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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Plant Sci.</journal-id>
<journal-title>Frontiers in Plant Science</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Plant Sci.</abbrev-journal-title>
<issn pub-type="epub">1664-462X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fpls.2023.1018984</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Plant Science</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Role of autophagy-related proteins ATG8f and ATG8h in the maintenance of autophagic activity in <italic>Arabidopsis</italic> roots under phosphate starvation</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Lin</surname>
<given-names>Li-Yen</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn003">
<sup>&#x2020;</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1958863"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Chow</surname>
<given-names>Hong-Xuan</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn003">
<sup>&#x2020;</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Chen</surname>
<given-names>Chih-Hao</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Mitsuda</surname>
<given-names>Nobutaka</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/101504"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Chou</surname>
<given-names>Wen-Chun</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1959377"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Liu</surname>
<given-names>Tzu-Yin</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/321813"/>
</contrib>
</contrib-group>
<aff id="aff1">
<sup>1</sup>
<institution>Institute of Bioinformatics and Structural Biology, College of Life Sciences and Medicine, National Tsing Hua University</institution>, <addr-line>Hsinchu</addr-line>, <country>Taiwan</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST)</institution>, <addr-line>Tsukuba</addr-line>, <country>Japan</country>
</aff>
<aff id="aff3">
<sup>3</sup>
<institution>Department of Life Science, College of Life Sciences and Medicine, National Tsing Hua University</institution>, <addr-line>Hsinchu</addr-line>, <country>Taiwan</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>Edited by: Petra Bauer, Heinrich Heine University of D&#xfc;sseldorf, Germany</p>
</fn>
<fn fn-type="edited-by">
<p>Reviewed by: Toshiro Shigaki, The University of Tokyo, Japan; Agnieszka Sirko, Polish Academy of Sciences, Poland</p>
</fn>
<fn fn-type="corresp" id="fn001">
<p>*Correspondence: Tzu-Yin Liu, <email xlink:href="mailto:tzliu@life.nthu.edu.tw">tzliu@life.nthu.edu.tw</email>
</p>
</fn>
<fn fn-type="equal" id="fn003">
<p>&#x2020;These authors have contributed equally to this work and share first authorship</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>26</day>
<month>06</month>
<year>2023</year>
</pub-date>
<pub-date pub-type="collection">
<year>2023</year>
</pub-date>
<volume>14</volume>
<elocation-id>1018984</elocation-id>
<history>
<date date-type="received">
<day>14</day>
<month>08</month>
<year>2022</year>
</date>
<date date-type="accepted">
<day>23</day>
<month>05</month>
<year>2023</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2023 Lin, Chow, Chen, Mitsuda, Chou and Liu</copyright-statement>
<copyright-year>2023</copyright-year>
<copyright-holder>Lin, Chow, Chen, Mitsuda, Chou and Liu</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>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.</p>
</license>
</permissions>
<abstract>
<p>Nutrient starvation-induced autophagy is a conserved process in eukaryotes. Plants defective in autophagy show hypersensitivity to carbon and nitrogen limitation. However, the role of autophagy in plant phosphate (Pi) starvation response is relatively less explored. Among the core autophagy-related (<italic>ATG</italic>) genes, <italic>ATG8</italic> encodes a ubiquitin-like protein involved in autophagosome formation and selective cargo recruitment. The <italic>Arabidopsis thaliana ATG8</italic> genes, <italic>AtATG8f</italic> and <italic>AtATG8h</italic>, are notably induced in roots under low Pi. In this study, we show that such upregulation correlates with their promoter activities and can be suppressed in the <italic>phosphate response 1</italic> (<italic>phr1</italic>) mutant. Yeast one-hybrid analysis failed to attest the binding of the <italic>At</italic>PHR1 transcription factor to the promoter regions of <italic>AtATG8f</italic> and <italic>AtATG8h</italic>. Dual luciferase reporter assays in <italic>Arabidopsis</italic> mesophyll protoplasts also indicated that <italic>At</italic>PHR1 could not transactivate the expression of both genes. Loss of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h leads to decreased root microsomal-enriched ATG8 but increased ATG8 lipidation. Moreover<italic>, atg8f/atg8h</italic> mutants exhibit reduced autophagic flux estimated by the vacuolar degradation of ATG8 in the Pi-limited root but maintain normal cellular Pi homeostasis with reduced number of lateral roots. While the expression patterns of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> overlap in the root stele, <italic>AtATG8f</italic> is more strongly expressed in the root apex and root hair and remarkably at sites where lateral root primordia develop. We hypothesize that Pi starvation-induction of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h may not directly contribute to Pi recycling but rely on a second wave of transcriptional activation triggered by PHR1 that fine-tunes cell type-specific autophagic activity.</p>
</abstract>
<kwd-group>
<kwd>
<italic>Arabidopsis</italic>
</kwd>
<kwd>phosphate starvation</kwd>
<kwd>autophagy</kwd>
<kwd>autophagy-related protein 8 (ATG8)</kwd>
<kwd>lateral root</kwd>
</kwd-group>
<contract-num rid="cn001">MOST 105-2621-M-007-001-MY3 , 108-2311-B-007-003-MY3</contract-num>
<contract-sponsor id="cn001">Ministry of Science and Technology, Taiwan<named-content content-type="fundref-id">10.13039/501100004663</named-content>
</contract-sponsor>
<counts>
<fig-count count="8"/>
<table-count count="0"/>
<equation-count count="0"/>
<ref-count count="96"/>
<page-count count="15"/>
<word-count count="8814"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-in-acceptance</meta-name>
<meta-value>Plant Nutrition</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1" sec-type="intro">
<title>Introduction</title>
<p>Autophagy is a highly conserved catabolic process in eukaryotes that maintains cellular homeostasis and contributes to stress adaptation (<xref ref-type="bibr" rid="B47">Marshall and Vierstra, 2018</xref>; <xref ref-type="bibr" rid="B24">Gross and Graef, 2020</xref>). It begins with the induction and nucleation of isolation membranes, followed by the formation of cup-shaped pre-autophagosome structures called phagophores, which eventually mature into closed double-membrane autophagosomes (<xref ref-type="bibr" rid="B92">Yoshimoto and Ohsumi, 2018</xref>; <xref ref-type="bibr" rid="B86">Wun et&#xa0;al., 2020</xref>). During the process, damaged or dispensable cytoplasmic components, protein aggregates, and dysfunctional organelles are enclosed in the autophagosome (<xref ref-type="bibr" rid="B92">Yoshimoto and Ohsumi, 2018</xref>; <xref ref-type="bibr" rid="B86">Wun et&#xa0;al., 2020</xref>). As the autophagosome reaches the vacuole or the lysosome, its outer membrane fuses with the vacuolar/lysosomal membrane and releases the autophagic bodies for degradation (<xref ref-type="bibr" rid="B92">Yoshimoto and Ohsumi, 2018</xref>; <xref ref-type="bibr" rid="B86">Wun et&#xa0;al., 2020</xref>). The breakdown products are then recycled for energy production or usage in biosynthetic pathways (<xref ref-type="bibr" rid="B92">Yoshimoto and Ohsumi, 2018</xref>; <xref ref-type="bibr" rid="B86">Wun et&#xa0;al., 2020</xref>). The biogenesis of autophagosome is stepwise and dynamic, and is driven by a large number of autophagy-related (ATG) genes that can be categorized into four functional groups (<xref ref-type="bibr" rid="B92">Yoshimoto and Ohsumi, 2018</xref>; <xref ref-type="bibr" rid="B86">Wun et&#xa0;al., 2020</xref>). The ATG1/ATG13 kinase complex stimulates autophagosome formation in response to the phosphorylation status of ATG13 (<xref ref-type="bibr" rid="B34">Kamada et&#xa0;al., 2000</xref>; <xref ref-type="bibr" rid="B71">Suttangkakul et&#xa0;al., 2011</xref>). The class III phosphatidylinositol 3-kinase (PI3K) complex containing VACUOLAR PROTEIN SORTING 34 (VPS34), ATG6 and ATG14, incorporates the phosphatidylinositol 3-phosphate (PI3P) phospholipids into the expanding phagophore (<xref ref-type="bibr" rid="B61">Russell et&#xa0;al., 2013</xref>). The ATG2-ATG18-ATG9 complex localizes to the edge of phagophore and delivers the lipid molecules for its expansion (<xref ref-type="bibr" rid="B45">Mari and Reggiori, 2007</xref>; <xref ref-type="bibr" rid="B96">Zhuang et&#xa0;al., 2017</xref>). The ATG12 and ATG8 ubiquitin-like conjugation systems, which consist of the E1-like ATG7, the E2-like ATG3 and ATG10 and the E3-like ATG12-ATG5 conjugate together with ATG16, participate in autophagosome maturation (<xref ref-type="bibr" rid="B22">Geng and Klionsky, 2008</xref>). Of note, the ubiquitin-like protein ATG8, through its covalent conjugation to the lipid phosphatidylethanolamine (&#x200b;PE), plays a central role in both bulk and selective autophagy (<xref ref-type="bibr" rid="B47">Marshall and Vierstra, 2018</xref>; <xref ref-type="bibr" rid="B9">Bu et&#xa0;al., 2020</xref>). Although ATG8 interacts with diverse receptors or adaptor proteins to recruit specific cargos for degradation, autophagy-independent function of ATG8 has also been reported (<xref ref-type="bibr" rid="B47">Marshall and Vierstra, 2018</xref>; <xref ref-type="bibr" rid="B9">Bu et&#xa0;al., 2020</xref>). In addition, ATG8 is used as a reliable marker to monitor autophagic degradation activity upon the inhibition of vacuolar/lysosomal degradation by protease inhibitors (<xref ref-type="bibr" rid="B37">Klionsky et&#xa0;al., 2021</xref>).</p>
<p>Unlike a single-copy <italic>ATG8</italic> gene in yeast and algae, the plant <italic>ATG8</italic> gene family has significantly expanded and some members are upregulated under various biotic and abiotic stresses (<xref ref-type="bibr" rid="B36">Kellner et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B9">Bu et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B56">Qi et&#xa0;al., 2021</xref>). Selective interaction of various ATG8 isoforms (ATG8s) with their protein targets may contribute to the diversification of autophagy pathways in plants (<xref ref-type="bibr" rid="B72">Svenning et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B36">Kellner et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B8">Boycheva Woltering and Isono, 2020</xref>; <xref ref-type="bibr" rid="B33">Jung et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B84">Wu et&#xa0;al., 2021</xref>). In the model plant <italic>Arabidopsis thaliana</italic>, nine <italic>ATG8</italic> genes were identified and classified into three separate groups. Intriguingly, the <italic>AtATG8h-i</italic> group have a characteristic C-terminal exposed glycine residue that does not require ATG4 protease-dependent cleavage prior to their lipidation (<xref ref-type="bibr" rid="B66">Seo et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B36">Kellner et&#xa0;al., 2017</xref>). Although the analysis of the <italic>At</italic>ATG8 gene family is incomplete, the expression of several <italic>At</italic>ATG8 genes showed different yet partially overlapping patterns (<xref ref-type="bibr" rid="B67">Sl&#xe1;vikov&#xe1; et&#xa0;al., 2005</xref>; <xref ref-type="bibr" rid="B8">Boycheva Woltering and Isono, 2020</xref>), supporting that different ATG8s share redundant roles while individual ATG8 members may have distinct and specific functions. Therefore, it remains challenging to distinguish the impact of each ATG8 isoform merely based on characterization of single knockouts due to functional redundancy.</p>
<p>Although most of the plant <italic>ATG</italic> genes are expressed at a ubiquitous and basal level, they can be induced by various developmental cues and environmental stimuli (<xref ref-type="bibr" rid="B91">Yoshimoto et&#xa0;al., 2004</xref>; <xref ref-type="bibr" rid="B67">Sl&#xe1;vikov&#xe1; et&#xa0;al., 2005</xref>; <xref ref-type="bibr" rid="B74">Thompson et&#xa0;al., 2005</xref>; <xref ref-type="bibr" rid="B59">Rose et&#xa0;al., 2006</xref>; <xref ref-type="bibr" rid="B53">Peng et&#xa0;al., 2007</xref>; <xref ref-type="bibr" rid="B15">Chung et&#xa0;al., 2009</xref>; <xref ref-type="bibr" rid="B3">Avin-Wittenberg et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B58">Rodriguez et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B56">Qi et&#xa0;al., 2021</xref>). Ectopic overexpression of certain <italic>ATGs</italic> in plants successfully upregulated autophagy for plant fitness and stress tolerance (<xref ref-type="bibr" rid="B87">Xia et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B39">Li et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B82">Wang et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B81">Wang et&#xa0;al., 2017a</xref>; <xref ref-type="bibr" rid="B83">Wang et&#xa0;al., 2017b</xref>; <xref ref-type="bibr" rid="B3">Avin-Wittenberg et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B48">Minina et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B69">Sun et&#xa0;al., 2018a</xref>; <xref ref-type="bibr" rid="B70">Sun et&#xa0;al., 2018b</xref>). Compared to the extensive identification of transcription factors (TFs) regulating <italic>ATGs</italic> in animal and yeast cells, only a few TFs were discovered for their role in activation or repression of <italic>ATGs</italic> in plants. In cassava, WRKY20 was identified as a transcriptional activator of <italic>ATG8a</italic> (<xref ref-type="bibr" rid="B89">Yan et&#xa0;al., 2017</xref>). In nitrogen (N)-starved tomato leaves, the brassinosteroid (BR)-activated TF BRASSINAZOLE-RESISTANT1 (BZR1) binds to the promoters of <italic>ATG2</italic> and <italic>ATG6</italic> and induces autophagosome formation (<xref ref-type="bibr" rid="B77">Wang et&#xa0;al., 2019</xref>). The tomato heat shock TF HsfA1a was shown to upregulate the expression of <italic>ATG10</italic> and <italic>ATG18f</italic> and thereby inducing autophagy for drought tolerance (<xref ref-type="bibr" rid="B76">Wang et&#xa0;al., 2015</xref>). Recently, a study using yeast one-hybrid (Y1H) screening has revealed the binding of 225 TFs to the promoter of several <italic>AtATG8s</italic> (<xref ref-type="bibr" rid="B79">Wang et&#xa0;al., 2020</xref>). However, only the basic leucine-zipper protein TF TGA9 was further validated to transcriptionally upregulate the expression of <italic>AtATG8b</italic> and <italic>AtATG8e</italic> (<xref ref-type="bibr" rid="B79">Wang et&#xa0;al., 2020</xref>).</p>
<p>Inorganic phosphate (Pi) is an essential nutrient to plants for their growth and reproduction, but is poorly accessible to plants in most soils (<xref ref-type="bibr" rid="B44">Manning, 2008</xref>). To cope with the low availability of Pi, plants acquire a series of metabolic and morphological strategies, including enhancing Pi acquisition and remobilization, increasing exudation of organic acid and phosphatase, and remodeling of root architecture (<xref ref-type="bibr" rid="B16">Crombez et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B78">Wang et&#xa0;al., 2021</xref>; <xref ref-type="bibr" rid="B52">Paz-Ares et&#xa0;al., 2022</xref>). Several TFs were identified to be responsible for the regulation of Pi starvation-responsive (PSR) genes (<xref ref-type="bibr" rid="B29">Jain et&#xa0;al., 2012</xref>). Among them, PHOSPHATE STARVATION RESPONSE1 (PHR1) has been extensively studied and shown to act as a master regulator of PSR genes (<xref ref-type="bibr" rid="B60">Rubio et&#xa0;al., 2001</xref>; <xref ref-type="bibr" rid="B10">Bustos et&#xa0;al., 2010</xref>). In <italic>Arabidopsis</italic>, nearly 2,000 PSR genes are controlled by PHR1, perhaps <italic>via</italic> binding to the PHR1-binding sites (P1BS) (<xref ref-type="bibr" rid="B11">Castrillo et&#xa0;al., 2017</xref>). Although PHR1 is weakly transcriptionally responsive to low Pi stress, its activity is regulated by the nuclear SPX (SYG1/Pho81/XPR1) domain proteins (<xref ref-type="bibr" rid="B5">Bari et&#xa0;al., 2006</xref>; <xref ref-type="bibr" rid="B55">Puga et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B80">Wang et&#xa0;al., 2014</xref>). Moreover, an increased number of lateral roots is often regarded as a typical adaptive response to Pi limitation in <italic>Arabidopsis</italic> and in species that produce cluster roots (<xref ref-type="bibr" rid="B18">Desnos, 2008</xref>; <xref ref-type="bibr" rid="B16">Crombez et&#xa0;al., 2019</xref>). Such phenotypic change may generate a greater number of root tips to enlarge the potential hotspots for Pi uptake (<xref ref-type="bibr" rid="B35">Kanno et&#xa0;al., 2016</xref>). Nevertheless, the results from many other studies in <italic>Arabidopsis</italic> as well as in other species were occasionally in disagreement with the increased lateral root response upon Pi starvation (<xref ref-type="bibr" rid="B16">Crombez et&#xa0;al., 2019</xref>).</p>
<p>Compared to the wealth of investigations on carbon (C) and N starvation-induced autophagy (<xref ref-type="bibr" rid="B2">Avila-Ospina et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B26">Hav&#xe9; et&#xa0;al., 2017</xref>), the mechanism by which plant cells sense Pi limitation and induce autophagy is relatively less explored. An early study using tobacco BY-2 cells expressing aggregate-prone fluorescent proteins showed that Pi deprivation induced autophagy to remove the aggregates (<xref ref-type="bibr" rid="B75">Toyooka et&#xa0;al., 2006</xref>; <xref ref-type="bibr" rid="B73">Tasaki et&#xa0;al., 2014</xref>). Recent analysis of GFP-<italic>At</italic>ATG8a-labeled autophagic structures also suggested that low P induced the autophagosome formation in <italic>Arabidopsi</italic>s root tips and such responses were exaggerated in the <italic>pdr2</italic> but attenuated in the <italic>pdr2/ire1a</italic> mutants, thereby linking Pi limitation-induced autophagy to the ER stress-dependent signaling pathway (<xref ref-type="bibr" rid="B50">Naumann et&#xa0;al., 2019</xref>). In addition, when Pi limitation was combined with a reduced C/N ratio, Rubisco-containing body (RCB)-mediated chlorophagy was induced (<xref ref-type="bibr" rid="B93">Yoshitake et&#xa0;al., 2021</xref>). Our recent study revealed that low Pi preferentially increased the autophagic flux in the differential zone of the <italic>Arabidopsis</italic> root and most <italic>AtATG</italic> genes are highly induced by N starvation but moderately upregulated by Pi starvation (<xref ref-type="bibr" rid="B12">Chiu et&#xa0;al., 2023</xref>). Among the <italic>AtATG8</italic> family, <italic>AtATG8a</italic>, <italic>AtATG8f</italic>, <italic>AtATG8g</italic> and <italic>AtATG8h</italic> were upregulated by Pi starvation in the shoot, but only <italic>AtATG8f</italic> and <italic>AtATG8h</italic> were strikingly upregulated in the Pi-deprived root (<xref ref-type="bibr" rid="B12">Chiu et&#xa0;al., 2023</xref>). In this study, we further investigated the Pi starvation-induced transcriptional regulation of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> and their spatial expression patterns. We also explored the physiological implication of Pi starvation-induced upregulation of <italic>AtATG8f</italic> and <italic>AtATG8h</italic>. Characterization of the <italic>atg8f</italic>/<italic>atg8h</italic> double mutants showed that loss of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h reduces the autophagic activities of root under Pi starvation but does not affect the cellular Pi levels. In addition, the <italic>atg8f</italic>/<italic>atg8h</italic> double mutants exhibited decreased number of lateral roots under both Pi-replete and Pi-deplete conditions but not under N-starved conditions. Although Pi starvation-induced upregulation of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> is PHR1-dependent, the results of Y1H and dual luciferase analyses indicated that PHR1 may not directly transactivate these two genes. As <italic>AtATG8f</italic> and <italic>AtATG8h</italic> are strongly expressed in the root stele tissues and involved in the lateral root development, we hypothesize that PHR1 may act upstream of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h to fine-tune the root cell type-specific autophagic activity under Pi starvation.</p>
</sec>
<sec id="s2" sec-type="results">
<title>Results</title>
<sec id="s2_1">
<title>Pi deficiency induces the expression of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in a <italic>At</italic>PHR1-dependent manner</title>
<p>Our recent study has revealed that Pi limitation upregulated the expression of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> among the <italic>ATG8</italic> family (<xref ref-type="bibr" rid="B12">Chiu et&#xa0;al., 2023</xref>). We further monitored the expression of these two genes in the wild-type (WT) plants at 24-, 48-, and 72-hour time points following Pi deprivation as well as in the <italic>pho1-2</italic> mutant, which exhibits extremely low shoot levels of Pi (<xref ref-type="bibr" rid="B54">Poirier et&#xa0;al., 1991</xref>). The progressive increase of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> transcripts during Pi limitation (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1A</bold>
</xref>) as well as the exacerbated upregulation of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in the shoot and/or root of <italic>pho1-2</italic> under Pi limitation (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1B</bold>
</xref>) suggested that <italic>AtATG8f</italic> and <italic>AtATG8h</italic> are induced according to the magnitude of Pi deficiency. We were then prompted to determine which TFs are involved in such upregulation. To find out whether <italic>AtATG8f</italic> and <italic>AtATG8h</italic> could be upregulated by <italic>At</italic>PHR1, we set out to search for potential <italic>cis</italic>-elements in the promoter region of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> that may be recognized by <italic>At</italic>PHR1. By using the PlantPan3.0 server (<xref ref-type="bibr" rid="B13">Chow et&#xa0;al., 2019</xref>), we found two and three putative P1BS elements in the proximal promoter of <italic>AtATG8f</italic> and <italic>AtATG8h</italic>, respectively (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2A</bold>
</xref>; <xref ref-type="supplementary-material" rid="SM1">
<bold>Table S1</bold>
</xref>). To validate whether <italic>At</italic>PHR1 participates in the regulation of <italic>AtATG8f</italic> and <italic>AtATG8h</italic>, we examined the expression of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in the <italic>phr1-3</italic> mutant (<xref ref-type="bibr" rid="B60">Rubio et&#xa0;al., 2001</xref>; <xref ref-type="bibr" rid="B57">Ren et&#xa0;al., 2012</xref>). The Pi starvation upregulation of <italic>AtATG8f AtATG8h</italic> was suppressed in both the shoot and root of <italic>phr1-3</italic> (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2B</bold>
</xref>), indicating that Pi limitation induces the expression of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in a PHR1-dependent manner.</p>
<fig id="f1" position="float">
<label>Figure&#xa0;1</label>
<caption>
<p>Low Pi induction of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> is progressive and exacerbated in the <italic>pho1-2</italic> mutant. <bold>(A)</bold> Fold-change of expression of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in the shoot (S) and root (R) of 11-day-old <italic>Arabidopsis</italic> WT seedling following 24, 48 and 72 hours of Pi starvation (&#x2013;P, 0 &#xb5;M KH<sub>2</sub>PO<sub>4</sub>) conditions as determined by qRT-PCR. Error bars represent SE (n = 3, biological replicate pools of 20 seedlings collected from three independent experiments). <sup>+++</sup>P&lt; 0.001 <sup>++</sup>P&lt; 0.01, <sup>+</sup>P&lt; 0.05 compared to Pi-sufficient conditions; Student&#x2019;s <italic>t</italic>-test; two-tailed. <bold>(B)</bold> Fold-change of expression of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> expression in the shoot and root of 11-day-old <italic>Arabidopsis</italic> WT and <italic>pho1-2</italic> seedling under Pi-sufficient (+P, 250 &#xb5;M KH<sub>2</sub>PO<sub>4</sub>) and Pi-deficient (&#x2013;P, 0 &#xb5;M KH<sub>2</sub>PO<sub>4</sub>, 3 days of starvation) conditions as determined by qRT-PCR. <italic>AtATG8b</italic> expression was used for comparison. Error bars represent SE (n = 3, biological replicate pools of 20 seedlings collected from three independent experiments). <sup>+++</sup>P&lt; 0.001 <sup>++</sup>P&lt; 0.01 <sup>+</sup>P&lt; 0.05 compared to Pi-sufficient conditions within the same genotype; Student&#x2019;s <italic>t</italic>-test; two-tailed.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-14-1018984-g001.tif"/>
</fig>
<fig id="f2" position="float">
<label>Figure&#xa0;2</label>
<caption>
<p>PHR1-dependent Pi starvation-induced upregulation of <italic>AtATG8f</italic> and <italic>AtATG8h.</italic> <bold>(A)</bold> Putative PHR1 binding sites (P1BS) predicted by the matrix TF_motif_seq_0434 (white diamond) and TFmatrixID_0351 (black diamond) in the proximal promoter of <italic>AtATG8f, AtATG8h</italic>, and <italic>AtIPS1</italic>. TSS, transcription start site; UTR, untranslated region. <bold>(B)</bold> Fold-change of expression of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in the shoot and root of 11-day-old <italic>Arabidopsis</italic> WT and <italic>phr1-3</italic> seedlings grown under Pi-sufficient (+P, 250 &#xb5;M KH<sub>2</sub>PO<sub>4</sub>) and Pi-deficient (&#x2013;P, 0 &#xb5;M KH<sub>2</sub>PO<sub>4</sub>, 3 days of starvation) conditions as determined by qRT-PCR. Error bars represent SE (n = 3, biological replicate pools of 20 seedlings collected from three independent experiments). <sup>+++</sup>P&lt; 0.001 <sup>++</sup>P&lt; 0.01 <sup>+</sup>P&lt; 0.05 compared to Pi-sufficient conditions within the same genotype; <sup>**</sup>P&lt; 0.01, compared to Pi-deficient WT; Student&#x2019;s <italic>t</italic>-test; two-tailed.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-14-1018984-g002.tif"/>
</fig>
</sec>
<sec id="s2_2">
<title>
<italic>At</italic>PHR1 does not directly transactivate the expression of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in <italic>Arabidopsis</italic> mesophyll protoplasts</title>
<p>In our initial attempt to search for potential TFs that bind to the promoter region of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> by Y1H, we surprisingly failed to identify <italic>At</italic>PHR1 as a positive candidate (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S1</bold>
</xref>). In parallel, we performed transient dual-luciferase reporter assays using <italic>Arabidopsis</italic> mesophyll protoplasts to test whether <italic>At</italic>PHR1 transactivates the expression of <italic>AtATG8f</italic> and <italic>AtATG8h in planta</italic>. For the reporter constructs encoding firefly luciferase (LUC) and <italic>Renilla</italic> luciferase (REN), the genomic sequences of each promoter were cloned into the pGreenII-0800-Luc vector (<xref ref-type="bibr" rid="B27">Hellens et&#xa0;al., 2005</xref>) (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3A</bold>
</xref>). For the effector construct, we used the &#xdf;-estradiol-inducible XVE expression system in the pGPTVII backbone to express TFs (<xref ref-type="bibr" rid="B64">Schl&#xfc;cking et&#xa0;al., 2013</xref>) (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3A</bold>
</xref>). In addition, the reporter construct carrying the promoter sequences of <italic>AtIPS1</italic> containing two P1BS elements was used as the positive control (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3A</bold>
</xref>) (<xref ref-type="bibr" rid="B10">Bustos et&#xa0;al., 2010</xref>). When <italic>At</italic>PHR1 was co-expressed with <italic>P<sub>IPS1</sub>
</italic>:LUC/<italic>P<sub>35S</sub>
</italic>:REN, the ratio of LUC : REN was increased to 2.9-fold as compared to the negative control in which GFP was co-expressed (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3B</bold>
</xref>). When we co-expressed the NAC domain TF <italic>At</italic>ATAF2 as a positive control with <italic>P<sub>ATG8h</sub>
</italic>:LUC/<italic>P<sub>35S</sub>
</italic>:REN (<xref ref-type="bibr" rid="B79">Wang et&#xa0;al., 2020</xref>), the ratio of LUC : REN was increased by 1.8-fold (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3B</bold>
</xref>). In comparison, when <italic>At</italic>PHR1 was co-expressed with <italic>P<sub>ATG8f</sub>
</italic>:LUC/<italic>P<sub>35S</sub>
</italic>:REN or <italic>P<sub>ATG8h</sub>
</italic>:LUC/<italic>P<sub>35S</sub>
</italic>:REN, the ratio of LUC : REN was similar to that of the GFP control (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3B</bold>
</xref>). These results indicated that <italic>At</italic>PHR1 may not directly transactivate <italic>AtATG8f</italic> and <italic>AtATG8h.</italic>
</p>
<fig id="f3" position="float">
<label>Figure&#xa0;3</label>
<caption>
<p>Transactivation of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> promoters not by <italic>At</italic>PHR1 in <italic>Arabidopsis</italic> mesophyll protoplasts. <bold>(A)</bold> Schematic design of the reporter and effector constructs used for dual-luciferase assay (not drawn in scale). 35S promoter: CaMV 35S promoter; POI, promoter of interest; P16&#x394;S: a constitutive promoter; XVE: a chimeric transcription activator; LexA: an operator; min.35S: minimal 35S promoter; TF, transcription factor. <bold>(B)</bold> The relative LUC: REN ratios for the co-expression of the reporter construct containing the <italic>AtIPS1</italic>, <italic>AtATG8f</italic>, or <italic>AtATG8h</italic> promoter with the effector construct containing the transcription factor <italic>At</italic>PHR1 or <italic>At</italic>ATAF2. The co-expression of the effector construct expressing GFP and the corresponding reporter construct was taken as the negative control (NC). Data represent mean &#xb1; S.E. of biological replicates from independent experiments (n = 4 for the <italic>AtATG8f</italic> promoter and n = 3 for the <italic>AtIPS1</italic> and <italic>AtATG8h</italic> promoters). <sup>*</sup>P&lt; 0.05, compared to NC; Student&#x2019;s <italic>t</italic>-test; two-tailed.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-14-1018984-g003.tif"/>
</fig>
</sec>
<sec id="s2_3">
<title>Loss of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h does not impair cellular Pi homeostasis</title>
<p>To investigate the physiological role of <italic>AtATG8f</italic> and <italic>AtATG8h</italic>, we obtained the homozygous T-DNA lines for each gene: <italic>atg8f-2</italic>, <italic>atg8f-3</italic>, <italic>atg8f-5</italic>, and <italic>atg8f-6</italic> for <italic>AtATG8f</italic> and <italic>atg8h-2</italic> and <italic>atg8h-3</italic> for <italic>AtATG8h</italic> (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S2</bold>
</xref>; <xref ref-type="supplementary-material" rid="SM1">
<bold>Table S2</bold>
</xref>). By reverse transcription polymerase chain reaction (RT-PCR), we validated that the full-length transcripts of <italic>AtATG8f</italic> were absent in the <italic>atg8f-2</italic> and <italic>atg8f-5</italic> homozygotes (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S2</bold>
</xref>), indicating that these two mutants carry null alleles. We only chose <italic>atg8f-5</italic> (hereafter referred to as <italic>atg8f</italic>) for further study because the T-DNA insertion site in this mutant was closer to the 5&#x2019; untranslated region (UTR) of <italic>AtATG8f</italic>, which likely resulted in complete disruption of the transcription. On the other hand, the full-length transcripts of <italic>AtATG8h</italic> were not detected in both the <italic>atg8h-2</italic> and <italic>atg8h-3</italic> mutants. Nevertheless, we were able to detect some truncated transcripts in <italic>atg8h-2</italic> (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S2</bold>
</xref>), and therefore <italic>atg8h-3</italic> (hereafter referred to as <italic>atg8h</italic>) was used. Through crosses we also successfully generated the <italic>atg8f-5/atg8h-3</italic> double mutant (hereafter referred to as <italic>atg8f/atg8h</italic>). The expression of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> was induced in the WT Pi-starved roots but not detectable in <italic>atg8f/atg8h</italic> under both Pi-replete and Pi-deplete conditions (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S2</bold>
</xref>). Of note, the transcript expression of the other <italic>AtATG8</italic> genes was comparable in WT and <italic>atg8f/atg8h</italic> (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S3</bold>
</xref>), suggesting no compensatory upregulation of the other <italic>AtATG8</italic> members for the loss of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in the double mutant. To investigate whether <italic>At</italic>ATG8f and <italic>At</italic>ATG8h could be involved in the maintenance of cellular Pi homeostasis, we measured the shoot and root Pi levels of <italic>atg8f, atg8h</italic>, and <italic>atg8f/atg8h</italic>. All of them showed no difference from WT under both Pi-replete and Pi-deplete conditions (<xref ref-type="fig" rid="f4">
<bold>Figures&#xa0;4A, B</bold>
</xref>), suggesting that defective <italic>AtATG8f</italic> and <italic>AtATG8h</italic> do not affect cellular Pi levels.</p>
<fig id="f4" position="float">
<label>Figure&#xa0;4</label>
<caption>
<p>Pi levels of <italic>atg8f</italic>, <italic>atg8h</italic> and <italic>atg8f/atg8h</italic> mutants. <bold>(A, B)</bold> The shoot and root Pi levels of 11-day-old <italic>Arabidopsis</italic> seedlings of WT, <italic>atg8f</italic>, and <italic>atg8h</italic> <bold>(A)</bold> and <italic>atg8f/atg8h</italic> <bold>(B)</bold> under Pi-sufficient (+P, 250 &#xb5;M KH<sub>2</sub>PO<sub>4</sub>) and Pi-deficient (&#x2013;P, 0 &#xb5;M KH<sub>2</sub>PO<sub>4</sub>, 3 days of starvation) conditions. Error bars represent SE (n = 9, biological replicate pools of 10 seedlings collected from three independent experiments).</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-14-1018984-g004.tif"/>
</fig>
</sec>
<sec id="s2_4">
<title>
<italic>At</italic>ATG8f and <italic>At</italic>ATG8h account for the maintenance of autophagic flux in the root under Pi starvation</title>
<p>To evaluate whether the low Pi induction of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> may change autophagic activities, we attempted to compare autophagic flux between WT and <italic>atg8f/atg8h</italic>. The GFP-ATG8 cleavage assay is a widely accepted tool to measure autophagic flux by calculating the ratio of the amount of cleaved GFP to the amount of full-length GFP-ATG8 (<xref ref-type="bibr" rid="B37">Klionsky et&#xa0;al., 2021</xref>). However, this approach would unfortunately introduce additional ATG8s into <italic>atg8f/atg8h</italic>. We therefore conducted the ATG8 degradation assay to estimate the autophagic flux in the root of <italic>atg8f/atg8h</italic>. As the steady-state abundance of ATG8s can be influenced by autophagy activation or blockage of downstream steps such as inefficient vacuolar fusion or decreased degradation (<xref ref-type="bibr" rid="B94">Zhang et&#xa0;al., 2013</xref>), the vacuolar H<sup>+</sup>-ATPase inhibitor concanamycin A (Conc A) was applied to prevent ATG8s from vacuolar degradation (<xref ref-type="bibr" rid="B21">Dr&#xf6;se et&#xa0;al., 1993</xref>; <xref ref-type="bibr" rid="B7">Bowman and Bowman, 2005</xref>). Without the availability of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h-specific antibodies, we performed immunoblotting with a polyclonal anti-ATG8s antibody that recognize all the <italic>At</italic>ATG8 isoforms (ATG8s). Regardless of Pi status, ATG8s were found to accumulate in the WT root upon Conc A treatment (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5A</bold>
</xref>). In the absence of Conc A, the abundance of ATG8s in the total root proteins was comparable in <italic>atg8f/atg8h</italic> and WT (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5A</bold>
</xref>). This may be because only a small proportion of ATG8s were contributed by <italic>At</italic>ATG8f and <italic>At</italic>ATG8h transcripts (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S3</bold>
</xref>). Nonetheless, the relative autophagic flux in the WT root calculated based on the changes of ATG8s between DMSO control and Conc A treatment showed no differences between Pi-replete and Pi-depleted conditions (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5B</bold>
</xref>). These results were in good agreement with our recent findings (<xref ref-type="bibr" rid="B12">Chiu et&#xa0;al., 2023</xref>). Notably, the autophagic flux was comparable in the Pi-repleted root of <italic>atg8f/atg8h</italic> and WT but reduced in the Pi-depleted root of <italic>atg8f/atg8h</italic> (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5A, B</bold>
</xref>). Given that the abundance of membrane-associated ATG8s would correlate with autophagic activity, we then compared the amount of ATG8s in the root microsomal fraction between WT and <italic>atg8f/atg8h</italic>. While the microsomal-enriched ATG8s was missing in the autophagy-defective <italic>atg7-3</italic> mutant, it was slightly reduced in the Pi-deplete root of WT (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S4</bold>
</xref>). There was a substantial decrease of microsomal-enriched ATG8s in the root of <italic>atg8f/atg8h</italic> as compared to WT, but no significant difference was found between Pi-replete and Pi-deplete root of <italic>atg8f/atg8h</italic> (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S4</bold>
</xref>). We then further examined ATG8s lipidation in WT and <italic>atg8f/atg8h</italic> by immunoblot. Because to distinguish lipidated ATG8s from non-lipidated ATG8s using immunoblot analyses was reported to be technically challenging due to the multiple variants in plants (<xref ref-type="bibr" rid="B91">Yoshimoto et&#xa0;al., 2004</xref>; <xref ref-type="bibr" rid="B14">Chung et&#xa0;al., 2010</xref>), we applied phospholipase D (PLD) treatment, which hydrolyzes the terminal phosphodiester bonds of phospholipids to produce phosphatidic acid (PA). The PLD-mediated cleavage of ATG8-PE yields ATG8-ethanolamine and PA, thus helping identify bands that correspond to lipidated ATG8s. The lipidated ATG8s migrated faster than the unmodified form during SDS-PAGE in the presence of urea and were sensitive to PLD digestion and absent in the <italic>atg5</italic> and <italic>atg7</italic> backgrounds (<xref ref-type="bibr" rid="B91">Yoshimoto et&#xa0;al., 2004</xref>; <xref ref-type="bibr" rid="B14">Chung et&#xa0;al., 2010</xref>; <xref ref-type="bibr" rid="B71">Suttangkakul et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B40">Li et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B96">Zhuang et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B43">Luo and Zhuang, 2018</xref>). Our results indicated that Pi starvation did not change the abundance of lipidated ATG8s in the WT root, but the lipidated ATG8s was unexpectedly increased in the Pi-replete root of <italic>atg8f/atg8h</italic> and remained a similar level or slightly declined following Pi starvation (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S5</bold>
</xref>).</p>
<fig id="f5" position="float">
<label>Figure&#xa0;5</label>
<caption>
<p>
<italic>AtATG8f</italic> and <italic>AtATG8h</italic> are required for the maintenance of autophagic flux in the Pi-starved roots. <bold>(A)</bold> Immunoblot analysis of the expression of ATG8s in the root of 11-day-old <italic>Arabidopsis</italic> WT and <italic>atg8f/atg8h</italic> seedlings under Pi-sufficient (+P, 250 &#xb5;M KH<sub>2</sub>PO<sub>4</sub>) and Pi-deficient (&#x2013;P, 0 &#xb5;M KH<sub>2</sub>PO<sub>4</sub>, 3 days of starvation) conditions with or without Conc A treatment (1 &#xb5;M, 6&#xa0;h). Representative images are shown. Arrowhead indicates the bands of ATG8s. <bold>(B)</bold> The expression change of ATG8s and the autophagic flux in the root of <italic>Arabidopsis</italic> WT and DM (<italic>atg8f/atg8h</italic>) seedlings. The expression level of ATG8s was normalized with the corresponding actin. Error bars represent SE (n = 3, biological replicate pools of 20 seedlings collected from three independent experiments). <sup>++</sup>P&lt; 0.01, compared to Pi-sufficient DM with Conc A treatment; Student&#x2019;s <italic>t</italic>-test; two-tailed. The relative autophagic flux was calculated by dividing the normalized ATG8s signal intensity of Conc A-treated samples by that of DMSO controls. Amido black staining was used for total protein detection.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-14-1018984-g005.tif"/>
</fig>
<p>Besides ATG8s, NBR1 known as a selective autophagy receptor is itself a substrate degraded in the vacuole (<xref ref-type="bibr" rid="B72">Svenning et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B95">Zhou et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B31">Ji et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B33">Jung et&#xa0;al., 2020</xref>). Disruption of <italic>At</italic>NBR1 conferred increased sensitivity to heat, drought, and salt stresses (<xref ref-type="bibr" rid="B95">Zhou et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B31">Ji et&#xa0;al., 2020</xref>). However, <italic>At</italic>NBR1 does not play an essential role in regulating N deprivation-induced autophagy (<xref ref-type="bibr" rid="B41">Lin et&#xa0;al., 2020</xref>). To answer whether <italic>At</italic>NBR1 is involved in Pi starvation-induced autophagy and thus its degradation could be used as an alternative method for measuring autophagic flux in the root, we monitored the expression changes of <italic>At</italic>NBR1 in the WT root following 12, 24, 48, 72 hours of Pi deprivation. The specificity of anti-NBR1 antibodies was validated with the <italic>nbr1-2</italic> and <italic>atg7-3</italic> mutants by the absence and accumulation of <italic>At</italic>NBR1 proteins, respectively (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S6</bold>
</xref>). Either with 6 or 12 hours of Conc A treatment, <italic>At</italic>NBR1 accumulated in the WT root to a similar extent at different time point of Pi starvation (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S6</bold>
</xref>). Of note, the expression changes of <italic>At</italic>NBR1 in the WT root upon Conc A treatment appeared to be smaller than that of ATG8s (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5A</bold>
</xref>, <xref ref-type="supplementary-material" rid="SM1">
<bold>S6</bold>
</xref>). It is possible that <italic>At</italic>NBR1 is subjected to selective autophagic degradation only under certain stress conditions. Accordingly, Pi deprivation did not alter the vacuolar degradation of <italic>At</italic>NBR1 in the WT root (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S6</bold>
</xref>). There was also no difference of <italic>At</italic>NBR1 degradation between <italic>atg8f/atg8h</italic> and WT (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S7</bold>
</xref>), indicating that <italic>At</italic>NBR1 may not participate in Pi starvation-induced autophagy. Overall, these results revealed that <italic>At</italic>ATG8f and <italic>At</italic>ATG8h contribute to a substantial proportion of microsomal-enriched ATG8s and may regulate the autophagic flux under Pi starvation through a mechanism other than promoting ATG8s lipidation.</p>
</sec>
<sec id="s2_5">
<title>Expression of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in the root stele and at the sites where lateral root primordia develop</title>
<p>To examine the spatial expression patterns of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> under Pi starvation, we generated GFP reporter lines, designated <italic>P<sub>ATG8f</sub>
</italic>:GFP and <italic>P<sub>ATG8h</sub>
</italic>:GFP. The promoter sequence of <italic>AtATG8f</italic> we used starts from 2386 bp upstream of the putative transcription start site (TSS) to 606 bp downstream of the TSS within the second exon as shown (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6A</bold>
</xref>). This is much longer than the one used by Di Berardino et&#xa0;al., which contains the 1651 bp upstream of the TSS and the 176 bp downstream of the TSS (<xref ref-type="bibr" rid="B19">Di Berardino et&#xa0;al., 2018</xref>). The upstream region of the TSS in our construct is also longer than the one used by Sl&#xe1;vikov&#xe1; et&#xa0;al., which includes the 1906 bp upstream of ATG codon plus the entire coding regions of <italic>AtATG8f</italic>, a total of 3125-bp genomic sequence containing the exons and introns (<xref ref-type="bibr" rid="B67">Sl&#xe1;vikov&#xe1; et&#xa0;al., 2005</xref>). While the study of Di Berardino et&#xa0;al. indicated the expression of <italic>AtATG8f</italic> in the veins of the pericarp and in the seed embryo, the study of Sl&#xe1;vikov&#xe1; et&#xa0;al. displayed the expression of <italic>AtATG8f</italic> in the root of seedlings with relatively poor resolution at the cell-type level. As for <italic>AtATG8h</italic>, due to the short intergenic region between <italic>AtATG8h</italic> and the upstream gene At3g06430, two <italic>AtATG8h</italic> promoter regions were considered in our study. The shorter one contains a total of 553 bp, starting from 221 bp upstream of the TSS to 312 bp downstream of the TSS. The longer one contains the partial genomic sequences of At3g06430 and extending to 312 bp downstream of the TSS within the second exon (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6A</bold>
</xref>). Overall, there were no differences in the expression levels and patterns of GFP between the <italic>AtATG8h</italic> reporter lines with different promoter lengths (data not shown), so we chose the transgenic lines with the longer <italic>AtATG8h</italic> promoter for our further investigation. Confocal analysis of the root of <italic>P<sub>ATG8f</sub>
</italic>:GFP lines showed that the expression of <italic>AtATG8f</italic> was in the root apical meristem, root cap, stele tissues, and root hairs of the primary root under Pi sufficiency (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6B</bold>
</xref>). By comparison, the GFP signals in <italic>P<sub>ATG8h</sub>
</italic>:GFP lines were much weaker and mainly detected in the root stele tissues (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6C</bold>
</xref>). Of note, GFP signals were hardly detected in the root cap and root hairs of <italic>P<sub>ATG8h</sub>
</italic>:GFP lines under Pi sufficiency (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6C</bold>
</xref>). Under Pi deficiency, the GFP expression patterns of <italic>P<sub>ATG8f</sub>
</italic>:GFP and <italic>P<sub>ATG8h</sub>
</italic>:GFP lines were similar as those under Pi sufficiency and the signals in the root hair showed stronger intensities (data not shown). Further quantitative real-time PCR analysis of GFP expression in the Pi-starved root of <italic>P<sub>ATG8f</sub>
</italic>:GFP and <italic>P<sub>ATG8h</sub>
</italic>:GFP lines also supported the upregulation of GFP expression by low Pi (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6D</bold>
</xref>), which was in good agreement with the increased endogenous <italic>AtATG8f</italic> and <italic>AtATG8h</italic> transcripts in these reporter lines (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S8</bold>
</xref>). These results suggested that <italic>AtATG8f</italic> and <italic>AtATG8h</italic> can be upregulated by Pi starvation at the transcriptional level.</p>
<fig id="f6" position="float">
<label>Figure&#xa0;6</label>
<caption>
<p>Expression patterns of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in <italic>Arabidopsis</italic> GFP reporter lines. <bold>(A)</bold> Schematic design of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> promoter-fused GFP reporter constructs. The upstream region of the putative transcription starts site (TSS) in <italic>AtATG8f</italic> and <italic>AtATG8h</italic> were indicated by rightwards thick arrows. Black, light gray and dark gray boxes represent exons, 5&#xb4;UTR and the gene At3g06430, respectively. Thick and thin lines indicate introns and linkers, respectively. The schematic structure is drawn according to scale. <bold>(B, C)</bold> GFP expression in the root of 3-day-old seedlings of <italic>P<sub>ATG8f</sub>
</italic>:GFP <bold>(B)</bold> and <italic>P<sub>ATG8h</sub>
</italic>:GFP <bold>(C)</bold> germinated under Pi-sufficient (a, c, and e; +P, 250 &#x3bc;M KH<sub>2</sub>PO<sub>4</sub>) and Pi-deficient (b, d, and f; &#x2013;P, 0 &#x3bc;M KH<sub>2</sub>PO<sub>4</sub>) conditions. GFP signals in the root apical meristem (a, b), the vascular tissue <bold>(C, D)</bold> and the root hair <bold>(E, F)</bold>. Scale bars = 50 &#x3bc;m. At least two independent lines were examined for each construct and representative images are shown. Propidium iodide (PI) was used as a root cell wall stain. <bold>(D)</bold> qRT-PCR analysis of GFP expression in the root of 11-day-old <italic>P<sub>ATG8f</sub>
</italic>:GFP and <italic>P<sub>ATG8h</sub>
</italic>:GFP seedlings grown under Pi-sufficient (+P, 250 &#xb5;M KH<sub>2</sub>PO<sub>4</sub>) and Pi-deficient (&#x2013;P, 0 &#xb5;M KH<sub>2</sub>PO<sub>4,</sub> 3 days of starvation) conditions. Error bars represent SE (n = 3, biological replicate pools of 20 seedlings from three independent experiments).</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-14-1018984-g006.tif"/>
</fig>
<p>To assess the promoter activities of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in the shoot, we also generated <italic>P<sub>ATG8f</sub>
</italic>:GUS and <italic>P<sub>ATG8h</sub>
</italic>:GUS lines, in which the promoter sequences used were the same as those used in the GFP lines. The expression of <italic>AtATG8f</italic> was mainly found in the shoot vascular tissues and mesophylls (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7A</bold>
</xref>). Similarly, the GUS staining of <italic>P<sub>ATG8h</sub>
</italic>:GUS lines was predominantly in the similar shoot tissues yet with weaker signals (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7B</bold>
</xref>). Moreover, the <italic>P<sub>ATG8f</sub>
</italic>:GUS lines showed the expression patterns of <italic>AtATG8f</italic> in the root stele tissues of both primary and lateral roots as well as in fully emerged lateral root primordia (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7A</bold>
</xref>). Of note, the promoter activity of <italic>AtATG8f</italic> was detected throughout the development of lateral root (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7A</bold>
</xref>), which was consistent with <italic>P<sub>ATG8f</sub>
</italic>:GFP lines (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S9</bold>
</xref>). By comparison, the promoter activity of <italic>AtATG8h</italic> was absent in the primary root apical meristem but detectable in the basal meristem (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7B</bold>
</xref>). Importantly, the GUS staining of <italic>AtATG8h</italic> reporter lines was neither detectable in the lateral root primordia nor at early stages of lateral root development (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7B</bold>
</xref>). Only after the establishment of lateral root meristem, we could detect the expression of <italic>AtATG8h</italic> in the stele and columella of lateral root (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7B</bold>
</xref>), which was also consistent with <italic>P<sub>ATG8h</sub>
</italic>:GFP lines (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S9</bold>
</xref>).</p>
<fig id="f7" position="float">
<label>Figure&#xa0;7</label>
<caption>
<p>Expression patterns of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h in <italic>Arabidopsis</italic> GUS reporter lines. <bold>(A, B)</bold> GUS staining in the 8-day-old seedlings of <italic>P<sub>ATG8f</sub>
</italic>:GUS <bold>(A)</bold> and <italic>P<sub>ATG8h</sub>
</italic>:GUS <bold>(B)</bold> seedlings under full nutrient (+PN, 250 &#x3bc;M KH<sub>2</sub>PO<sub>4</sub> and 7.5 mM NO<sub>3-</sub>) conditions. GUS signals in the cotyledon (a), root apical meristem (b), lateral root primordia (c), and lateral root tip (d). Scale bars, 50 &#x3bc;m. The time of GUS staining for <italic>P<sub>ATG8f</sub>
</italic>:GUS and <italic>P<sub>ATG8h</sub>
</italic>:GUS, was 1 and 2 hours, respectively. At least two independent lines were examined for each construct and representative images are shown.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-14-1018984-g007.tif"/>
</fig>
</sec>
<sec id="s2_6">
<title>Loss of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h suppresses the lateral root number</title>
<p>Next, we focused to characterize the root phenotypes of <italic>atg8f/atg8h</italic> mutants and used the <italic>atg7-3</italic> mutant for comparison (<xref ref-type="bibr" rid="B28">Huang et&#xa0;al., 2019</xref>). Pi starvation is known to induce the synthesis of extracellular acid phosphatases and organic acids for P mobilization (<xref ref-type="bibr" rid="B46">Marschner, 1995</xref>). Considering that the phytochemical or metabolite crosstalk between plants under nutrient deficiency may affect the root phenotypes of different genotypes when grown on the same plate, we grew four seedlings for each genotype <italic>per</italic> plate to avoid the mutual effect of root exudates from different genotypes. Under our full nutrient and Pi- and N-deprived conditions, the primary root length showed no difference between WT and <italic>atg8f/atg8h</italic> but was shorter in <italic>atg7-3</italic> (<xref ref-type="fig" rid="f8">
<bold>Figures&#xa0;8A, B</bold>
</xref>). These results suggested that unlike the impairment of the single-copy <italic>ATG</italic> gene, loss of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h does not retard the primary root growth. Because strong <italic>AtATG8f</italic> and <italic>AtATG8h</italic> expression was observed during the lateral root development, we set out to analyze the number of lateral roots for <italic>atg8f/atg8h</italic>. Similar to the results of previous studies showing the inhibition of lateral growth under severe N starvation (<xref ref-type="bibr" rid="B38">Krouk et&#xa0;al., 2010</xref>; <xref ref-type="bibr" rid="B25">Gruber et&#xa0;al., 2013</xref>), we observed a reduction of lateral root number <italic>per</italic> seedling in all genotypes grown on N-limited media (<xref ref-type="fig" rid="f8">
<bold>Figure&#xa0;8C</bold>
</xref>). The lateral root number was strikingly reduced in the autophagy-defective <italic>atg7-3</italic> mutant under all the growth conditions, implying that functional autophagy is required for the lateral root development (<xref ref-type="fig" rid="f8">
<bold>Figure&#xa0;8C</bold>
</xref>). Intriguingly, the lateral root number was also significantly reduced in the <italic>atg8f/atg8h</italic> relative to the WT under Pi-rich and Pi-starved conditions (<xref ref-type="fig" rid="f8">
<bold>Figure&#xa0;8C</bold>
</xref>), indicating that <italic>ATG8f</italic> and <italic>ATG8h</italic> are involved in the regulation of lateral root growth. While under N starvation the lateral root number of <italic>atg7-3</italic> was reduced relative to the WT, no significant differences were found between WT and <italic>atg8f/atg8h</italic> (<xref ref-type="fig" rid="f8">
<bold>Figure&#xa0;8C</bold>
</xref>), indicating that the other <italic>At</italic>ATG8 may share redundant roles in lateral root development during N starvation.</p>
<fig id="f8" position="float">
<label>Figure&#xa0;8</label>
<caption>
<p>Loss of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h suppresses lateral root development. <bold>(A)</bold> Representative images of 10-day-old WT, <italic>atg7-3</italic>, and <italic>atg8f/atg8h</italic> seedlings under full nutrient (+PN, 250 &#x3bc;M KH<sub>2</sub>PO<sub>4</sub> and 7.5 mM <inline-formula>
<mml:math display="inline" id="im1">
<mml:mrow>
<mml:msubsup>
<mml:mrow>
<mml:mtext>NO</mml:mtext>
</mml:mrow>
<mml:mn>3</mml:mn>
<mml:mo>&#x2212;</mml:mo>
</mml:msubsup>
</mml:mrow>
</mml:math>
</inline-formula>), Pi-deficient (&#x2013;P, 0 &#x3bc;M KH<sub>2</sub>PO<sub>4</sub>, 5 days of starvation) and N-deficient (&#x2013;N, 0.1 mM <inline-formula>
<mml:math display="inline" id="im2">
<mml:mrow>
<mml:msubsup>
<mml:mrow>
<mml:mtext>NO</mml:mtext>
</mml:mrow>
<mml:mn>3</mml:mn>
<mml:mo>&#x2212;</mml:mo>
</mml:msubsup>
</mml:mrow>
</mml:math>
</inline-formula>, 5 days of starvation) conditions. Scale bars = 1&#xa0;cm. <bold>(B, C)</bold> Data are presented for the primary root (PR) length <bold>(B)</bold> and the lateral root (LR) number <bold>(C)</bold> of WT, <italic>atg7-3</italic> and <italic>atg8f/atg8h</italic> seedlings. Error bars represent SE (n = 35&#x2013;40, collected from three independent experiments). <sup>+++</sup>P&lt; 0.001 <sup>++</sup>P&lt; 0.01, compared to Pi-sufficient conditions within the same genotype; <sup>***</sup>P&lt; 0.001 <sup>**</sup>P&lt; 0.01, compared to WT under the same conditions; Student&#x2019;s <italic>t</italic>-test; two-tailed.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-14-1018984-g008.tif"/>
</fig>
</sec>
</sec>
<sec id="s3" sec-type="discussion">
<title>Discussion</title>
<sec id="s3_1">
<title>PHR1 acts upstream of the transcriptional regulation of <italic>AtATG8f</italic> and <italic>AtATG8h</italic>
</title>
<p>A chromatin immunoprecipitation sequencing (ChIP-seq) study has revealed <italic>AtATG8f</italic> to be a direct target of <italic>At</italic>PHR1 (<xref ref-type="bibr" rid="B11">Castrillo et&#xa0;al., 2017</xref>), while a previous Y1H screen discovered that <italic>At</italic>PHR1 was not among the 32 TFs interacting with the <italic>AtATG8h</italic> promoter (<xref ref-type="bibr" rid="B79">Wang et&#xa0;al., 2020</xref>). In our Y1H assay, we failed to identify <italic>At</italic>PHR1 as a positive TF binding to the promoter of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S1</bold>
</xref>). Results of our dual luciferase reporter assays also did not support a direct transactivation of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> by <italic>At</italic>PHR1. Recently, a chromatin remodeling analysis of <italic>Arabidopsis</italic> Pi-starved roots suggested that <italic>At</italic>PHR1 activates a set of TFs triggering a second wave of epigenetic changes required for upregulation of PSR genes (<xref ref-type="bibr" rid="B6">Barrag&#xe1;n-Rosillo et&#xa0;al., 2021</xref>). Intriguingly, the association of <italic>AtATG8h</italic> with increased chromatin accessibility (upDARs) was found in Pi-limited root of WT but not <italic>phr1/phl2</italic> (<xref ref-type="bibr" rid="B6">Barrag&#xe1;n-Rosillo et&#xa0;al., 2021</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Table S3</bold>
</xref>), indicating that PHR1 and/or its paralogues may engage transcriptional activation of <italic>AtATG8h</italic> in response to Pi limitation by enhancing chromatin accessibility. According to the ChIP-seq data (<xref ref-type="bibr" rid="B11">Castrillo et&#xa0;al., 2017</xref>), we found that only less than 20% of PHR1/PHL2-dependent Pi starvation-induced genes are direct targets of PHR1 (<xref ref-type="bibr" rid="B6">Barrag&#xe1;n-Rosillo et&#xa0;al., 2021</xref>). This could in part explain the discrepancy among conclusions due to different methods or test systems. Nevertheless, it warrants further investigation as to whether other TFs are responsible for the transcriptional regulation of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> under Pi limitation and whether low Pi induction of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> involves the coordination of epigenetic and transcriptional changes.</p>
</sec>
<sec id="s3_2">
<title>
<italic>AtATG8f</italic> and <italic>AtATG8h</italic> finetune the autophagic flux in response to Pi starvation</title>
<p>ATG8 itself is degraded together with cargos and serves as a faithful proxy for autophagy activity readout (<xref ref-type="bibr" rid="B37">Klionsky et&#xa0;al., 2021</xref>). In this study, we showed that the expression changes of endogenous ATG8s were not prominent in the Pi-depleted root of WT and <italic>atg8f/atg8h</italic> relative to their respective Pi-replete controls (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5A, B</bold>
</xref>). This may be explained, at least in part, by the modest upregulation of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in certain root cell types. It is also likely that the abundance of the other ATG8s masks the small expression changes of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h by Pi starvation (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S3</bold>
</xref>). On the other hand, we could detect the reduction of microsomal-enriched ATG8s in the root of <italic>atg8f/atg8h</italic>, indicating that loss of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h indeed decreased the abundance of membrane-associated ATG8s (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S4</bold>
</xref>). Intriguingly, neither the autophagic flux nor the <italic>At</italic>ATG8s lipidation was increased by Pi starvation in the WT root, which reinforced the view of the two recent studies that nutrient starvation-induced autophagy is likely tissue- or cell type-specific (<xref ref-type="bibr" rid="B20">Dong et&#xa0;al., 2022</xref>; <xref ref-type="bibr" rid="B12">Chiu et&#xa0;al., 2023</xref>). While based on the expression changes of ATG8s between DMSO control and Conc A treatment, the relative autophagic flux was reduced in the Pi-starved root of <italic>atg8f/atg8h</italic> (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5A, B</bold>
</xref>), the autophagic flux estimated by NBR1 degradation failed to support this conclusion (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S7</bold>
</xref>). We thought that NBR1 may not play a direct role in Pi starvation-induced autophagy, at least at the whole-root level. Nevertheless, we found the increased amount of lipidated ATG8s in the <italic>atg8f/atg8h</italic> root (<xref ref-type="supplementary-material" rid="SM1">
<bold>Figure S5</bold>
</xref>). The discrepancy between the reduced autophagic flux and the increased lipidated ATG8s in the Pi-depleted root of <italic>atg8f/atg8h</italic> might hint that the lipidation/de-lipidation of the other isoforms is altered and/or that <italic>At</italic>ATG8f and <italic>At</italic>ATG8h fine-tune the autophagic flux under Pi starvation through an unknown ATG8s lipidation-independent pathway. In mammalian cells, knockout of all the ATG8 family members suggested that the ATG8s are dispensable for autophagosome formation but crucial for autophagosome&#x2013;lysosome fusion (<xref ref-type="bibr" rid="B51">Nguyen et&#xa0;al., 2016</xref>). However, it remains to discover whether the plant ATG8s are also important at this step to regulate the autophagic flux. It is worth noting that there are intrinsic limitations in measuring autophagic flux changes based on the steady-state abundance of ATG8s in the whole root. Even in the presence of vacuolar inhibitors that isolate autophagy induction from inhibition of autophagic degradation, this assay obscures estimates of substrate clearance &#x2013; the most ideal measure of autophagic flux (<xref ref-type="bibr" rid="B37">Klionsky et&#xa0;al., 2021</xref>) and thus cannot determine the autophagic flux at the cellular level. The development of tool that allows to quantify autophagic responses at cell-type specific resolution as well as generation of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h-specific antibodies may advance these issues (<xref ref-type="bibr" rid="B68">Stephani and Dagdas, 2020</xref>).</p>
</sec>
<sec id="s3_3">
<title>Role of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in the lateral root development</title>
<p>In our recent study of <italic>Arabidopsis</italic> autophagy-defective mutants, we showed that the <italic>atg5-1</italic>, <italic>atg7-3</italic> and <italic>atg10-1</italic> mutants exhibited impaired Pi homeostasis and compromised plant fitness in response to fluctuating Pi availability (<xref ref-type="bibr" rid="B12">Chiu et&#xa0;al., 2023</xref>). However, we did not observe similar phenotypes for <italic>atg8f, atg8h</italic>, and <italic>atg8f/atg8h</italic> (<xref ref-type="fig" rid="f4">
<bold>Figures&#xa0;4A, B</bold>
</xref>). Regardless of nutrient conditions, the primary root length showed no difference between WT and <italic>atg8f/atg8h</italic> but was shorter in <italic>atg7-3</italic> (<xref ref-type="fig" rid="f8">
<bold>Figures&#xa0;8A, B</bold>
</xref>). Rather, we found a reduction in the lateral root number of both <italic>atg7-3</italic> and <italic>atg8f/atg8h</italic> (<xref ref-type="fig" rid="f8">
<bold>Figure&#xa0;8C</bold>
</xref>). The development of lateral root primordia is sensitive to the availability of N (<xref ref-type="bibr" rid="B4">Banda et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B63">Santos Teixeira and ten Tusscher, 2019</xref>). Under severe N starvation, the primary root length, the lateral root length, and the number of lateral roots <italic>per</italic> primary root were reported to be inhibited in <italic>atg4a4b-1</italic> (<xref ref-type="bibr" rid="B91">Yoshimoto et&#xa0;al., 2004</xref>). We also found that compared to WT, <italic>atg7-3</italic> but not <italic>atg8f/atg8h</italic> had a decreased lateral root number under relatively mild N deficiency (<xref ref-type="fig" rid="f8">
<bold>Figure&#xa0;8C</bold>
</xref>). We reasoned that while <italic>At</italic>ATG8f and <italic>At</italic>ATG8h are critical for lateral root development under full nutrient and Pi-starved conditions, some other ATG8s are induced under N limitation and thus compensate for the loss of <italic>At</italic>ATG8f and <italic>At</italic>ATG8h. It is known that nutrient cues can affect lateral root formation <italic>via</italic> crosstalk with hormone signaling at four key developmental steps: initiation, primordium establishment, emergence, and elongation (<xref ref-type="bibr" rid="B32">Jia et&#xa0;al., 2021</xref>). As <italic>AtATG8f</italic> is present throughout the lateral root formation and <italic>AtATG8h</italic> starts to express likely after vascular tissue differentiation, we speculate that <italic>AtATG8f</italic> and <italic>AtATG8h</italic> may be involved in the lateral root development at different stages, which needs to be further studied. Intriguingly, we found that <italic>AtATG8f</italic> but not <italic>AtATG8h</italic> is expressed in the root cap. The periodicity of lateral root formation is driven by programmed cell death of the root cap (<xref ref-type="bibr" rid="B88">Xuan et&#xa0;al., 2016</xref>). Prior to the root cap cell death, autophagy has been shown to be required for organelle clearance and organized cell separation (<xref ref-type="bibr" rid="B23">Goh et&#xa0;al., 2022</xref>). In addition, selective autophagy was previously proposed to promote the lateral root development upon Pi starvation through ARK2-PUB9 module-dependent auxin accumulation (<xref ref-type="bibr" rid="B17">Deb et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B62">Sankaranarayanan and Samuel, 2015</xref>). However, the underlying mechanism remains to be elucidated on a molecular basis.</p>
</sec>
</sec>
<sec id="s4" sec-type="materials|methods">
<title>Materials and methods</title>
<sec id="s4_1">
<title>Plant material and growth conditions</title>
<p>Seeds of the <italic>Arabidopsis thaliana atg7-3</italic> (SAIL_11_H07), <italic>atg8f-2</italic> (SALK_052510C), <italic>atg8f-3</italic> (SALK_039231), <italic>atg8f-5</italic> (SALK_133008), <italic>atg8f-6</italic> (SALK_004370), <italic>atg8h-2</italic> (SALK_021495), <italic>atg8h-3</italic> (SALK_136493), <italic>phr1-3</italic> (SALK_067629), <italic>nbr1-2</italic> (GK-246H08), and <italic>pho1-2</italic> (<xref ref-type="bibr" rid="B54">Poirier et&#xa0;al., 1991</xref>) mutants used in this study were in the Columbia (Col) background and obtained from the Arabidopsis Biological Resource Center (ABRC). The <italic>Arabidopsis</italic> seeds were surface-sterilized and germinated on agar plates with one-half modified Hoagland&#x2019;s solution containing 1% Suc and 0.8% Bacto agar (BD Difcom 204010), and grown in the growth chamber at 22&#xb0;C with a 16&#xa0;h light/8&#xa0;h dark cycle. The full nutrient (+PN) or Pi-sufficient (+P) and Pi-deficient (&#x2212;P) media were supplemented with 250 &#x3bc;M and 0 or 10 &#xb5;M KH<sub>2</sub>PO<sub>4</sub>, respectively, unless specified otherwise. The full nutrient (+PN) and N-deficient (&#x2212;N) media were supplemented with 7.5 mM and 0 or 0.1 mM &#xb5;M Ca(NO<sub>3</sub>)<sub>2</sub>/KNO<sub>3</sub>, respectively, unless specified otherwise.</p>
</sec>
<sec id="s4_2">
<title>Construct design</title>
<p>All the insert fragments of interest were amplified by polymerase chain reaction (PCR) and cloned into pJET1.2/blunt vector for sequencing and then subcloned into the desired vectors. For the constructs used for dual-luciferase assay, the promoter sequences of <italic>AtATG8f</italic>, <italic>AtATG8h</italic> and <italic>AtIPS1</italic> were subcloned into the pGreenII-0800-Luc vector (<xref ref-type="bibr" rid="B27">Hellens et&#xa0;al., 2005</xref>). The full-length coding sequences of <italic>At</italic>ATAF2 and <italic>At</italic>PHR1 were subcloned into the &#x3b2;-estradiol-inducible P16&#x394;S:sXVE:S10 vector (<xref ref-type="bibr" rid="B42">Liu et&#xa0;al., 2018</xref>). For the constructs used for Y1H analysis, the promoter sequences of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> were as same as those used for dual-luciferase reporter constructs and were cloned into the pHISi2 vector in which extra start codons of pHISi (Clontech/Takara bio Inc.) residing within 5&#x2019; untranslated region of the reporter gene HIS3 are mutated. For the GFP or GUS reporter constructs, the <italic>P<sub>ATG8f</sub>
</italic>:GFP or <italic>P<sub>ATG8h</sub>
</italic>:GFP constructs were obtained by inserting the promoter sequences of <italic>AtATG8f</italic> and <italic>AtATG8h</italic> in the binary vector pMDC111. The <italic>P<sub>ATG8f</sub>
</italic>:GUS or <italic>P<sub>ATG8h</sub>
</italic>:GUS constructs were made by inserting the genomic sequences into the binary vector pMDC163. Primer sequences used for gene cloning are listed in <xref ref-type="supplementary-material" rid="SM1">
<bold>Table S4</bold>
</xref>.</p>
</sec>
<sec id="s4_3">
<title>Yeast one-hybrid analysis</title>
<p>The yeast strain YM4271 was employed for Y1H analysis of the <italic>AtATG8f</italic> and <italic>AtATG8h</italic> promoters, which was performed as described previously (<xref ref-type="bibr" rid="B49">Mitsuda et&#xa0;al., 2010</xref>) but with some modifications. The promoter-cloned pHISi construct was linearized with the restriction enzyme ApaI (for <italic>AtATG8f</italic>) or NcoI (for <italic>AtATG8h</italic>) and the promoter::<italic>HIS3</italic> fusion was then integrated into the YM4271 genome. A total of 1,736 <italic>Arabidopsis</italic> transcription factor genes were cloned into pGADT7 vector (Clontech/Takara bio Inc.), divided into 384 mini pools and individual interactions between each promoter and mini pool were examined by the yeast growth on the selective media lacking leucine (L), uracil (U) or histidine (H) with or without the addition of 3-amino-1,2,4-triazole (3-AT) as indicated.</p>
</sec>
<sec id="s4_4">
<title>
<italic>Arabidopsis</italic> mesophyll protoplast isolation and transfection</title>
<p>Leaves of 4-week-old <italic>Arabidopsis</italic> plants grown under 12&#xa0;h light/12&#xa0;h dark were harvested and protoplasts were isolated following the tape-<italic>Arabidopsis</italic> sandwich method (<xref ref-type="bibr" rid="B85">Wu et&#xa0;al., 2009</xref>) with minor modifications. About 2.5&#xd7;10<sup>4</sup> cells were transfected by the PEG/calcium-mediated method (<xref ref-type="bibr" rid="B90">Yoo et&#xa0;al., 2007</xref>). An equal volume of the freshly-prepared PEG 4000 solution containing 40% (w/v) PEG, 0.1 M CaCl<sub>2</sub>, and 0.2 M mannitol was added, completely mixed, and incubated at RT for 10&#xa0;min. A 600 &#x3bc;L of modified W5 solution (154 mM NaCl, 125 mM CaCl<sub>2</sub>, 5 mM KCl, 5 mM glucose, and 2 mM MES) was added and gently mixed to stop the transfection. Transfected protoplasts were collected by centrifugation at 100&#xa0;g for 2&#xa0;min and were re-suspended in 0.5 mL of W5 solution. The final protoplasts were incubated in a 1% BSA pre-coated 12-well plate at 22&#xb0;C for 16 hours in light. 10 &#x3bc;g/mL (36.7 &#x3bc;M) <italic>&#x3b2;</italic>-estradiol in ethanol was added 8 hours before performing the dual-luciferase assay.</p>
</sec>
<sec id="s4_5">
<title>Dual-luciferase assay in <italic>Arabidopsis</italic> protoplasts</title>
<p>Dual-luciferase assays were carried out as described with slight modifications (<xref ref-type="bibr" rid="B27">Hellens et&#xa0;al., 2005</xref>). Briefly, after 8 hours of induction, the transfected protoplast suspension was transferred to a 1.5 mL centrifugation tube and centrifuged at 100&#xa0;g for 10&#xa0;min. The supernatant was discarded and the pellets were re-suspended in 100 &#x3bc;L of 1X passive lysis buffer (PLB) provided in the Dual Luciferase Reporter Assay System kit (Promega). Protoplasts were disrupted by vortex for 10 s followed by centrifugation at 10,000 g for 2&#xa0;min. A 5 &#x3bc;L of the supernatant sample was loaded into a well of a white flat bottom Costar 96 well plate (Corning). Dual-luciferase assays were performed in Synergy&#x2122; HTX Multi-Mode Microplate Reader (BioTek). A 40 &#x3bc;L luciferase assay reagent and a 40 &#x3bc;L Stop and Glo reagent (Promega) were injected <italic>per</italic> well. The ratio of LUC to REN was measured to represent the activity of the corresponding promoter when the effector plasmid DNA was co-transfected.</p>
</sec>
<sec id="s4_6">
<title>Phosphate concentration analysis</title>
<p>Pi concentrations were analyzed as described (<xref ref-type="bibr" rid="B1">Ames, 1966</xref>) with minor modifications. For the measurement of Pi concentrations, fresh tissue was frozen with liquid nitrogen and homogenized with 1% glacial acetic acid and incubated at 42&#xb0;C for 30&#xa0;min followed by centrifugation at 13,000 g for 5&#xa0;min. The supernatant aliquot was mixed with the assay solution (0.35% NH<sub>4</sub>MoO<sub>4</sub>, 0.86&#xa0;N H<sub>2</sub>SO<sub>4</sub>, and 1.4% ascorbic acid) and incubated at 42&#xb0;C for 30&#xa0;min. Pi content determined by colorimetric assay based on the formation of phosphomolybdate was measured at A<sub>750</sub>.</p>
</sec>
<sec id="s4_7">
<title>RNA isolation, reverse transcription PCR, quantitative real-time RT-PCR</title>
<p>Total RNA from samples was isolated using GENEzol&#x2122; TriRNA Pure Kit with DNase (Geneaid, GZXD200). The first strand cDNA was synthesized from 0.5 &#x3bc;g total RNA using PrimeScript&#x2122; 1st strand cDNA Synthesis Kit (TaKaRa, 6110A) with oligo(dT) primer. qRT-PCR was performed using KAPA SYBR<sup>&#xae;</sup> FAST qPCR Master Mix (2X) Kit on StepOnePlus&#x2122; Real-Time PCR System (Applied Biosystems) according to the manufacturer&#x2019;s instructions. Relative expression levels were normalized to that of an internal control <italic>ACT8</italic> (At1g49240). Sequences of primers used are listed in <xref ref-type="supplementary-material" rid="SM1">
<bold>Table S5</bold>
</xref>.</p>
</sec>
<sec id="s4_8">
<title>Immunoblot analysis</title>
<p>For extraction of total root protein, the roots of WT and mutant seedlings were ground in liquid nitrogen and dissolved in protein lysis buffer containing 60 mM 2&#x2010;amino&#x2010;2&#x2010;(hydroxymethyl)&#x2010;1,3&#x2010;propanediol (Tris)-HCl (pH 8.5), 2% Sodium dodecyl sulfate (SDS), 2.5% glycerol, 0.13 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) and Protease Inhibitor Cocktail (Sigma-Aldrich P9599). A total of 25 &#xb5;g root protein from each sample was loaded onto 12% Q-PAGE&#x2122; Bis-Tris Precast Gel (SMOBIO) or NuPAGE 4&#x2013;12% Bis-Tris Gels (Thermo Fisher Scientific) and transferred to polyvinylidene difluoride (PVDF) membranes. The membrane was blocked with 1 or 2% BSA in 1X PBS solution with 0.2% Tween 20 (PBST, pH 7.2) at room temperature for 1&#xa0;h and hybridized with primary antibodies of ATG8 (1:1000; Agrisera AS14 2811), NBR1 (1:4000; Agrisera AS14 2805) and actin (1:4000; Abcam, ab197345) for 1&#xa0;h at room temperature in blocking solution. The membrane was washed four times with 1X PBST for 5&#xa0;min followed by hybridization with the horseradish peroxidase&#x2013;conjugated secondary antibody (1:10,000&#x2013;20,000 dilution; GeneTex GTX213110-01) in blocking solution for 1&#xa0;h. After four washes in 1&#xd7; PBST for 5&#xa0;min and a rinse with distilled water, chemiluminescent substrates (Advansta, WesternBright ECL) for signal detection were applied.</p>
</sec>
<sec id="s4_9">
<title>Isolation of root microsomal protein and ATG8 lipidation assay</title>
<p>Root microsomal protein was isolated with the Minute Plant Microsomal Membrane Extraction Kit (Invent, MM-018) according to the manual instruction. The resultant pellets (microsomal protein) were resuspended in the solubilization buffer containing 350 mM sucrose, 0.5% Triton X-100, 10 mM Tris-MES (pH 7.0), 1 mM Dithiothreitol (DTT) and Protease Inhibitor Cocktail (Sigma-Aldrich P9599). A total of 10 &#xb5;g root microsomal protein from each sample was loaded onto NuPAGE 4&#x2013;12% Bis-Tris Gels (Thermo Fisher Scientific) and transferred to PVDF membranes for further immunoblot analysis as described above, except with 3% BSA-containing blocking solution. Phospholipase D (PLD; Enzo Lifesciences BML-SE301) treatment was performed by mixing 10 &#xb5;g root microsomal protein with 80 U PLD in reaction buffer containing 10 mM Tris-HCl (pH 8.0), 1% glycerol, 0.01% Triton X-100 and incubated at 37&#xb0;C for 1&#xa0;h. Each sample was loaded onto 15% mPAGE<sup>&#xae;</sup> TurboMix Bis-Tris Gel (TMKIT, Merck) with 6 M urea for electrophoresis according to the manual instruction and transferred to PVDF membranes for further immunoblot analysis as described above.</p>
</sec>
<sec id="s4_10">
<title>
<italic>Arabidopsis</italic> transformation and transgenic plant selection</title>
<p>The binary plasmid was introduced into <italic>A. tumefaciens</italic> strain GV3101:pMP90 and selected on 5 &#x3bc;g ml<sup>-1</sup> rifampicin, 50 &#x3bc;g ml<sup>-1</sup> gentamycin and 50 &#x3bc;g ml<sup>-1</sup> kanamycin. The <italic>Arabidopsis</italic> plants were transformed using standard floral dip method, and T1 transgenic plants were selected on half-strength MS medium supplemented with 1% sucrose plates containing appropriate antibiotics. T2 transgenic lines with a segregation ratio of 3 resistant: 1 sensitive were used for further study as presumably having single insertion of T-DNA.</p>
</sec>
<sec id="s4_11">
<title>GUS staining</title>
<p>GUS activity was detected as previously described with modifications (<xref ref-type="bibr" rid="B30">Jefferson et&#xa0;al., 1987</xref>). Briefly, seedlings were placed in 90% acetone on ice after sampling and vacuum infiltrated in freshly prepared GUS assay buffer containing 500 mM NaH<sub>2</sub>PO<sub>4</sub>, 500 mM Na<sub>2</sub>HPO<sub>4</sub> 7H<sub>2</sub>O, 1mM K<sub>3</sub>Fe(CN)<sub>6</sub>, 1 mM K<sub>4</sub>Fe(CN)<sub>6</sub>, 10 mM EDTA, 0.1% Triton X-100, and 2.25 mM X-Gluc (5-bromo-4-chloro-3-indoyl-&#x3b2;-D-glucuronide sodium salt; Cyrusbioscience) for 20&#xa0;min followed by incubation at 37&#xb0;C, 1 and 2 hours for <italic>P<sub>ATG8f</sub>
</italic>:GUS and <italic>P<sub>ATG8h</sub>
</italic>:GUS reporter lines, respectively. Destaining was made with ethanol to remove chlorophyll. GUS staining was observed under the stereomicroscope and Leica DM2000 microscope.</p>
</sec>
<sec id="s4_12">
<title>Confocal microscopy</title>
<p>Confocal microscopy images were acquired using Zeiss LSM 800 with objectives Plan-Apochromat 40x/1.3 Oil DIC M27 in multi-track mode with line switching and averaging of two &#x2013; four readings. The excitation/emission wavelengths for GFP and propidium iodide (PI) were 488 nm/530 nm and 548 nm/561 nm, respectively.</p>
</sec>
<sec id="s4_13">
<title>Analysis of root morphology</title>
<p>Seedlings were germinated on one-half modified Hoagland&#x2019;s media containing full nutrient (+PN) for 5 days and then transferred for vertical growth under full nutrient (+PN), Pi-deficient (0 &#x3bc;M KH<sub>2</sub>PO<sub>4</sub>) or N-deficient (0.1 mM <inline-formula>
<mml:math display="inline" id="im3">
<mml:mrow>
<mml:msubsup>
<mml:mrow>
<mml:mtext>NO</mml:mtext>
</mml:mrow>
<mml:mn>3</mml:mn>
<mml:mo>&#x2212;</mml:mo>
</mml:msubsup>
</mml:mrow>
</mml:math>
</inline-formula>) conditions for another 5 days. For each independent experiment, the plates were prepared with the same volume of medium from the same batch. For the lateral root analyses, at least 9 plates were taken for the total sample collection. Photos were taken by PowerShot G16 Camera. The length of the primary roots and the number of lateral roots with length longer than 0.25&#xa0;cm <italic>per</italic> seedlings were calculated or counted using ImageJ (<xref ref-type="bibr" rid="B65">Schneider et&#xa0;al., 2012</xref>).</p>
</sec>
<sec id="s4_14">
<title>Chemical treatments</title>
<p>The Concanamycin A (Conc A; 1 mM; Cayman 11050) and Acetosyringone (150 mM; Sigma-Aldrich D134406) stock solutions were prepared in dimethyl sulfoxide (DMSO). The PI working solution (20 &#xb5;g/ml) was prepared from the stock solution (1 mg/ml; Invitrogen P3566). A six-hour of 1&#xb5;M Conc A or DMSO treatment was applied in the sample preparation for immunoblot analysis of ATG8s and NBR1 proteins. &#x3b2;-estradiol (36.7 mM; Sigma-Aldrich E2758) and acetosyringone (150 mM; Sigma-Aldrich D134406) stock solutions were prepared in ethanol and DMSO, respectively.</p>
</sec>
</sec>
<sec id="s5" sec-type="data-availability">
<title>Data availability statement</title>
<p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p>
</sec>
<sec id="s6" sec-type="author-contributions">
<title>Author contributions</title>
<p>T-YL designed the research. L-YL, H-XC, C-HC, W-CC, T-YL and NM performed experiments. T-YL, L-YL, H-XC, C-HC and NM analyzed data. T-YL, L-YL, H-XC and NM wrote the manuscript. All authors contributed to the article and approved the submitted version.</p>
</sec>
</body>
<back>
<sec id="s7" sec-type="funding-information">
<title>Funding</title>
<p>This work was supported by grants from the Ministry of Science and Technology of the Republic of China (MOST 105-2621-M-007-001-MY3 and 108-2311-B-007-003-MY3).</p>
</sec>
<ack>
<title>Acknowledgments</title>
<p>We thank Dr Tzyy-Jen Chiou at Academia Sinica, Taiwan, for kindly providing the <italic>pho1-2</italic> and <italic>phr1-3</italic> seeds and the technical support from Ms. Fumie Tobe at AIST and Ms. Ya-Hsien Chou at the confocal imaging core in National Tsing Hua University (sponsored by MOST 108-2731-M-007-001 and MOST 110-2731-M-007-001). We also thank Dr Wen-Chi Chang at National Cheng Kung University, Taiwan, for personal advice and additional help on using PlantPAN3.0.</p>
</ack>
<sec id="s8" sec-type="COI-statement">
<title>Conflict of interest</title>
<p>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.</p>
</sec>
<sec id="s9" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s10" sec-type="supplementary-material">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fpls.2023.1018984/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fpls.2023.1018984/full#supplementary-material</ext-link>
</p>
<supplementary-material xlink:href="DataSheet_1.pdf" id="SM1" mimetype="application/pdf"/>
<supplementary-material xlink:href="Table_3.xlsx" id="SM2" mimetype="application/vnd.openxmlformats-officedocument.spreadsheetml.sheet"/>
</sec>
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