes. We observed that the transcriptional levels of YUC2, YUC3 and YUC5 were significantly decreased in the er-105 and er erl1 erl2 triple mutants, whereas increased in the 35S::ERECTA transgenic plants (**Figure 4F**). Our data suggest that the ERECTA gene family positively regulates auxin biosynthesis by activating the key auxin biosynthesis genes.

Previous study has shown that the overexpression of YUC5 in the wild type, er-103 and er-105 cause the same extent of increase of hypocotyls regardless of their genotypes (Woodward et al., 2005), which raises the question whether the ERECTA family genes regulate the cell elongation in the hypocotyl via auxin biosynthesis. By careful selection of a very low concentration, 50 nM of IAA fully rescued the short hypocotyl and cell elongation defects in the er-105 and er erl1 erl2 mutants, but have no significant effects in the wild type (**Figures 5**, **6**), suggesting that these defects of the erf mutants are caused by the auxin deficiency in the hypocotyl. Further support for this idea came from the observation that the elongated hypocotyl cells in the 35S::ERECTA seedlings are completely suppressed by using the 5 µM yucasin treatments to inhibit YUCCA genes (Supplementary Figure S4). Our data demonstrate that the ERECTA gene family controls cell elongation by positively regulating auxin biosynthesis. Likely, during leaf margin morphogenesis, the ligand-receptor pair of EPFL2-ER has been shown to be essential for maintaining an appropriate auxin concentration (Tameshige et al., 2016).

The small-secreted peptide PSY1, together with its LRR receptor, positively regulates cell elongation in hypocotyls by triggering a signaling cascade (Mahmood et al., 2014), suggesting a crucial role of peptide ligand-receptor pair in the hypocotyl regulation. ERECTA with its ligands EPF/EPFL peptides family, participate multiple functions during plant development, for example, EPF1, EPF2 and EPFL9 in stomatal regulation, EPFL4, EPFL5 and EPFL6 in inflorescence development and EPFL2 in tooth development (Abrash et al., 2011; Uchida and Tasaka, 2013; Tameshige et al., 2016; Lin et al., 2017). Moreover, in the secondary growth of Arabidopsis hypocotyls, ERECTA and ERL1 are very important in the xylem and phloem to prevent premature during sequential events in the secondary growth, suggesting that ERECTA family plays an important role in the morphogenesis of hypocotyls (Ikematsu et al., 2017). Therefore, we speculate that ERECTA family might regulate the cell elongation in hypocotyls with some members of the EPF/EPFL family or other ligands.

During the reproductive stage, the short internodes and pediceels phenotypes of the erf mutants in the inflorescences were mainly due to reduced cell proliferation (Shpak et al., 2003, 2004; Woodward et al., 2005). However, in the hypocotyl, mutations of the ERECTA gene family result in the defects specifically in the cell elongation rather than the cell division (**Figure 3**). This idea is further support by the early observation that, in Arabidopsis, hypocotyl elongation is mainly due to the cell elongation, while the number of cells is rather stable (Gendreau et al., 1997). These data suggest that the functions of ERECTA gene family in controlling of cell proliferation and cell elongation is highly tissue-specific, however, it remains to be shown how the functions of ERECTA gene family are specified in different tissues to fulfill their biological functions.

### AUTHOR CONTRIBUTIONS

fpls-08-01688 September 26, 2017 Time: 16:47 # 10

ZT, XQ, and ZZ designed the experiments, analyzed the data and wrote the paper. XQ performed the experiments.

### FUNDING

This work was supported by the grant to ZT from the National Natural Science Foundation of China (31300248), and the grant to ZZ from the Ministry of Science and Technology of China (2013CB967300).

### REFERENCES


### ACKNOWLEDGMENT

We thank Prof. Masao Tasaka, Prof. Zuhua He, Prof. Yuling Jiao, and Prof. Yunde Zhao for sharing seeds or plasmid.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.01688/ full#supplementary-material


protein kinase with extracellular leucine-rich repeats. Plant Cell 8, 735–746. doi: 10.1105/tpc.8.4.735


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Qu, Zhao and Tian. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fpls-08-01688 September 26, 2017 Time: 16:47 # 11

# Transcriptome Analysis of Calciumand Hormone-Related Gene Expressions during Different Stages of Peanut Pod Development

Yan Li<sup>1</sup> , Jingjing Meng<sup>1</sup> , Sha Yang<sup>1</sup> , Feng Guo<sup>1</sup> , Jialei Zhang<sup>1</sup> , Yun Geng<sup>1</sup> , Li Cui<sup>1</sup> , Shubo Wan<sup>2</sup> \* and Xinguo Li<sup>1</sup> \*

<sup>1</sup> Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan, China, <sup>2</sup> Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Shandong Academy of Agricultural Sciences, Jinan, China

Edited by: Yi Ma,

University of Connecticut, United States

#### Reviewed by:

Mei Yuan, Shandong Peanut Research institute, China Kunling Chen, Institute of Genetics and Developmental Biology (CAS), China Jianwei Gao, Institute of Vegetables and Flowers, Shandong Academy of Agricultural Sciences, China

#### \*Correspondence:

Shubo Wan wansb@saas.ac.cn Xinguo Li xinguol@163.com

#### Specialty section:

This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science

Received: 21 March 2017 Accepted: 30 June 2017 Published: 14 July 2017

#### Citation:

Li Y, Meng J, Yang S, Guo F, Zhang J, Geng Y, Cui L, Wan S and Li X (2017) Transcriptome Analysis of Calcium- and Hormone-Related Gene Expressions during Different Stages of Peanut Pod Development. Front. Plant Sci. 8:1241. doi: 10.3389/fpls.2017.01241 Peanut is one of the calciphilous plants. Calcium serves as a ubiquitous central hub in a large number of signaling pathways. In the field, free calcium ion (Ca2+)-deficient soil can result in unfilled pods. Four pod stages were analyzed to determine the relationship between Ca2<sup>+</sup> excretion and pod development. Peanut shells showed Ca2<sup>+</sup> excretion at all four stages; however, both the embryo of Stage 4 (S4) and the red skin of Stage 3 (S3) showed Ca2<sup>+</sup> absorbance. These results showed that embryo and red skin of peanut need Ca2<sup>+</sup> during development. In order to survey the relationship among calcium, hormone and seed development from gene perspective, we further analyzed the seed transcriptome at Stage 2 (S2), S3, and S4. About 70 million high quality clean reads were generated, which were assembled into 58,147 unigenes. By comparing these three stages, total 4,457 differentially expressed genes were identified. In these genes, 53 Ca2<sup>+</sup> related genes, 40 auxin related genes, 15 gibberellin genes, 20 ethylene related genes, 2 abscisic acid related genes, and 7 cytokinin related genes were identified. Additionally, a part of them were validated by qRT-PCR. Most of their expressions changed during the pod development. Since some reports showed that Ca2<sup>+</sup> signal transduction pathway is involved in hormone regulation pathway, these results implied that peanut seed development might be regulated by the collaboration of Ca2<sup>+</sup> signal transduction pathway and hormone regulation pathway.

Keywords: peanut, calcium, hormone, pod development, transcriptome

### INTRODUCTION

Peanut (Arachis hypogaea L.) is an important crop member of the legume family and a major source of plant oil, proteins, essential vitamins and minerals that can be used for human consumption, animal feed, bioenergy, and health products (Higgs, 2002; Li et al., 2010, 2011). Seed formation of peanut is a central stage of pod development. Some reports illustrated that seed development depends on the highly coordination between endogenous signal and environment stimuli (Sun, 2008). For instance, several plant hormones have long been known to play a significant role in peanut gynophore elongation and embryo differentiation, such as auxin (Jacobs, 1951; Moctezuma and Feldman, 1996), the ration of NAA and kinetin (Ziv and Zamski, 1975),

**161**

(dry soil). (A) Applied

ABA (Ziv and Kahana, 1988), ethylene (Shlamovitz et al., 1995), and so on. In addition, peanut needs more calcium relative to other plants. Free Ca2<sup>+</sup> concentration in soil serious affects peanut fruiting and yield (Cox et al., 1982). During pod development, more than 90% calcium was directly absorbed from the soil by pod (Beringer and Taha, 1976). For plants, Ca2<sup>+</sup> not only play roles as a nutrient element, but also as a second messenger in the regulation of diverse metabolic processes (Poovaih and Ready, 1993). As to the possible relationship between calcium signal transduction and plant hormones, Yang et al. (2015) reported that the expressions of most calmodulin-binding transcription activators of M. Truncatula (MtCAMTAs) were responsive to the four hormones, including IAA, salicylic acid (SA), jasmonic acid (JA), and ABA, which play critical roles in the regulation of nodule organogenesis in legumes. Additionally, it seems that the ripening of strawberry fruit are co-regulated by phytohormone and calcium signal transduction (Chen et al., 2016). Recent years, although there is a comprehensive understanding of calcium physiology related to peanut abiotic stress resistance, it is of vitally important to isolate and characterize more candidate genes for understanding some mechanisms regulating peanut pod development, especially Ca2<sup>+</sup> and hormone regulating pathway.

. (B) Without CaO application.

With the development of molecular biological techniques, the genomic research has been conducted and made a considerable progress in model legume, e.g., soybean (Severin et al., 2010; Wilson and Grant, 2010; Woody et al., 2011) and Medicago (Cannon et al., 2005), but relatively less progress in peanut (Pandey et al., 2012; Zhang et al., 2012). The advent of rapid and high-throughput technology for quantification of the transcriptome (Malone and Oliver, 2011) benefited the peanut genomics research, and was used in the seed development and tissue expression of peanut (Zhang et al., 2012; Chen et al., 2013; Wang et al., 2013; Zhu et al., 2014). These researches make it probability to study the relationship of between calcium and peanut pod development by transcriptome method, and explore valuably candidate genes. In the present study, to better understand the roles of Ca2<sup>+</sup> in pod development, we compared the transcriptome profile of peanut pod at different developmental stages, through which some potential candidate genes was identified to be related to calcium and hormone.

### MATERIALS AND METHODS

### Plant Materials and Treatments

Peanut cultivar, 'Huayu 22,' was provided by Biotechnology Research Center, Shandong Academy of Agricultural Science (SAAS, China). Free Ca2<sup>+</sup> content in soil is 14 g (free Ca2+) kg−<sup>1</sup> (dry soil). The pods were collected at the 1st (Stage 1, abbr. S1), 5th (Stage 2, abbr. S2), 10th (Stage 3, abbr. S3), 20th (Stage 4, abbr. S4) day after the peg elongation into the soil. Four stage materials were used for Ca2<sup>+</sup> excretion determination. The materials of S2, S3, and S4 were collected and immediately frozen in liquid nitrogen, and then

120 kg CaO ha−<sup>1</sup>

fpls-08-01241 July 14, 2017 Time: 9:37 # 2

stored in a freezer at −80◦C for comparative transcriptome analysis.

### Calcium oxide (CaO) Application in Soil

Field experiments was conducted to observe effects of free Ca2<sup>+</sup> in soil on pod development in Sanzhuang, Rizhao, Shandong Province in spring 2014 and 2015. Free Ca2<sup>+</sup> content in soil is 4 g (free Ca2+) kg−<sup>1</sup> (dry soil). The 120 kg CaO ha−<sup>1</sup> was applied in soil as Ca2<sup>+</sup> treatment, and non-CaO treatment was as control. Each treatment had three replicates. CaO was applied before sowing.

### Ca2<sup>+</sup> Excretion Measured by Non-invasive Micro-test Technology (NMT)

Ca2<sup>+</sup> excretion in pods of peanut were measured with NMT system (NMT100 Series, YoungerUSA LLC, Amherst, MA, United States; Xuyue (Beijing) Science and Technology Co., Ltd., Beijing, China) and iFluxes/imFluxes 1.0 (YoungerUSA, LLC, Amherst, MA, United States) Software. Steady-state ion fluxes were measured for 5–20 min. After that, the test treatment was applied and the ion flux in the meristematic zone (120 µm from the root/pod tip) was measured for further 10 min and recorded the data (Yue et al., 2012). And the roots and pods were treated with 40 µm Ca2<sup>+</sup> solution. Each treatment was repeated at least three times and all tests were repeated at least six times.

### Total RNA Isolation and mRNAs Purification

Total RNA was isolated and integrity confirmed using a 2100 Bioanalyzer (Agilent Technologies). Beads with oligo(dT) were used to isolate poly(A) mRNA from total RNA (Qiagen GmbH, Hilden, Germany).

### Synthesis of cDNA and Sequencing

Following purification, the mRNA was fragmented using divalent cations under elevated temperature. Taking these short fragments as templates, the first-strand cDNA was synthesized using random hexamer primers and SuperscriptTM III (InvitrogenTM, Carlsbad, CA, United States). The second strand cDNA was synthesized using buffer, dNTPs, RNaseH, and DNA polymerase I. Short fragments were purified with a QiaQuick PCR extraction kit (Qiagen) and resolved with EB buffer for end reparation and poly(A) addition. The short fragments were then connected using sequencing adapters. After agarose gel electrophoresis, suitable fragments were used as templates for PCR amplification. Finally, the library was sequenced from both directions on HiSeq 2000 System (illumina, San Diego, CA, United States) with 100 bp of data collected per run by applying TruSeq PE Cluster and TruSeq SBS Kits (illumina, San Diego, CA, United States). Data analysis and base calling were achieved by applying the illumina instrument software.

### Transcriptome De Novo Assembly

The reference transcripts of a progenitor of cultivated peanut (Arachis ipaensis) were used to generate an integrated reference library. The processing and assembly of transcriptome data were performed with the application of modified Velvet to construct unique consensus sequences (Zerbino and Birney, 2008). The gene expression profile was developed by mapping trimmed transcriptome reads onto the unique consensus sequences by using SOAP2 (Li et al., 2009).

### Annotation and Classification of Unigenes

Comparative transcriptomics analysis was conducted among the three different developmental stages. The intensity values of each sample were further transformed on log2-scale and used for differential expression analysis. The probe sets with a P-value < 0.01 and > two-fold changes in at least one of the comparisons were considered as differentially expressed genes (DEGs) for further analysis. Unigenes were used for BLAST searches and annotation against an NCBI Nr protein database (NCBI non-redundant sequence database) by using an E-value cut-off of 10−<sup>5</sup> (E-value < 0.00001). Unigenes sequences were further aligned by BLASTX to protein databases such as Swiss-Prot, KEGG, and COG, retrieving proteins with the highest sequence similarity with the given unigenes along with their protein functional annotations. If results of different databases conflicted, a priority order of Nr, Swiss-Prot, KEGG, and COG was followed. For unigenes that did not align to any of the above databases, EST Scan software (Iseli et al., 1999) was used to predict their coding regions and determine sequence

direction. Unigenes aligned to databases with higher priority did not enter the next circle. The alignments were considered complete when all circles were finished. The coding region sequences were then determined for proteins with the highest ranks using BLAST. Unigenes that could not be aligned to any database were scanned by EST Scan (Iseli et al., 1999) to determine the nucleotide (50–30) and amino acid sequences of the coding regions. The Blast2 GO was used to obtain Gene Ontology (GO) annotations for the unigenes, as well as for KEGG and COG analysis (Conesa et al., 2005). The WEGO software was then used to perform GO functional classification of all unigenes to view the distribution of gene functions of the species at the macro level (Ye et al., 2006). It was mapped all of the annotated unigenes to GO terms in the database and calculated the number of unigenes associated with every term.

## Real-Time Fluorescent Quantitative PCR Analysis

Total RNA was isolated and purified using TRIzol Reagent (Tiangen, China) according to the manufacture's instruction. The first-strand cDNA was synthesized by using Reverse Transcriptase M-MLV (Takara, Japan) according to the manufacturer's protocol. The PCR was amplified following the instruction of SYBR Premix Ex TaqTM (Takara, Japan) with the fluorescent quantitative PCR amplification instrument (ABI 7500, United States). The target gene primers were used to detect the sample mRNA. Control reactions were carried out by using primers Tua5-F and Tua5-R to detect the transcript encoding ubiquitously expressed tua5 (Chi et al., 2012). All assays for a target gene were performed in triplicate synchronously under identical conditions.

### RESULTS

### Effects of Calcium on Peanut Pod Development and Ca2<sup>+</sup> Excretion of Different Pod Tissues

Peanut is one of the important oil crops for China, and its yield is often affected by all kinds of environmental factors, including free Ca2<sup>+</sup> content in soil. When free Ca2<sup>+</sup> is deficient in soil, it will cause unfilling of pods. **Figure 1** showed that pods were unfilled under 4 g (free Ca2+) kg−<sup>1</sup> (dry soil), and CaO application can benefit pod filling.

To assess the role of Ca2<sup>+</sup> on pod development, hair root and four stages of pods were analyzed to determine Ca2<sup>+</sup> excretion where: the peg just entered the soil (S1), tip of the peg initiated expansion (S2), beginning of pod reticulation (S3), and when the pod was clearly reticulated (S4), respectively, used to analyze (**Figure 2A**). The non-invasive microtest technique was used to detect net fluxes of Ca2<sup>+</sup> at different seed tissues of different stages. Besides hair roots, peanut shells showed Ca2<sup>+</sup> excretion at all four stages; however, both the embryo of S4 and the red skin of S3 and S4 showed Ca2<sup>+</sup> absorbance. These results showed that embryo alone and embryo with red skin of peanut need Ca2<sup>+</sup> during development (**Figure 2B**).

### Assembly and Functional Annotations of Peanut Seed Transcriptomes

To further detect some mechanisms relate to the roles of Ca2<sup>+</sup> in pod development. About 70 million high quality reads were obtained from the library. The average read size, Q30 percentage (sequencing error rate, 0.1%), and GC percentage for each library was 90 bp, >95%, and >40%, respectively. Clean reads from each library were used for assembly separately. And 263,110 unigenes were generated from library. The de novo assembly of all sequencing data using the modified Velvet was followed by the application of the SOAP2 program for unigene identification. The average sequence length of the unigenes was 1,235 bp and its range was 201–4,000 bp (**Figure 3A**).

The GO assignments were used to categorize the functions of the predicted peanut Unigenes. Based on the sequence homologies, the sequences were categorized into 45 functional groups (**Figure 3B**), contained three main categories of the GO classification (biological processes, cellular components, and molecular functions). The genes involved in cellular process and metabolic process were dominant in the "Biological Process" category. "Cell," "Cell part," and the "Organelle" are the top three abundant categories in "Cellular component," While "Binding," and "catalytic activity" are dominant in the "Molecular function" category (**Figure 3B**).

### Comparative Analysis of Three Pod Developmental Stages

To investigate expression levels of stage-specific genes during pods development, we conducted comparative analysis of the transcriptome profiles among three pod development stages at 5th (S2), 10th (S3), 20th (S4) day after the peg elongation into the soil. As shown in **Figure 4A**, totally 1,169 DEGs were obtained between S2 and S3, including 50 up-regulated and 1,119 down-regulated genes. Similarly, 1,172 DEGs (including 53 up-regulated and 1,119 downregulated genes), 7 DEGs (including two up-regulated and five down-regulated genes) were identified between S3 and S4, S2 and S4, respectively. In addition, some other stagespecific expression genes were also analyzed. Approximately 39, 2157, and 1 stage-specific expression genes were identified between S2 and S3, S3 and S4, S2 and S4, respectively (**Figure 4B**).

### Calcium and Ca2<sup>+</sup> Signal Related Unigenes and Their Functional Categories

Since deficient free Ca2<sup>+</sup> in soil can cause unfilling of pods, some DEGs involved in biological process of Ca2<sup>+</sup> binding and Ca2<sup>+</sup>

TABLE 1 | The annotation of candidate genes related to calcium during pods development.


TABLE 2 | The annotation of candidate genes related to hormone during pods development.


(Continued)

### TABLE 2 | Continued

fpls-08-01241 July 14, 2017 Time: 9:37 # 8


signaling were identified based on GO analysis. These potential candidate genes contained 53 Ca2<sup>+</sup> or Ca2<sup>+</sup> signal pathway related genes (**Table 1**). These genes were significant differentially expressed during seed and pod development, suggesting that Ca2<sup>+</sup> and calcium related protein might be involved in seed development.

### Hormone Related Unigenes and Their Functional Categories

Genes related to hormone response were screened out by GO analysis. There were 40 auxin related genes, 15 gibberellin related genes, 20 ethylene related genes, 2 abscisic acid related genes, and 7 cytokinin related genes (**Table 2**) showed significant differentially expression during seed and pod development.

### Real-Time Fluorescent Quantitative PCR Analysis

In order to confirm the transcriptome sequencing results, Ca2<sup>+</sup> and hormone related genes were selected from DEGs based on the GO analysis and used to real-time fluorescent quantitative PCR analysis. As shown in **Figure 5**, the expression pattern of 8 selected DEGs, which are related to Ca2<sup>+</sup> signal pathway, were consistent with their respective microarrays data. Among these genes, two genes, which includes one CDPK gene (comp33153) and one uncharacterized calciumbinding protein At1g02270 (comp46176), were up-regulated at S4. The Serine/threonine-protein kinase SRK2E (comp45239) was up-regulated at S2, down-regulated at S3, and then upregulated at S4. Three probable calmodulin like protein CMLs (comp33933, comp43665, and comp50658), one calcineurin

B-like protein (comp47042) and one calcium-binding protein PBP1 (comp43681) were up-regulated at S2 and then downregulated during the later stages.

Among the 12 selected auxin related genes, six genes, i.e., auxin-induced protein 5NG4 (comp43478), auxinresponsive protein IAA31 (comp35718), auxin-induced in root cultures protein 12 (comp50484), auxin-induced protein 15A (comp35357), auxin-induced protein 10A5 (comp36019), and auxin transporter-like protein 3 (comp36785), were downregulated in S2 and then up-regulated during the later stages. Three auxin response factor 8 (comp52994, comp45289, and comp46658), one auxin-responsive protein IAA26 (comp36524) were down-regulated at S4. One auxin-induced protein 22E (comp45084) was down-regulated at S2, up-regulated at S3, and then down-regulated at S4 (**Figure 6**).

Among 11 selected gibberellin related genes, four genes, i.e., two gibberellin-regulated protein (comp41469, comp27867), two gibberellin 2-beta-dioxygenase (comp45036, comp43715), were down-regulated at S2 and then up-regulated at S4. One transcription factor MYB44 (comp40799), one gibberellin 20 oxidase (comp44953) and one gibberellin 3-beta-dioxygenase (comp44039) were up-regulated at S4. One gibberellin 20 oxidases (comp53643), one gibberellin 2-beta-dioxygenase (comp47582), one gibberellin 3-beta-dioxygenase (comp45509) and one gibberellin-regulated protein (comp13386) were upregulated at S2, down-regulated at S3 and then up-regulated at S4 (**Figure 7**).

Seventeen selected ethylene related genes were analyzed. Two ethylene-responsive transcription factor RAP2 (comp53691 and comp42029), one ethylene-responsive transcription factor WRI1 (comp48452), three ethylene-responsive transcription factor ERF (comp34495, comp37722, and comp11865) and one AP2-like ethylene-responsive transcription factor AIL (comp47962) were down-regulated at S2 then up-regulated at S4. One ethylene-responsive transcription factor ERF (comp35300), one ethylene-responsive transcription factor (comp44242), two ethylene-responsive transcription factor CRF (comp47316 and comp43675) and one AP2-like ethylene-responsive transcription factor ANT (comp43442) were only down-regulated at S4. The AP2-like ethylene-responsive transcription factor TOE (comp47833) and ethylene-responsive transcription factor ERF (comp45116) were up-regulated at S4. AP2-like ethyleneresponsive transcription factor At2g41710 (comp42987) and AP2-like ethylene-responsive transcription factor AIL1 (comp48832) were down-regulated at S2, up-regulated at S3, and then down-regulated at S4. The ethylene-responsive transcription factor 13 (comp37530) was up-regulated at S2 and S3, then down-regulated at S4 (**Figure 8**).

Among the four cytokinin related genes, cytokinin dehydrogenase (comp42347) was only up regulated at S4, cytokinin hydroxylase (comp44683) was down-regulated at S2 then up-regulated in later stage, cytokinin riboside 5<sup>0</sup> monophosphate phosphoribohydrolase LOG3 (comp53620) was down-regulated from S3 and the Cytokinin riboside 5<sup>0</sup> monophosphate phosphoribohydrolase LOG8 (comp38806) up-regulated from S3 (**Figure 9A**).

For the two abscisic acid related genes, the abscisic acid 8<sup>0</sup> hydroxylase 3 (comp29840) was up-regulated at S3 and then

down-regulated, however, the abscisic acid insensitive 5-like protein 1 (comp38442) was down-regulated at S2 (**Figure 9B**).

Calcium is not only an essential macroelement for plant growth, but also a second messenger involved in regulating diverse physiological processes (Poovaih and Ready, 1993). Peanut remains one of the most important oil-crops in the world, and Calcium is by far the most critical nutrient for this crop to achieve (**Figure 1**), and the pod yield would decreased by 20% or more. For pods, it seems that embryo alone or embryo with red skin are the main tissues need more Ca2<sup>+</sup> (**Figure 2B**). It attracts us to pay more attention to investigate the mechanisms of how Ca2<sup>+</sup> is involved in pod development.

As a powerful technique, transcriptome sequencing was used to observe global gene expression profiles and the physiological processes involved in various plants (Clarke and Zhu, 2006). As to fruit development, it has been applied to strawberry (Aharoni and O'Connell, 2002), tomato (Alba et al., 2005), pear (Fonseca et al., 2004), apple (Lee et al., 2007), and peanut seed development (Zhang et al., 2012; Zhu et al., 2014). But the relationship between calcium and peanut seed at different pods developmental stages was seldom mentioned. In the present work, 263,110

DISCUSSION

fpls-08-01241 July 14, 2017 Time: 9:37 # 10

unigenes were obtained from library. We identified 4,457 DEGs via sequencing analysis, and got 2,197 stage-specific expressed genes at different pod developmental stages (**Figure 4B**). It has been reported that free Ca2<sup>+</sup> concentration in soil seriously affected peanut fruiting and yield, ovary handle and pods could absorb Ca2<sup>+</sup> from soil, and transport it to both the developing seed and the vegetative organs, such as stem, leaves, etc. (Beringer and Taha, 1976). In this study, through Gene Ontology analysis, many DEGs involved in the Ca2<sup>+</sup> responding and Ca2<sup>+</sup> signal transduction, which were identified as potential candidate genes responsible for development of pods (**Table 1**). That is to say, calcium was related to pod development at the genetic levels.

Besides an important essential element, Ca2<sup>+</sup> also acts as a secondary messenger in various biological processes. It has been shown that various Ca2<sup>+</sup> transporters in plants (Dayod et al., 2010) and Ca2<sup>+</sup> signal transduction protein were involved in gametogenesis, fertilization and early pollen tube growth (Tirlapur and Cresti, 1992). Interestingly, in the present study, we found some calcium-related unigenes at different pod developmental stages, and eight Ca2<sup>+</sup> signaling-related genes were analyzed. These genes were calcium-dependent protein kinase (CDPK), calcineurin B-like protein (CBL), calmodulin like protein (CML), and some other Ca2<sup>+</sup> signaling related genes. CDPKs were thought be involved in embryogenesis, seed development and germination of sandalwood (Anil et al., 2000) and in rice seed development (Morello et al., 2000). OsCDPK2 protein had a low level during early seed development, but had a high level which maintained in 20 days after fertilization (Frattini et al., 1999). Overexpression of the CDPK OsCDPK2 in transgenic rice disrupted seed development (Laura et al., 2000). In peanut, CDPK genes might be involved in pod development since they were up-regulated at 20th day after the peg elongation into

FIGURE 8 | Real-time fluorescent quantitative PCR analysis on mRNA transcription of the selected differentially expressed ethylene related genes. These were the 5th (S2), 10th (S3), 20th (S4) day after the peg elongation into the soil. The ordinate axis greater than one means up-regulated, less than one was down-regulated.

the soil (**Figure 5**). However, some other calcium signal related genes, such as CBL, CML, and PBP1 were all down-regulated. These results implied that Ca2+-signaling transduction pathway might be involved in pod development.

Auxin, gibberellin, abscisic acid, cytokinin, and ethylene have been implicated in regulating the peanut seed development and pod maturation (Jacobs, 1951; Ziv and Kahana, 1988; Shlamovitz et al., 1995; Moctezuma and Feldman, 1996; Ozga and Reinecke, 2003). Meanwhile, genes encoding key enzymes related to hormone metabolism showed significant changes in transcriptional level during peanut early pod development (Xia et al., 2013). In this study, a number of DEGs related to hormone response were identified at different pods developmental stages, e.g., auxin response factor, auxin-induced protein, auxin response factor, gibberellin-regulated protein, gibberellin 2-beta-dioxygenase, gibberellin 3-beta-dioxygenase, ethylene-responsive transcription factor, AP2-like ethyleneresponsive transcription factor, cytokinin hydroxylase, cytokinin dehydrogenase, abscisic acid insensitive 5-like protein, and abscisic acid 8<sup>0</sup> -hydroxylase (**Table 2**). All these revealed that hormone response genes potentially played a crucial role in peanut seed development.

Seed development is a complex process, many physiological processes occurred during seed development (**Figure 3**), calcium and hormone seems to play important roles in this progress, respectively. It has been known that hormonal controls on cell division and expansion are active in the development of fruit. Many of phytohormonal pathways utilize changes in cytoplasmic calcium concentration ([Ca2C]cyt) as a secondary signal messenger (e.g., ABA, JA, auxin, GA, ethylene, brassinosteroids, and cytokinins) (Fortes et al., 2015). Recently, more and more reports showed that Ca2<sup>+</sup> signal transduction pathway is involved in phytohormonal pathways. In Arabidopsis, a CPK4 is involved in modulating ABA signaling (Wang et al., 2017), meanwhile, CPK28 plays a role in balancing the phytohormones JA and GA in development (Matschi et al., 2015), and affects ethylene biosynthesis (Jin et al., 2017). Additionally, the calmodulin-like protein CML20, a functional Ca2<sup>+</sup> sensor, negatively regulates ABA-induced stomatal movement in Arabidopsis (Wu et al., 2017), StCDPK3 in involved in the cross-talk between ABA and GA signaling at the onset of potato tuber development (Grandellis et al., 2016).

The present study provided basis for finding and screening calcium related gene and hormone response genes during peanut seed development. We speculated that besides being components of cell, such as membrane or something else, peanut seed development might be regulated by the collaboration of Ca2<sup>+</sup> signal transduction pathway and hormone regulation pathway, or Ca2<sup>+</sup> regulates pod development through modulating hormone since Ca2<sup>+</sup> signal transduction pathway can modulate plant hormone (Yang and Komatsu, 2000; Ullanat and Jayabaskaran, 2002; Mori et al., 2006), and it also might be related to the utilization calcium of auxin and ABA pathways as both a protein binding secondary messenger and in membrane transport mechanisms that modify turgor and solute accumulation to drive cell expansion and ripening (Hocking et al., 2016).

### AUTHOR CONTRIBUTIONS

SW and XL planned and designed the research. YL, JM, FG, SY, JZ, YG, and LC performed experiments. YL collected data and conducted analysis. YL and XL wrote the manuscript.

### REFERENCES


### ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (31571581, 31571605, 31601261, 31601261), the Natural Science Foundation of Shandong Province (ZR2015YL077 and BS2015SW020), the Postdoctoral Science Foundation of China (2016M592236), the Project of Significant Application of Agricultural Technology Innovation in Shandong Province, International Science and Technology Cooperation Program of China (2015DFA31190), the Supporting Plan of National Science and Technology of China (2014BAD11B04), the earmarked fund for Modern Agroindustry Technology Research System (CARS14).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Li, Meng, Yang, Guo, Zhang, Geng, Cui, Wan and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fpls-08-01241 July 14, 2017 Time: 9:37 # 15

## Both Light-Induced SA Accumulation and ETI Mediators Contribute to the Cell Death Regulated by BAK1 and BKK1

Yang Gao, Yujun Wu, Junbo Du, Yanyan Zhan, Doudou Sun, Jianxin Zhao, Shasha Zhang, Jia Li and Kai He\*

*Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China*

Edited by:

*Yi Ma, University of Connecticut, USA*

### Reviewed by:

*Jianfeng Li, Sun Yat-sen University, China Xiaofeng Wang, Northwest A&F University, China Honghui Lin, Sichuan University, China*

> \*Correspondence: *Kai He hekai@lzu.edu.cn*

### Specialty section:

*This article was submitted to Plant Traffic and Transport, a section of the journal Frontiers in Plant Science*

Received: *01 March 2017* Accepted: *06 April 2017* Published: *25 April 2017*

#### Citation:

*Gao Y, Wu Y, Du J, Zhan Y, Sun D, Zhao J, Zhang S, Li J and He K (2017) Both Light-Induced SA Accumulation and ETI Mediators Contribute to the Cell Death Regulated by BAK1 and BKK1. Front. Plant Sci. 8:622. doi: 10.3389/fpls.2017.00622* Receptor-like kinases BAK1 and BKK1 modulate multiple cellular processes including brassinosteroid signaling and PRR-mediated PTI in Arabidopsis. Our previous reports also demonstrated that *bak1 bkk1* double mutants exhibit a spontaneous cell death phenotype under normal growth condition. With an unknown mechanism, the cell death in *bak1 bkk1* is significantly suppressed when grown in dark but can be quickly induced by light. Furthermore, little is known about intrinsic components involved in *BAK1* and *BKK1*-regulated cell death pathway. In this study, we analyzed how light functions as an initiator of cell death and identified ETI components to act as mediators of cell death signaling in *bak1 bkk1*. Cell death suppressed in *bak1 bkk1* by growing in dark condition recurred upon exogenously treated SA. SA biosynthesis-related genes *SID2* and *EDS5*, which encode chloroplast-localized proteins, were highly expressed in *bak1-4 bkk1-1*. When crossed to *bak1-3 bkk1-1*, *sid2* or *eds5* was capable of efficiently suppressing the cell death. It suggested that overly produced SA is crucial for inducing cell death in *bak1 bkk1* grown in light. Notably, *bak1-3* or *bkk1-1* single mutant was shown to be more susceptible but *bak1-3 bkk1-1* double mutant exhibited enhanced resistance to bacterial pathogen, suggesting immune signaling other than PTI is activated in *bak1 bkk1*. Moreover, genetic analyses showed that mutation in *EDS1* or *PAD4*, key ETI mediator, significantly suppressed the cell death in *bak1-3 bkk1-1*. In this study, we revealed that light-triggered SA accumulation plays major role in inducing the cell death in *bak1 bkk1*, mediated by ETI components.

Keywords: Arabidopsis, BAK1, BKK1, cell death, light, SA, PTI, ETI

## INTRODUCTION

Plant cells utilize plasma membrane (PM)-localized receptor-like kinases (RLKs) to communicate with each other and respond to environmental challenges in coordinating plant growth, development and stress adaptions. RLKs, the single-pass transmembrane proteins with an extracellular domain and a cytoplasmic kinase domain, play essential roles in transducing apoplastic signals into intracellular responses. Leucine-rich repeat (LRR)-RLK family is the largest RLK subfamily containing at least 223 members (Gou et al., 2010), a number of which were found to regulate a variety of physiological processes, such as phytohormone signaling (Li and Chory, 1997; Li et al., 2002; Nam and Li, 2002), innate immunity (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006), meristem maintenance (Clark et al., 1997), pollen development (Zhao et al., 2002; Albrecht et al., 2005; Colcombet et al., 2005), stomatal development (Shpak et al., 2005), pollen tube-egg recognition (Escobar-Restrepo et al., 2007) and cell death control (He et al., 2007, 2008; Kemmerling et al., 2007) etc. Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, containing SERK1-SERK5, has become one of the best functionally analyzed LRR-RLK subfamilies (Li, 2010). SERK was originally isolated in carrot and identified as a marker gene of somatic embryogenesis (Schmidt et al., 1997). In Arabidopsis, SERKs were found to regulate various signaling pathways, in which they often play redundant roles. SERK1 and SERK2 regulate porogenesis by maintaining a normal tapetum during pollen maturation (Albrecht et al., 2005; Colcombet et al., 2005); SERK1, SERK3/BRI1-ASSOCIATED KINASE 1 (BAK1), and SERK4/BAK1-LIKE 1 (BKK1) act as co-receptors of the brassinosteroid (BR) receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) to perceive BRs and initiate cellular signaling cascade (Li et al., 2002; Nam and Li, 2002; Karlova et al., 2006; He et al., 2007; Gou et al., 2012).

BAK1 is also engaged in plant innate immunity. A twotiered system has been proposed to regulate plant innate immunity (Dodds and Rathjen, 2010). The first layer of immunity is activated after perceiving pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) localized at the surface of a plant cell, known as PAMPtriggered immunity (PTI) (Macho and Zipfel, 2014). The second layer of immunity relies on recognition of pathogen-derived effectors by intracellular resistance (R) proteins in plants, known as effector-triggered immunity (ETI) (Jones and Dangl, 2006). PTI confers plant basal resistance to pathogens and ETI enables faster and stronger defense responses in plants, which is often accompanied with hypersensitive response (HR), a type of programmed cell death (PCD) (Jones and Dangl, 2006; Macho and Zipfel, 2014). Canonical R proteins are nucleotide-binding/leucine-rich repeat (NLR) receptors which fall into two groups, including CNLs, possessing a coiledcoil (CC) domain, and TNLs, containing a Toll-Interleukin-1 receptor (TIR) domain (Meyers et al., 1999; Cannon et al., 2002). EDS1 and PAD4, two lipase-like proteins, are required for TNL-mediated ETI; while NDR1, a PM-associated protein, functions as key mediator in CNL signaling (Aarts et al., 1998; Wiermer et al., 2005; Knepper et al., 2011). BAK1 was found to be involved in PAMP recognition and mediating PTI via associating with several PRR RLKs such as FLAGELLIN-SENSITIVE 2 (FLS2), the receptor for bacterial flagellin, EF-TU RECEPTOR (EFR), the receptor for bacterial elongation factor Tu (EF-Tu), and PEP1 RECEPTOR 1 (PEPR1), the receptor for plant endogenous PROPEP-derived Pep epitopes (Chinchilla et al., 2007; Heese et al., 2007; Schulze et al., 2010). BAK1, therefore, positively regulates PTI, the first tier of plant innate immunity.

Intriguingly, our previous studies revealed BAK1/SERK3 and BKK1/SERK4 also mediate a cell death pathway that is independent of the BR signaling pathway (He et al., 2007). A null double mutant, bak1-4 bkk1-1, exhibits an extremely strong cell lesion phenotype even on the sterilized medium under normal growth condition. The cell death symptom of bak1-4 bkk1- 1 emerged about 7 days after germination and eventually led to lethality within 2 weeks (He et al., 2007). The cell death phenotype we reported was in line with a simultaneous study of a different group showing that bak1-4 single mutant exhibited a runaway cell death upon microbial pathogen inoculation (Kemmerling et al., 2007).

Salicylic acid (SA) was identified as an essential signal molecule in mediating plant defense responses (Vlot et al., 2009). Serving as a crucial signal for activating disease resistance including PTI and ETI, SA is rapidly synthesized upon pathogen invasions. Arabidopsis mutants that fail to sufficiently synthesize endogenous SA exhibit impaired innate immunity and enhanced susceptibility to pathogen treatments (Rogers and Ausubel, 1997; Nawrath and Métraux, 1999; Wildermuth et al., 2001; Nawrath et al., 2002). Given that PR1 is a key marker gene of SA signaling, the extremely high expression of PR1 suggested an exceedingly activated SA signaling in bak1-4 bkk1-1 (He et al., 2007). In Arabidopsis, SA is synthesized through two distinct routes, the phenylalanine ammonia-lyase (PAL) pathway and the isochorismate (IC) pathway (Dempsey et al., 2011). Chorismate, the end product of the shikimate pathway, is used as the SA precursor in both pathways (Dempsey et al., 2011). In Arabidopsis, four PAL proteins (PAL1-4) are essential for catalyzing the reaction from phenylalanine to trans-cinnamic acid, a critical step for SA production (Mauch-Mani and Slusarenko, 1996; Chen et al., 2009; Huang et al., 2010). The PAL pathway occurs in cytoplasm and was proposed to account for only 5% of SA production (Métraux, 2002; Chen et al., 2009). The IC pathway, by contrast, takes place in chloroplast and contributes 95% of total cellular SA production (Wildermuth et al., 2001; Métraux, 2002; Garcion et al., 2008; Chen et al., 2009). Two redundant genes ISOCHORISMATE SYNTHASE 1 (ICS1) and ICS2 were identified to encode essential enzymes localized in chloroplasts to catalyze the conversion of chorismate to isochorismate, a key step of SA biosynthesis in the IC pathway (Garcion et al., 2008). Since ICS2 only plays a marginal role in this reaction, ICS1, also known as SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2), functions as the determinate catalyzator of SA synthesis in IC pathway (Wildermuth et al., 2001; Garcion et al., 2008). Distinct from WT, ics1/sid2 single mutant plants showed no obvious SA accumulation upon a variety of stress treatments such as UV light, ozone, or pathogen incubations (Nawrath and Métraux, 1999; Garcion et al., 2008). The majority of SA, therefore, appears to be produced in chloroplasts and subsequently transported to cytosol and other cellular compartments to fulfill its physiological roles. It has been previously reported that ENHANCED DISEASE SUSCEPTIBILITY 5 (EDS5), a member of Multidrug and Toxic Compound Extrusion (MATE) transporter family, also impacts SA accumulation (Nawrath et al., 2002). EDS5 was suggested to be localized on plastid membrane and presumably function as an SA transporter in translocating SA from chloroplasts to cytosol (Serrano et al., 2013). Like ics1/sid2, eds5 mutants also failed to efficiently accumulate SA when exposed to pathogens (Nawrath and Métraux, 1999).

Little progress has been made since BAK1 and BKK1 were discovered to be essential in mediating a cell death pathway. One earlier study indicated that another LRR-RLK, BAK1-INTERACTING RECEPTOR-LIKE KINASE 1 (BIR1), can interact with BAK1, and that bir1 mutants displayed a cell death phenotype similar to that of the bak1 bkk1 double mutants (Gao et al., 2009). A PM-localized copine-like protein, BONZAI1 (BON1), was identified as BAK1- and BIR1-interacting protein (Wang et al., 2011). The bon1 mutant plants also exhibited a cell-death phenotype (Yang et al., 2006). However, the detailed mechanisms of BIR1 and BON1 in regulating cell death pathways and their connections to the BAK1 and BKK1-regulated cell death pathway remain elusive. Key open questions still await answers. For instance, what are the primary factors responsible for initiating cell death in bak1 bkk1 double mutant at early stage? Which biological processes are related to the cell death of bak1 bkk1? Our previous report suggested that light likely serves as a key factor in triggering cell death in bak1-4 bkk1- 1 (He et al., 2008). Distinct from the bak1-4 bkk1-1 plants starting to exhibit lesion symptom at 7 days when grown under a long-day condition, bak1-4 bkk1-1 grown in darkness did not display a cell death phenotype at 8 days (He et al., 2008). The mechanism regarding how light contributes to triggering cell death in bak1 bkk1 has not been depicted. Despite the well establishment of BAK1 as an essential co-receptor of key PRRs in positively regulating PTI, the cell death occurring in bak1 bkk1 seems to be unrelated to PTI. Overexpression or knocking-out of known BAK1-associated PRRs does not cause cell death in plants. Instead, the cell death phenotype of bak1-4 bkk1-1 resembles HR, a typical defense response in ETI. It implies BAK1 and BKK1 play roles beyond PTI. However, whether ETI regulatory components are involved in the cell death pathway mediated by BAK1 and BKK1 remains unknown.

In this study, we carefully analyzed the light-triggered cell death in bak1 bkk1 mutants. Our results indicated that SA accumulation in bak1 bkk1 mutants may function as a primary initiator to trigger cell death. Genes encoding chloroplastlocalized proteins SID2 and EDS5 that are essential for SA biosynthesis and accumulation were highly up-regulated in bak1 bkk1 and played critical roles in cell death formation. Further analyses revealed that, despite maintaining their insensitivity to bacterial PAMP, bak1 bkk1 mutants exhibited overly activated immunity, suggesting BAK1 and BKK1 mediate a different immunity signaling besides PTI. At last, our results showed that the loss-of-function mutation of PAD4 or EDS1, key component mediating ETI, was capable of significantly suppressing the cell death of a weak double mutant, bak1-3 bkk1-1. These results provide genetic evidence suggesting the involvement of ETI in BAK1 and BKK1-mediated cell death pathway.

### MATERIALS AND METHODS

### Plant Materials and Growth Conditions

All of the Arabidopsis lines used in this study are in the Col-0 ecotype except pif1-1 (Col-3). T-DNA insertion lines bak1-4 (SALK\_116202), bak1-3 (SALK\_034523), bkk1-1 (SALK\_057955), sid2-3 (SALK\_088254), eds5- 2 (SALK\_091541C), pad4-2 (SALK\_089936), eds1-3 (SALK\_057149), pif1-1 (CS66041), pif3-1 (SALK\_030753), pif4-3 (SALK\_140393C), pif5-3 (SALK\_087012C) were obtained from the Arabidopsis Biological Resource Center (ABRC). Multiple mutants were generated by genetic crossings. Homozygous plants were isolated via PCR with genotyping primers designed by http://signal.salk.edu/tdnaprimers.2.html. The wildtype and mutant plants were grown in greenhouse or illumination incubator at 22◦C in dark condition or under long day cycle (16 h light/8 h dark). For SA treatment assay, seedlings were grown on 0.5× Murashige and Skoog medium with or without SA in dark condition and were analyzed at indicated days.

### Trypan Blue Staining and DAB Staining

Tissue stainings with trypan blue and DAB were carried out as previously described (He et al., 2007).

### Determination of Subcellular Distribution

The full length CDS of SID2 and EDS5 were amplified by RT-PCR and cloned into pBIB-BASTA-35S-GWR-GFP and pBIB-BASTA-35S-GWR-YFP, respectively. The two constructs were transformed into Agrobacterium tumefaciens strain GV3101. The transformed GV3101 were cultured in LB medium (supplemented 20 µM acetosyringone) about 12–16 h. Centrifuged and re-suspended the cultured cells with injection buffer (liquid MS medium containing 150 µM acetosyringone, 10 mM MgCl2, 10 mM MES, pH 5.7) and adjusted to OD<sup>600</sup> = 0.5. After quiescence for 2 h, the cultures were injected into the N. benthamiana mesophyll tissue. N. benthamiana protoplasts were isolated after 3 days of injection. YFP or GFP fluorescence signal was observed by Olympus FluoView FV1000 confocal microscope.

### Gene Expression Analyses

For quantitative RT-PCR (qRT-PCR) assays, total RNA was extracted from the whole seedlings harvested under different time points. Two micrograms of total RNA was reversely transcribed in a 20µl volume with M-MLV reverse transcriptase (Invitrogen). The fragments of target genes were amplified using SYBR Premix Ex Taq (TaKaRa). The thermal cycling program was 95◦C for 30 s, followed by 40 cycles of 95◦C for 5 s, 60◦C for 30 s. ACTIN2 (At3G18780) was used for normalization the relative expression level of each transcript, and the comparative 11C<sup>T</sup> method was employed to calculate the relative quantities of each amplified product. For identification of mutant alleles, total RNA was isolated from the leaves of 11-day-old plants grown in soil. Two micrograms of total RNA was reversely transcribed in a 20µl volume using M-MLV reverse transcriptase (Invitrogen). The transcripts of specific genes were amplified with gene-specific primers. ACTIN2 was used as a reference. The primers used for qRT-PCR and semi-quantitative RT-PCR are listed in Table S1.

### Measurements of SA Content

SA content measurements were carried out as previously described (Du et al., 2016).

### Bacterial Infection Assays

Pst DC3000-LUX strain expressing luxCDABE operon was cultured in KB medium for 12 h. Centrifuged and re-suspended the cultured cells with 10 mM MgCl<sup>2</sup> and adjusted to OD<sup>600</sup> = 0.2. The Pst DC3000-LUX suspension was sprayed onto the 3-week-old Arabidopsis plant lines indicated. All the sprayed plants were kept under a transparent lid to keep high humidity conditions. The plants were checked by a CCD camera to obtain the images of luciferase fluorescence at 30 h after inoculation. The experiments were repeated three times.

### flg22 Treatments

Diluted flg22 with sterilized 0.5× Murashige and Skoog solution (pH5.7) to the concentration of 0.2 µM. Nine-day-old Col-0, bak1-4 bkk1-1, bak1-4 and bkk1-1 grown on 0.5× Murashige and Skoog medium were immersed with flg22 solution for 5, 15, and 30 min. Total protein was extracted from the seedlings harvested under different time points. The total protein extracts were separated by a 10% SDS-PAGE gel for western blot assay. P-p44/42 (Cell Signaling Technology) was used as the antibody. Equal loadings were tested by staining the SDS-PAGE gel with Coomassie brilliant blue G250.The experiments were repeated three times.

### RESULTS

### Initiation of the Cell Death in bak1-4 bkk1-1 Was Light-Dependent

In order to elucidate the mechanism of light-induced cell death in bak1 bkk1, we analyzed bak1-4 bkk1-1 mutant plants under a variety of light conditions. Consistent with our earlier results, dark-grown bak1-4 bkk1-1 showed no clear cell death symptom 8 days after germination, similar to WT Col-0 plants (**Figure 1A**). Next, Col-0 and bak1-4 bkk1-1 plants were first grown in dark for 4 days then transferred to a long-day condition (16 h light/8 h dark). bak1-4 bkk1-1 started to exhibit cell death 2 days after transferring to light (**Figure 1A**). By contrast, no cell death was detected in dark-grown Col-0 subsequently treated by light (**Figure 1A**). These results indicated that dark condition prevented bak1-4 bkk1-1 from forming cell death, which was quickly triggered by light. Cell death is often accompanied with ROS burst, which could be either the cause or consequence of cell death (Chaouch and Noctor, 2010). DAB staining was used to visualize the accumulation of H2O2. Col-0 did not accumulate H2O<sup>2</sup> under either light or dark condition (**Figure 1B**). ROS burst was only detected in dark-grown bak1-4 bkk1-1 after exposure to light (**Figure 1B**), displaying patterns similar to cell death formation (**Figure 1A**). Under light condition, the initiation of cell death was found to be 1 day prior to overaccumulation of ROS in bak1-4 bkk1-1. These results suggested that, instead of causing cell death, ROS burst may be the physiological consequence of cell death in bak1-4 bkk1-1.

Next, we examined whether the defense response of bak1- 4 bkk1-1 was also related to light. Utilizing qRT-PCR, a SAresponsive gene, PR1, and a cell death marker gene, FOM1 (Bartsch et al., 2006; Li et al., 2013), were checked. Col-0 and bak1-4 bkk1-1 grown in dark for 4 days then transferred to long-day condition for 1 or 2 days were analyzed. After being transferred to a long-day condition for 1 or 2 days, PR1 in dark-grown bak1-4 bkk1-1 increased about 17 or 600-fold, respectively, compared to the one in Col-0 (**Figure 2A**). In Col-0, PR1 gene was kept at constantly low expression levels under both dark and light conditions (**Figure 2A**). FMO1 expression was also significantly induced when dark-grown bak1-4 bkk1- 1 was exposed to light for 1 or 2 days (**Figure 2B**), consistent with trypan blue straining results (**Figure 1A**). As control PR1 and FOM1 genes were also analyzed in the seedlings grown in continuous dark. In contrast to the significantly increased expression of PR1 and FMO1 in dark-grown bak1-4 bkk1-1 after light treatment, when grown in dark for 5 or 6 days, the expression of PR1 and FMO1 showed slight increases or almost no changes in bak1-4 bkk1-1 compared to the ones in Col-0 (**Figures 2A,B**). Since PR1, a marker gene of SA signaling pathway, was highly expressed in bak1-4 bkk1-1 upon light exposure, it was conjectured SA may contribute to the lightinduced cell death mediated by BAK1 and BKK1. The SA contents were measured in Col-0, bak1-4, bkk1-1, and bak1-4 bkk1-1 seedlings grown in dark for 4 days and the seedling grown in dark for 4 days followed by exposure to long-day condition for 1 day. The free SA in Col-0 and single mutant plants grown in dark for 4 days was unchanged after transferring to a long-day condition for 1 day. In bak1-4 bkk1-1 grown under dark for 4 days, free SA increased about 3-fold after growing the plants in long-day condition for 1 day, indicating a quick SA accumulation in bak1-4 bkk1-1 upon light treatment (**Figure 2C**). Similarly, total SA in bak1-4 bkk1-1 plants was also significantly increased when moved from dark to light condition (**Figure 2D**). Notably, high concentration of total SA was already detected in bak1-4 bkk1-1 plants grown in dark before light treatment (**Figure 2D**). This result suggested that BAK1 and BKK1 may also regulate a pathway inactivating SA.

The majority of SA is synthesized in chloroplasts where SID2/ICS1 serves as a determinate enzyme (Wildermuth et al., 2001; Métraux, 2002; Garcion et al., 2008; Chen et al., 2009). A minor portion of SA is produced in cytoplasm, which is catalyzed by PALs (Métraux, 2002; Chen et al., 2009). To explicate why total SA was accumulated in bak1-4 bkk1-1 even under a dark condition, we analyzed the expression levels of PAL1-4 genes. In dark, the expression of all four PAL genes increased in bak1- 4 bkk1-1 compared to those in Col-0 (Figure S1). The elevated expression of PAL genes in bak1-4 bkk1-1 may contribute to the accumulation of total SA in a dark condition, when free SA was still kept at low concentrations.

Interestingly, cell death eventually started to be detectable in bak1-4 bkk1-1 at 12 days when grown in dark (Figure S2). These results indicated that the cell death phenotype was extensively

(4D+1L, 4D+2L, 4D+3L, 4D+4L). Seedlings grown in dark (D) for the indicated days (4D+0L, 5D+0L, 6D+0L, 7D+0L, 8D+0L) are presented as control. The trypan blue staining assays indicate cell death symptom is induced in *bak1-4 bkk1-1* 2 days after the transfer from dark to light condition (A). The DAB staining assays indicate ROS accumulation is detected in *bak1-4 bkk1-1* 3 days after the transfer from dark to light condition (B). The scale bars represent 100 µm.

delayed but not completely abolished in bak1-4 bkk1-1 when grown in dark.

### Exogenously Applied SA Triggered Cell Death in bak1-4 bkk1-1 in Dark

Given that the initiation of light-dependent cell death in bak1- 4 bkk1-1 was likely SA-related, we further analyzed the detailed role of SA in cell death induction. Trypan blue staining was used to detect cell death in Col-0 and bak1-4 bkk1-1 grown on 1/2 MS medium supplemented with 0, 10, or 50µM SA in dark condition. Col-0 showed no cell death when treated with 10 or 50µM SA (**Figure 3A**). When grown on the medium supplemented with 50µM SA in a dark condition, bak1-4 bkk1- 1 started to show cell death symptom 5 days after germination. When treated with 10µM SA in dark, bak1-4 bkk1-1 also exhibited obvious but less severe cell death symptom compared to those treated with 50µM SA (**Figure 3A**). These results clearly demonstrated that cell death suppressed in bak1-4 bkk1- 1 by growing the seedlings in dark condition reappeared by exogenously treated SA. Therefore, SA seemed to reproduce the

effect of light with regard to cell death induction in bak1-4 bkk1-1. ROS accumulation was also examined in Col-0 and bak1-4 bkk1- 1 plants upon SA treatment. This result suggested that H2O2was also accumulated in bak1-4 bkk1-1 but not in Col-0 when SA was applied to the medium (**Figure 3B**). In dark condition, the ROS burst in bak1-4 bkk1-1 appeared 1 day after the emergence of cell death symptom when treated with SA (**Figure 3**), similar to the patterns of light-induced cell death and ROS burst in bak1-4 bkk1-1 (**Figure 1**).

### The Expression of SID2 and EDS5 Was Highly Elevated in bak1 bkk1

SA-induced cell death in bak1-4 bkk1-1 was light-independent, supporting light plays an indirect role in the induction of cell death in bak1-4 bkk1-1. SA accumulation may contribute to this process. We first analyzed whether light signaling pathways were involved in BAK1 and BKK1-mediated cell death. A weak double mutant bak1-3 bkk1-1 was crossed to several pif mutants to generate triple and quadruple mutant lines. PIF genes encode transcription factors mediating red light and blue light signaling pathways. PIF1 usually functions redundantly with PIF3, and PIF4 often plays an overlapping role with PIF5. Our genetic results demonstrated that none of bak1-3 bkk1-1 pif1-1, bak1-3 bkk1-1 pif3-1, or bak1-3 bkk1-1 pif1-1 pif3-1 mutant plants displayed a phenotypic difference in cell death symptom compared to their background bak1-3 bkk1-1 (Figure S3). Similarly, bak1-3 bkk1-1 pif4-3, bak1-3 bkk1-1 pif5- 3, or bak1-3 bkk1-1 pif4-3 pif5-3 mutant plants did not show alterations to bak1-3 bkk1-1 with regard to cell death phenotype (Figure S4). These results suggested that the PIF-mediated light signalings may not contribute to the cell death initiation in bak1 bkk1 under light condition. Thus, it was hypothesized that SA synthesis, mainly occurring in chloroplast and relying on lightdependent photomorphogenesis, plays a key role in the lightinduced cell death in bak1 bkk1. Through data mining of our earlier microarray results that compare global gene expression patterns between Col-0 and bak1-4 bkk1-1 seedlings, we noticed that two genes, SID2 and EDS5, encoding critical components involved in SA biosynthesis and cytoplasmic accumulation, were dramatically up-regulated in bak1-4 bkk1-1. The microarray data indicated that the transcripts of SID2 and EDS5 in bak1-4 bkk1- 1 increased 19.86 and 5.78-fold, respectively, compared to the ones in Col-0. Interestingly enough, the protein products of these two genes were both suggested to locate to chloroplast. SID2 encodes an isochorismate synthase catalyzing chorismate into isochorismate, a SA precursor, in chloroplast. EDS5 encodes a MATE transporter, locating to the chloroplast envelope, and is suggested to function as an SA transporter mediating the translocation of chloroplast-synthesized SA to cytosol. Using a quantitative RT-PCR assay, we analyzed the expression patterns of SID2 and EDS5 in Col-0 and bak1-4 bkk1-1 plants grown in dark condition for 4 days and the plants grown in dark for

4 days then exposed in long-day condition for 1 or 2 days. SID2 showed significantly high expression in bak1-4 bkk1-1 (**Figure 4A**), indicating a robustly negative regulation on SID2 expression by BAK1 and BKK1. Treated with or without light condition, the transcripts of SID2 increased about 30–40-fold in bak1-4 bkk1-1 compared to those in Col-0 (**Figure 4A**), suggesting the inhibition on SID2 expression by BAK1 and BKK1 was likely light-independent. In dark, the expression of EDS5 increased about 2-fold in bak1-4 bkk1-1 compared to the one in Col-0 (**Figure 4A**). Upon Light treatment, EDS5 expression decreased in Col-0 but increased in bak1-4 bkk1- 1 (**Figure 4A**), suggesting EDS5 is also suppressed by BAK1 and BKK1, especially under light condition. When grown in dark for 5 or 6 days, SID2 and EDS5 in bak1-4 bkk1-1 showed much lower levels of increases (**Figure 4A**). We next analyzed the subcellular locations of SID2 and EDS5 proteins. The cDNAs of SID2 and EDS5 were fused with GFP and YFP, respectively, and transiently expressed in N. benthamiana, driven by 35S promoter. SID2-GFP was detected to be localized in chloroplast (**Figure 4B**). EDS5-YFP mainly located to the surface of chloroplast (**Figure 4B**), consistent with the previous report that EDS5 may function as an SA transporter at the chloroplast envelope. Taken together, our results indicated SID2 and EDS5, encoding chloroplast-localized proteins essential for SA accumulation, were suppressed by BAK1 and BKK1 at transcriptional levels.

### Mutation of SID2 or EDS5 Partially Suppressed the Cell Death in bak1-3 bkk1-1

In order to further dissect the function of SID2 and EDS5 in initiating cell death in bak1 bkk1, genetic approaches were employed. bak1-3 bkk1-1 was crossed to sid2-3 and eds5-2 T-DNA insertion lines, respectively, to generate triple-mutants. RT-PCR results confirmed that SID2 and EDS5 were completely knocked out in bak1-3 bkk1-1 sid2-3 and bak1-3 bkk1-1 eds5- 2, respectively (Figure S5). Introduction of sid2-3 into bak1-3 bkk1-1 significantly suppressed the cell death symptom of bak1-3 bkk1-1 when grown in soil for 3-week-old (**Figure 5A**). Trypan blue and DAB staining results confirmed SID2 mutation greatly inhibited the cell death phenotype and ROS accumulation in 11-day-old bak1-3 bkk1-1 (**Figures 5B–E,H–K**). In addition, the highly expressed PR1 and FMO1 in bak1-3 bkk1-1 restored to WT-like levels in bak1-3 bkk1-1 sid2-3 triple mutant, indicating SID2 is essential for cell death induction in bak1-3 bkk1-1 (**Figures 5N,O**). Similarly, the absence of EDS5 also appeared to partially rescue the cell death phenotype of 3-week-old bak1- 3 bkk1-1 (**Figure 5A**). Trypan blue and DAB staining results verified that the cell death and ROS burst in 11-day-old bak1-3 bkk1-1 eds5-2 was largely prevented (**Figures 5B–M**). The qRT-PCR results showed that the expression of PR1 and FMO1 was greatly suppressed in bak1-3 bkk1-1 eds5-2 compared to the ones in bak1-3 bkk1-1 (**Figures 5N,O**). Collectively, our genetic results indicated SA biosynthesis-related genes SID2 and EDS5 act as crucial components in promoting cell death in bak1-3 bkk1-1.

### bak1 bkk1 Showed Enhanced Bacterial Resistance and Impaired PTI

Since it is established that BAK1 positively regulates PTI via interacting and collaborating with several PRRs, we further examined the basal defense in bak1 bkk1. Three-week-old soil-grown plants of Col-0, sid2-3, eds5-2, bak1-3, bkk1-1, bak1-3 bkk1-1, bak1-3 bkk1-1 sid2-3, and bak1-3 bkk1-1 eds5-2 were sprayed with P. syringae pv tomato DC3000 (Pst DC3000)-LUX for 30 h before the images of luciferase florescence were taken. Single mutant sid2-3 or eds5-2 showed enhanced susceptibility to the bacterial pathogen, indicating the essential role of SA in mediating basal defense (**Figure 6A**). Similarly, single mutant bak1-3 or bkk1-1 also exhibited markedly reduced resistance to Pst DC3000, consistent with the notion that BAK1 functions as a positive regulator in PTI (**Figure 6A**). Surprisingly, the double mutant bak1-3 bkk1-1 showed enhanced resistance to Pst DC3000, different from either bak1-3 or bkk1-1 (**Figure 6A**). Compared to bak1-3 bkk1-1, bak1-3 bkk1-1 sid2-3 or bak1-3 bkk1-1 eds5-2 showed reduced bacterial resistance, suggesting SA was involved in the defense activation in bak1-3 bkk1-1. To confirm these results, we analyzed the activation status of MPK3/6 in various plants lines. MPK3/6 were identified as mediators in basal defense and receptor triggered immunity including PTI and ETI (Rasmussen et al., 2012; Tsuda et al., 2013). Activated MPK3/6 via phosphorylation indicate enhanced immune response. By using

a P-p44/42 antibody, phosphorylated MPK3/6 were detected in various plant lines. Consistent to Pst DC3000 treatment results, bak1-3 bkk1-1 showed over-activated MPK3/6, which were less phosphorylated in sid2-3, eds5-2, bak1-3, bak1-4, or bkk1-1, compared to Col-0 (**Figure 6B**). In bak1-3 bkk1-1 sid2-3 and bak1-3 bkk1-1 eds5-2, phosphorylated MPK3/6 were reduced compared to bak1-3 bkk1-1, suggesting the autoimmunity in bak1-3 bkk1-1 is partially SA-dependent (**Figure 6B**). It was intriguing that bak1-3 and bkk1-1 single mutants were more susceptible but bak1-3 bkk1-1 double mutant were more resistant to bacterial pathogen. We next analyzed PTI signaling in bak1 bkk1 to examine whether PTI was responsible for the enhanced immunity in bak1-4 bkk1-1. Nine-day-old Col-0, bak1-4 bkk1-1, bak1-4, and bkk1-1 were incubated with flg22, a 22-amino-acid peptide conserved in bacterial flagellin that is efficient to trigger FLS2-mediated immune signaling, for 5, 15, and 30 min before examining phosphorylated MPK3/6. Both of the single mutants bak1-4 and bkk1-1 showed reduced response to flg22 (**Figure 6C**). The response of double mutant bak1-4 bkk1-1 to flg22 was dramatically reduced, demonstrating PTI in bak1-4 bkk1-1 was nearly blocked (**Figure 6C**). Our results supported the

previous findings that BAK1 is essential for PRR-mediated PTI and, more importantly, suggested that bak1 bkk1 double mutants undergo an enhanced immune signaling other than PTI.

### EDS1 and PAD4, Components Mediating ETI, Were Involved in the Cell Death in bak1 bkk1

EDS1-PAD4 complex acts as an essential component in mediating all tested TNL type R protein pathway in ETI (Wiermer et al., 2005). To understand whether ETI is involved in the cell death induction in bak1 bkk1, bak1- 3 bkk1-1 was crossed to eds1-3 and pad4-2, respectively eds1-3 was able to partially rescue the cell death of bak1- 3 bkk1-1; while pad4-2 dramatically suppressed the lesion symptom of bak1-3 bkk1-1 (**Figure 7A**). Our genetic results thus implicated the autoimmunity in bak1 bkk1 is likely ETIrelated.

Given that EDS1 and PAD4 also function as upstream of SA pathway via regulating ICS1-generated SA biogenesis, we next tested whether the suppression of bak1-3 bkk1-1 by eds1 or pad4 was SA-dependent. No clear cell death symptom was detected in 3-week-old bak1-3 bkk1-1 sid2-3 (**Figure 5**). By contrast, 26-day-old bak1-3 bkk1-1 sid2-3 started to exhibit detectable

lesion symptom, suggesting the cell death pathway was partially SA-dependent. The cell death of bak1-3 bkk1-1 sid2-3 was further suppressed by eds1 or pad4, suggesting EDS1 and PAD4 contributed to the BAK1 and BKK1-mediated cell death pathway other than, or in addition to, regulating SA accumulation (**Figure 7B**). Therefore, ETI mediated by EDS1 and PAD4 was proposed to be involved in BAK1-mediated cell death pathway. We also introduced a mutation of NDR1, a key mediator in CNL signaling, into bak1-3 bkk1-1. The triple mutant bak1-3 bkk1-1 ndr1-1 exhibited identical cell death phenotype to bak1- 3 bkk1-1, suggesting TNL type but not CNL type R proteins are likely involved in BAK1 and BKK1-mediated cell death pathway (Figure S6).

### DISCUSSION

BAK1 was originally identified as a co-receptor of BRI1, positively regulating BR signal transduction. Genetic analyses revealed that BAK1 and its closest homolog BKK1 can also modulate a cell death pathway that is independent of the BR signaling pathway. This unexpected finding suggested the versatile roles of BAK1 in regulating plant growth and development, as well as adaptations to environmental stresses. Removal of both BAK1 and BKK1 in a single plant leads to cell lesion and ultimately a lethality phenotype under a normal growth condition, indicating the irreplaceable roles BAK1 and BKK1 play in the physiological events essential for plants to fulfill their life cycles. However,

the detailed mechanisms of BAK1 and BKK1 in mediating cell death are still beyond what we understand. After the finding of BAK1-mediated cell death, BAK1 was reported to be engaged in plant innate immunity through associating with PRRs such as FLS2, EFR and PEPR1 (Chinchilla et al., 2007; Heese et al., 2007; Schulze et al., 2010). Nevertheless, loss-of-function mutants of the BAK1-interacting PRRs only exhibit impaired innate immunity but no cell death symptom, suggesting the cell death mediated by BAK1 and BKK1 is unlikely directly related to PTI mediated by PRRs. In addition, a BAK1 point mutation allele, bak1-5, was identified to display disrupted FLS2-mediated PTI, but bak1-5 bkk1-1 showed no cell death (Schwessinger et al., 2011), suggesting that BAK1-mediated PTI pathway and BAK1 mediated cell death pathway belong to distinct signaling events.

Our previous research suggested dark growth condition largely suppressed the cell death symptom in bak1-4 bkk1-1 and light treatment quickly induced lesion phenotype in dark-grown bak1-4 bkk1-1. How light serves as an initiator of cell death in bak1 bkk1 mutants is still unclear. Furthermore, the downstream signaling components involved in cell death induction in bak1 bkk1 are nearly unknown. Considering that PTI is not likely involved in the cell death of bak1 bkk1, ETI, the stronger immune response featured by quick cell death formation, is worth being investigated whether is related to BAK1 and BKK1-mediated cell death pathway.

In this study, we analyzed the formation of cell death in bak1 bkk1 upon light treatment. Our results indicated that cell death was undetectable in bak1-4 bkk1-1 when grown in dark up to 12 days, while evident lesion symptom was induced in bak1-4 bkk1-1 2 days after being transferred from dark to light condition, accompanied with highly accumulated SA and ROS. Fascinatingly, dark-grown bak1-4 bkk1-1 was capable of exhibiting obvious cell death when exogenous SA was applied, illustrating that light does not directly regulate cell death and SA might function downstream of light and play a central role in promoting the cell death in bak1 bkk1. qRT-PCR results revealed that two genes SID2 and EDS5, critical in modulating SA biosynthesis, were significantly up-regulated in bak1-4 bkk1- 1; and more interestingly, the protein products of both genes are located in chloroplasts. The mutation in either SID2 or EDS5 in bak1-3 bkk1-1 remarkably rescued the cell death phenotype of the double mutant, suggesting chloroplast-localized SID2 and EDS5 are crucial for cell death formation in bak1-3 bkk1-1, likely via affecting SA accumulation. Furthermore, in a pathogen treatment assay, both bak1-3 and bkk1-1 single mutants were more susceptible to Pst DC3000 compared to Col-0, consistent to the notion that BAK1 functions as major contributor to the basal resistance to several pathogens including Pst DC3000 (Roux et al., 2011). Surprisingly, bak1-3 bkk1-1 double mutant exhibited enhanced resistance to Pst DC3000, indicating the loss of either BAK1 or BKK1 causes impaired immunity but removing both BAK1 and BKK1 leads to triggering additional route of plant immunity instead. Genetic analyses supported this notion by showing that mutation in either EDS1 or PAD4, key regulator mediating ETI, resulted in suppression of cell death in bak1 bkk1.

Based on the aforementioned results, we propose a hypothetic model. SA is not efficiently synthesized in bak1 bkk1 in dark condition. Light-induced overexpression of SID2 and EDS5 in bak1 bkk1 causes highly accumulated SA. Positive feedback regulation on SA biosynthetic genes by high concentrations of SA further enhances the SA biosynthesis and leads to SA burst that ultimately induce cell death in bak1 bkk1. In addition, BAK1 and BKK1 regulate a signaling pathway to inhibit ETI, which is mediated by EDS1-PAD4 complex, under normal growth conditions. When BAK1 and BKK1 are both depleted, ETI is activated to initiate cell death in plants, a faster and stronger defense response (**Figure 7C**).

Light has been reported to cause cell death when it is excessive (Szechyn´ska-Hebda and Karpinski, 2013 ´ ); and UV light treatment often leads to lesions in plant tissues (Nawkar et al., 2013). Nevertheless, the light-dependent cell death found in bak1 bkk1 seems to be distinct from the known lightinduced cell death. The known light-induced lesion is caused by rapid ROS accumulation, a key cell death signal. In bak1 bkk1 double mutants, the cell death symptom appears prior to the accumulation of ROS, indicating ROS is not the cause of the cell death. Instead, ROS accumulation is likely an effect of cell death in bak1 bkk1. Nevertheless, since ROS also serves as a feedback signal for SA biosynthesis (Torres et al., 2006), the ROS accumulation in bak1 bkk1 may further deteriorate the cell death symptom. In addition, none of the multiple bak1-3 bkk1-1 pif mutant plants displayed altered lesion phenotype compared with bak1-3 bkk1-1 (Figures S3, S4), suggesting light-induced cell death in bak1 bkk1 is independent of PIF-mediated blue and red light signaling pathways (Castillon et al., 2007; Pedmale et al., 2016). Moreover, the effect of light on cell death initiation can be reproduced by SA treatment in bak1 bkk1 in dark (**Figure 3**), suggesting light acts as an indirect factor to trigger cell death in bak1 bkk1 and light signaling is not essential for this process.

Compared to those in Col-0, the transcripts of SID2 increased about 30–40-fold in bak1-4 bkk1-1, demonstrating SID2 expression is strongly inhibited by BAK1 and BKK1. Even though the expression of SID2 was indeed enhanced by light, the suppression of SID2 by BAK1 and BKK1 seemed to be lightindependent, indicated by similar multiple increases of SID2 in dark-grown Col-0 and bak1-4 bkk1-1 upon light treatment (**Figure 4A**). The expression of EDS5 is suppressed by BAK1 and BKK1. Compared to those in Col-0, EDS5 transcripts increased about 2-fold in dark condition, but elevated around 12-fold 1 day after relocating to light condition in bak1-4 bkk1-1. This result suggested the expression of EDS5 is synergistically controlled by light and BAK1and BKK1 (**Figure 4**).

In PAL pathway of SA synthesis, four PAL genes were identified to encode enzymes facilitating production of the SA precursor in cytosol. Our results suggested the expression levels of PAL genes were also inhibited by BAK1 and BKK1. It may explicate that high total SA content was detected under dark condition in bak1-4 bkk1-1. The PAL-dependent SA biosynthesis pathway may contribute to the cell death initiation in bak1-4 bkk1-1 when grown in dark for an elongated period of time (Figures S1, S2). It is also possible that additional BAK1 and BKK1-mediated pathway that is SA-independent may contributes to the cell death signaling. Despite high concentration of total SA in bak1-4 bkk1-1 in dark, the free SA content was almost equal to that in Col-0. Thus, BAK1 and BKK1 might control an unknown SA regulation pathway conjugating SA to other components, serving as a mechanism of preventing toxicity caused by highly accumulated free SA (Dempsey et al., 2011).

A recent study indicated that bak1 mutant exhibited enhanced resistance to Pst DC3000, through reprogramming PEPRmediated defense signaling. Upon Pst DC3000 treatment, PROPEP genes were up-regulated at 24 hpi (hours postinoculation) and bak1 was more resistant at 3 dpi (days postinoculation) (Yamada et al., 2016). It suggested the resistance to Pst DC3000 found in bak1 is caused by activation of an additional immune response, the PEPR-mediated signaling. To wipe out the potential interference by secondary response in bak1, we examined the early responses of various plant lines to Pst DC3000 at 30 hpi. Instead, the single mutant bak1-3 or bkk1-1 was more susceptible to Pst DC3000, which is in line with previous studies showing that BAK1 serves as a co-receptor for multiple PRRs and the mutation of BAK1 causes impaired PTI and reduced resistance (Chinchilla et al., 2007; Heese et al., 2007; Schulze et al., 2010; Roux et al., 2011; Schwessinger et al., 2011). It is interesting that bak1-3 bkk1-1 double mutant was more resistant to Pst DC3000 (**Figure 6A**). In an flg22 treatment assay, we confirmed that the sensitivity of flagellin PAMP was almost abolished in bak1-4 bkk1-1 (**Figure 6C**). Thus, bak1 bkk1 double mutant maintains a dampened PTI but exhibits enhanced pathogen resistance. Therefore, additional immune signaling must be activated in bak1-3 bkk1-1. Our results showed that eds1 or pad4 can significantly suppress the cell death phenotype of bak1-3 bkk1-1, supporting the notion that ETI is inhibited by BAK1 and BKK1-mediated signaling (**Figure 7A**). It explained why bak1-3 bkk1-1 was more resistant to pathogen. Although EDS1-PAD4 complex was known to regulate SA via affecting ICS1-generated SA biosynthesis, the suppression of bak1-3 bkk1-1 by eds1 or pad4 appears to be SA-independent. bak1-3 bkk1-1 sid2-3 showed lesion phenotype when grown for an extended period of time. eds1 or pad4 suppressed the cell death of bak1-3 bkk1-1 sid2-3, suggesting EDS1-PAD4 regulate BAK1-mediated cell death signaling via a SA-independent manner (**Figure 7B**). In a recent report, it was shown that the major function of EDS1 and PAD4 in mediating ETI is SA-independent (Rietz et al., 2011). This study was in line with our results showing that sid2 only partially suppressed the cell death of bak1-3 bkk1-1 and that eds1 or pad4 further enhanced the suppression of cell death in bak1-3 bkk1-1 sid2-3.

Pathogen-derived effectors target key components mediating PTI in plant cell. FLS2, EFR, or CERK1 (CHITIN ELICITOR RECEPTOR KINASE 1) was shown to be the substrate of several

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effectors such as Pto, a kinase inhibitor, PtoB, a ubiquitin ligase, and HopAO1, a tyrosine phosphatase (Göhre et al., 2008; Xiang et al., 2008; Gimenez-Ibanez et al., 2009; Macho et al., 2014). The association between PRRs and effectors leads to dysfunction or degradation of PRRs, a strategy of pathogens to suppress plant immunity and enhance pathogenesis. BAK1, the co-receptor of several key PRRs, is also the target of several effectors, including PtoB, HopF2, an ADP-ribosyltransferase, and HopB1, a protease (Shan et al., 2008; Zhou et al., 2014; Li et al., 2016). It seems to be an efficient way for invading pathogens to attack a co-receptor in order to interrupt multiple PRR-mediated immune signalings. In our model, BAK1 and BKK1 may serve as guard proteins to surveil pathogen invasions. Depletion of BAK1 and BKK1 caused by effector attacks activates ETI, the stronger immunity featured by induction of cell death (HR), to enhance plant resistance to pathogens (**Figure 7C**).

In this study, we investigated the mechanism of how light functions as a primary factor in initiating cell death in bak1 bkk1. Our results supported light-induced overly expressed SA biosynthetic genes play a central role in this process. We also found key ETI components contribute to the cell death formation in bak1 bkk1. Future studies will focus on whether R proteins, the initiators of ETI signaling, function as downstream substrates of BAK1 and BKK1 and are engaged in the cell death induction in bak1 bkk1.

### AUTHOR CONTRIBUTIONS

KH, JL, and YG designed the experiments. YG, YW, JD, YZ, DS, JZ, and SZ performed the experiments. KH and YG analyzed the data and wrote the manuscript.

### ACKNOWLEDGMENTS

We thank Dr. Guojing Li for providing technical support for SA measurements, and the Arabidopsis Biological Resource Center for other seed stocks. These studies were supported by the grant from the National Natural Science Foundation of China to KH (31471305), the Ministry of Agriculture of the People's Republic of China (2016ZX08009-003-002), the Fundamental Research Funds for the Central Universities to KH (lzujbky-2016-75), and the grant from the National Natural Science Foundation of China to JL (31530005 and 31470380).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017. 00622/full#supplementary-material


signaling in Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1 and the nudix hydrolase NUDT7. Plant Cell 18, 1038–1051. doi: 10.1105/tpc.105.039982


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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