A Novel Sweetpotato Transcription Factor Gene IbMYB116 Enhances Drought Tolerance in Transgenic Arabidopsis

Several members of the MYB transcription factor family have been found to regulate growth, developmental processes, metabolism, and biotic and abiotic stress responses in plants. However, the role of MYB116 in plants is still unclear. In this study, a MYB transcription factor gene IbMYB116 was cloned and characterized from the sweetpotato [Ipomoea batatas (L.) Lam.] line Xushu55-2, a line that is considered to be drought resistant. We show here that IbMYB116 is a nuclear protein and that it possesses a transactivation domain at the C terminus. This gene exhibited a high expression level in the leaf tissues of Xushu55-2 and was strongly induced by PEG6000 and methyl-jasmonate (MeJA). The IbMYB116-overexpressing Arabidopsis plants showed significantly enhanced drought tolerance, increased MeJA content, and a decreased H2O2 level under drought stress. The overexpression of IbMYB116 in Arabidopsis systematically upregulated jasmonic acid (JA) biosynthesis genes and activated the JA signaling pathway as well as reactive oxygen species (ROS)-scavenging system genes under drought stress conditions. The overall results suggest that the IbMYB116 gene might enhance drought tolerance by activating a ROS-scavenging system through the JA signaling pathway in transgenic Arabidopsis. These findings reveal, for the first time, the crucial role of IbMYB116 in the drought tolerance of plants.


INTRODUCTION
Drought seriously affects the productivity of agricultural crops in the world (Yang et al., 2010;Boyer et al., 2013). Improving the drought tolerance of crops has become important for food security (Tester and Langridge, 2010;Zhu, 2016). Plants adapt to drought stress by developing a variety of mechanisms, including growth and development regulation, osmotic adjustment, ion homeostasis, and detoxification (Bohnert et al., 1995;Zhu, 2002). In response to water deficits, plants increase water uptake by forming long roots to promote their survival (Yu et al., 2008;Wang et al., 2016a). At the phytohormone level, jasmonic acid (JA), abscisic acid (ABA), salicylic acid (SA), and ethylene (ETH) play important roles in plant growth and development as well as protective responses against biotic and abiotic stresses (Fujita et al., 2005;Dong et al., 2014;Vargas et al., 2014;Zhang et al., 2015a;Jin et al., 2016;Xu et al., 2017).
The productivity of sweetpotato, Ipomoea batatas (L.) Lam., as an important food crop is seriously affected by drought stress. It has been shown that drought tolerance of sweetpotato could be improved through gene engineering (Park et al., 2011;Wang et al., 2016b;Fan et al., 2017;Kang et al., 2018a, Kang et al., 2018bKim et al., 2018). However, MYB TFs from sweetpotato have not been functionally characterized to date. In this study, we cloned a novel MYB TF gene named IbMYB116 from a droughttolerant sweetpotato line Xushu55-2 and found its overexpression in Arabidopsis enhanced drought tolerance through the JA signaling pathway.

Plant Materials
The drought-tolerant sweetpotato line Xushu55-2 was applied to isolate the IbMYB116 gene. The expressed sequence tag (EST) was screened from the transcriptome sequencing data of Xushu55-2 (Zhu et al., 2018). The function of IbMYB116 was identified using Arabidopsis thaliana Columbia-0.

Cloning and Sequence Analysis of IbMYB116 and Its Promoter
The total RNA from in vitro-grown plants of Xushu55-2 was transcribed into the first-strand cDNA (Kang et al., 2018a). The rapid amplification of cDNA ends (RACE) procedure was used to amplify the full-length cDNA of IbMYB116. The genomic sequence of IbMYB116 was amplified from the genomic DNA of in vitro-grown Xushu55-2. Its promoter was obtained from the genomic DNA of in vitro-grown Xushu55-2 by homology cloning strategy. All of the specific primers are listed in Supplementary  Table S1. IbMYB116 was analyzed with online BLAST, ORF Finder, DNAMAN software, Splign tool, and MEGA 7.0, and the cis-acting regulatory elements in its promoter region were defined online (Kang et al., 2018b).

Subcellular Localization of IbMYB116
For subcellular localization, the coding sequence of IbMYB116 was inserted into the pMDC83 vector. The fusion construct (35S::IbMYB116::GFP) and the empty vector (35S::GFP) were separately transformed into the EHA105 strain of Agrobacterium tumefaciens and further infiltrated the leaves of Nicotiana benthamiana (Strasser et al., 2007;Li et al., 2017). After 48 h of growth in a greenhouse, agroinfiltrated leaf sections were imaged at room temperature using a laser scanning confocal microscope with an Argon laser (LSM710, Zeiss, Germany). GFP was excited at 488 nm, and the emitted light was captured at 505-555 nm.

Transactivation Activity Assay of IbMYB116
The transactivation activity of IbMYB116 was tested in yeast (Saccharomyces cerevisiae) (Jiang et al., 2014). The full-length (construct 1) and a series of deletion mutations of IbMYB116 (constructs 2-9) were fused to the yeast expression vector pGBKT7 (pBD). The expression vector pBD-IbMYB116, pGAL4 (positive control), and pBD (negative control) were transferred into the host strain AH109. After the cells were cultured on the SD/-Trp medium, the yeast was further cultured on medium with X-α-Gal (SD/-Trp/-His/X).

Expression Analysis of IbMYB116 in Sweetpotato
The transcript levels of IbMYB116 in the roots, stems, and leaves of the in vitro-grown Xushu55-2 plants were analyzed using quantitative real-time PCR (qRT-PCR), the expression levels were normalized to Ibactin (AY905538), and the relative expression levels in different tissues were calibrated using the roots (Schmittgen and Livak, 2008;Liu et al., 2014). Furthermore, in vitro-grown Xushu55-2 plants were treated in Hoagland solution with H 2 O, 30% PEG6000, and 100 μM MeJA; the plants were sampled 0, 1, 3, 6, 12, and 24 h after treatments and then analyzed for the expression of IbMYB116. The expression levels were normalized to Ibactin (AY905538), and the relative expression levels in different treatments were calibrated using the plant sampled 0 h after treatment. The specific primers were designed in the nonconserved domain ( Figure S1 and Supplementary Table S1).

Production of the Transgenic Arabidopsis Plants
The overexpression vector pC3301-35S-IbMYB116 was generated by inserting the expression cassette 35S-IbMYB116 into pCAMBIA3301, and then, it was transferred into the A. tumefaciens strain GV3101. The transgenic Arabidopsis plants were produced and further grown in pots to obtain T 3 seeds as described by Kang et al. (2018b). The transcript levels of IbMYB116 in wild-type and transgenic lines were analyzed using qRT-PCR, and the expression levels were normalized to Atactin (NM112764) (Supplementary Table S1). The relative expression levels in transgenic lines were calibrated using the transgenic line with the lowest IbMYB116 expression.

Assay for Drought Tolerance
For the germination rate assay, T 3 and wild-type (WT) seeds sterilized with 2% NaClO for 5 min were sown on 1/2 MS medium with mannitol (0, 100, 200, and 300 mM). Approximately 50 seeds per line were sown for each experiment, and their germination rates were investigated after 3 days. For the root length assay, 5-dayold seedlings of T 3 and WT formed on 1/2 MS basal medium were vertically cultured on 1/2 MS medium with 0 (as a control) or 300 mM mannitol, and their roots were took out, and the primary root lengths were measured with a ruler after 3 weeks.
Furthermore, the 7-day-old seedlings obtained on 1/2 MS basal medium were transplanted in pots; after 2 weeks, they were stressed by a 4-week drought followed by 2-day rewatering for investigating their phenotypes. The 6-week normal treatment with water was used as a control.

Expression Analysis of the Related Genes and Measurements of MeJA and H 2 O 2 Contents
T 3 and WT plants were pot grown for 2 weeks under drought stress conditions after 2 weeks of normal treatment with water, and they were employed to analyze the expression of the genes involved in JA biosynthesis and signaling pathways as well as the reactive oxygen species (ROS)-scavenging system with qRT-PCR and Atactin (NM112764) as an internal control (Supplementary Table S1), and the relative expression levels in T 3 and WT plants were calibrated using WT. Their MeJA and H 2 O 2 contents were determined using high-performance liquid chromatography (HPLC) and an H 2 O 2 Assay Kit (Comin Biotechnology Co., Ltd. Suzhou, China), respectively. The 4-week normal treatment with water was used as a control.

Statistical Analysis
Three biological replicates were performed for all experiments. Difference analysis of data presented as the mean ± SE was done with Student's t-test (two-tailed analysis) using SPSS 20.0 Statistic Program. Significance levels at P < 0.05, P < 0.01, and P < 0.001 were indicated with * , * * , and * * * , respectively.

Cloning and Sequence Analysis of IbMYB116 and Its Promoter
The IbMYB116 full-length cDNA was 958 bp in length and contained an 849-bp ORF that encoded a polypeptide of 282 residues with a predicted molecular weight of 31.998 kDa and a pI of 6.79. The IbMYB116 protein had two conserved MYB domains that belonged to R2R3-MYB TFs and shared a high-sequence identity with MYB TFs from Ipomoea nil (InMYB21, XP_019189643.1, 91.23%), Glycine max (GmMYB184, NP_001235837.1, 49.12% and GmMYB84, NP_001235789.1, 43.83%), and Arabidopsis thaliana (AtMYB116, AT1G25340.1, 43.24%) ( Figure 1A). The protein was characterized with a highly conserved DNA-binding sequence at the N-terminus ( Figure 1A). Phylogenetic analysis along with 125 Arabidopsis R2R3-MYB TFs belonging to 24 groups (Dubos et al., 2010) revealed that IbMYB116 belonged to group S20 of the MYB family and had a close relationship with AtMYB116 (At1G25340.1) ( Figure 1B). The genomic DNA of IbMYB116 was 1,498 bp with three exons and two introns ( Figure 1C). Its promoter region (~1,455 bp) contained the cis-acting regulatory elements associated with stresses and phytohormones, including LTR, HSE, MBS, GARE-motif, CGTCA-motif, TGACG-motif, P-box, and SARE ( Figure S2).

Subcellular Location of IbMYB116
IbMYB116-GFP showed fluorescence in the nuclei of Nicotiana benthamiana leaf hypodermal cells, while the fluorescence of GFP was distributed in the entire cell (Figure 2). These results clearly indicated that IbMYB116 is a nuclear protein.

Expression of IbMYB116 in Sweetpotato
A significantly higher expression level of IbMYB116 was found in the leaves of Xushu55-2 than that in the roots and stems ( Figure S3). Its expression in Xushu55-2 was strongly induced by 30% PEG6000 and 100 μM MeJA, and it reached the highest level at 12 h (4.78-fold) and 24 h (4.15-fold), respectively (Figure 4).

Production of the Transgenic Arabidopsis Plants
The transgenic Arabidopsis plants were obtained using the protocols of Kang et al. (2018b). Eight transgenic plants named L1, L2, …, L8 were randomly selected to analyze the expression level of IbMYB116 with qRT-PCR ( Figure S1). The results showed that all of the transgenic plants exhibited significantly higher expression levels of IbMYB116 than the WT (Figure S4).

Enhanced Drought Tolerance
Three transgenic Arabidopsis plants (L1, L2, and L3) with high expression levels of IbMYB116 were selected for investigating their drought tolerance. The transgenic and WT plants showed no differences in germination rates on 1/2 MS media with 0 (control) and 100 mM mannitol (Figure 5). However, the transgenic plants provided significantly higher germination rates than the WT under 200 and 300 mM mannitol stress conditions (Figure 5). The transgenic and WT plants showed similar growth on 1/2 MS media without mannitol, but the transgenic plants formed significantly longer roots than the WT at the level of 300 mM mannitol (Figure 6).
Furthermore, the transgenic and WT plants grown in pots had no differences in growth without drought stress ( Figure 7A). However, after being stressed by drought, the transgenic plants exhibited better growth, increased MeJA content, and decreased H 2 O 2 content compared with the WT (Figures 7A, B). These results indicated that the transgenic

Expression of the Related Genes
Systematic upregulation of the JA biosynthesis key enzyme genes AtLOX, AtAOS, AtAOC, AtOPR, AtOPCL, AtACOX3, AtfadA, and AtACAA1, except for AtACOX1 (no change) and AtMFP2 (downregulated), was found in the transgenic Arabidopsis plants under drought stress conditions (Figure 8A). In the JA signaling pathway, AtCOI1 and AtJAZ were downregulated, but AtMYC2 was upregulated under drought stress conditions ( Figure 8B). The genes encoding ROS scavenging enzymes superoxide dismutase (SOD), glutathione peroxidase (GPX), and peroxidase (POD) were also upregulated, and both AtCAT and AtDHAR apparently had not changed ( Figure 8C). FIGURE 5 | Germination assay of the transgenic Arabidopsis and WT seeds sown for 3 days on 1/2 MS medium with 0, 100, 200, and 300 mM mannitol, respectively. * and ** indicate significant differences from WT at P < 0.05 and P < 0.01, respectively, according to the Student's t-test.
FIGURE 6 | Primary root lengths of the transgenic Arabidopsis and WT seedlings cultured for 3 weeks on 1/2 MS medium without (control) and with 300 mM mannitol, respectively. All the plants were grown in a standard square plate of 12 × 12 cm. Data are presented as the mean ± SE (n = 3). * and ** indicate significant differences from WT at P < 0.05 and P < 0.01, respectively, according to the Student's t-test.

IbMYB116 Enhances Drought Tolerance
It has been shown that several MYB TFs play crucial roles in the response to drought stress as positive stress response TFs (Rusconi et al., 2013;Lee et al., 2014;Yuan et al., 2015;Butt et al., 2017;Wang et al., 2017b). To date, the role of MYB116 in plants is still unknown. In this study, we cloned the IbMYB116 gene from the drought-tolerant sweetpotato line Xushu55-2 and found that the IbMYB116 protein belonged to R2R3-MYB TFs (Figure 1) (Jia et al., 2004;He et al., 2016). This gene was strongly induced by PEG6000 and MeJA, and its overexpression significantly improved the drought tolerance of the transgenic Arabidopsis plants (Figures 4-7). This study reveals, for the first time, the crucial role of IbMYB116 in the drought tolerance of plants.

IbMYB116 Upregulates JA Signaling Pathway Genes
JAs are naturally occurring signaling compounds that regulate plant responses to abiotic stresses, such as drought, salt, and ozone exposure (Rojo et al., 2003;Ahmad et al., 2016;Dhakarey et al., 2017). Rohwer and Erwin (2008) found that JA and JA-induced protein levels were increased in sorbitol-treated barley leaves, which confirmed the role of JAs in osmotic stress. MeJA enhanced drought tolerance by improving the water status of wheat plants (Ma et al., 2004) or increasing antioxidant WT stressed by a 4-week drought followed by 2 days rewatering after 2 weeks of normal treatment with water. The 6-week normal treatment was used as a control. (B) Contents of MeJA and H 2 O 2 in the leaves of the transgenic and WT plants grown for 4 weeks under normal treatment (control) and 2 weeks under drought stress after 2 weeks of normal treatment, respectively. *, **, and *** indicate significant differences from WT at P < 0.05, P < 0.01, and P < 0.001, respectively, according to the Student's t-test. activities in soybean plants (Anjum et al., 2011). In Arabidopsis, four 13-LOX forms (AtLOX2, 3, 4, and 6) contributed to JA formation in the stress response (Caldelari et al., 2011;Chauvin et al., 2013). The upregulation of OsLOX, OsAOS2, and OsOPR7 led to massive accumulation of endogenous JA in the rice rim1 mutant (Yoshii et al., 2010). The tolerance to drought as well as osmotic and salinity changes were increased in the Arabidopsis plants overexpressing CaLOX1 from pepper (Lim et al., 2015). The TaAOC1-overexpressing wheat and Arabidopsis plants accumulated more JA and exhibited enhanced tolerance to changes in salinity, which indicated that JA is related to the plant salinity response . The expression of OsOPR7 was induced by drought stress and wounding, and the increase in OsOPR7 expression led to an elevated endogenous JA level (Tani et al., 2008).
The JA signaling pathway plays an important role in regulating the plant response to drought stress Ahmad et al., 2016). As components of the core JA signaling pathway, COI1 participates in removing repressors of JA transduction (Xie et al., 1998;Ahmad et al., 2016); JAZs are transcription repressors of JA-responsive genes (Chini et al., 2007), and MYC2 acts as a positive regulator of JA signals (Zhang et al., 2015b). The overexpression of AtMYC2 in Arabidopsis improved osmotic stress tolerance (Abe et al., 2003). The OsMYC2-overexpressing rice exhibited increased resistance against Xoo (Uji et al., 2016).
Our results showed that overexpression of IbMYB116 in Arabidopsis systematically upregulated the JA biosynthesis genes, including AtLOX, AtAOS, AtAOC, AtOPR, AtOPCL, AtCOX3, AtfadA, and AtACAA1, and the MeJA content increased under drought stress conditions (Figures 7, 8). AtCOI1 and AtJAZ were downregulated, and AtMYC2 was upregulated in the transgenic Arabidopsis plants under drought stress conditions (Figure 8). These findings suggest that IbMYB116 enhances drought tolerance through the JA signaling pathway in transgenic Arabidopsis (Figure 9).

IbMYB116 Activates the ROS-Scavenging System
In plants, ROS cause damage to the structure and function of biomolecules, which leads to oxidative stress. H 2 O 2 , as one of the prominent ROS, is considered to be an important signaling molecule in plant cells (Wang et al., 2017a). The ROS-scavenging system can detoxify ROS to reduce oxidative damage in plant cells (Gill and Tuteja, 2010;Liu et al., 2014). It has been shown that JA can activate the ROS-scavenging system in plants. The overexpression of CaLOX1 in Arabidopsis systematically upregulated the ROS-scavenging system genes, which resulted in the reduced H 2 O 2 level under drought and salt stress conditions (Lim et al., 2015). The JA-deficient tomato mutant def-1 showed higher ROS levels than the WT under salt stress conditions (Abouelsaad and Renault, 2018). JA influenced oxidative stress through its direct effect on the activities of ROSscavenging enzymes in raspberry and strawberry (Wang, 1999;Ghasemnezhad and Javaherdashti, 2008). Reduced levels of JA led to more GLN18:3-induced ROS production in Arabidopsis and tomato (Block et al., 2018).
The results of this study demonstrated that ROS-scavenging system genes, including AtSOD, AtGPX, and AtPOD, were upregulated and that the H 2 O 2 level was decreased in the IbMYB116-overexpressing Arabidopsis plants under drought stress conditions (Figures 7, 8). These results suggest that IbMYB116 might enhance drought tolerance by activating the ROS-scavenging system through the JA signaling pathway in transgenic Arabidopsis (Figure 9).

CONCLUSION
We found a crucial role for IbMYB116 in the drought tolerance of plants. The results suggest that the overexpression of IbMYB116 might enhance drought tolerance by activating the ROS-scavenging system through the JA signaling pathway in transgenic Arabidopsis. This gene has the potential to be used for improving the drought tolerance of sweetpotato and other plants.

AUTHOR CONTRIBUTIONS
QL and YZ conceived and designed the experiments. YZ and HZu performed the experiments. YZ, SH, and HZa analyzed the data. QL, NZ, SX, and ZW contributed reagents, materials, and analysis tools. QL and YZ wrote the paper. All authors read and approved the final manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.01025/ full#supplementary-material FIGURE 9 | Diagram showing a proposed model for the regulation of IbMYB116 in drought tolerance for transgenic Arabidopsis. ↑, ↓and | indicate upregulation, downregulation, and no obvious change of expression for genes coding these enzymes, respectively.