RICE SALT SENSITIVE3 Forms a Ternary Complex with JAZ ... · RICE SALT SENSITIVE3 Forms a Ternary...

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RICE SALT SENSITIVE3 Forms a Ternary Complex with JAZ and Class-C bHLH Factors and Regulates Jasmonate-Induced Gene Expression and Root Cell Elongation C W Yosuke Toda, a Maiko Tanaka, a Daisuke Ogawa, a Kyo Kurata, a Ken-ichi Kurotani, a Yoshiki Habu, b Tsuyu Ando, b Kazuhiko Sugimoto, b Nobutaka Mitsuda, c Etsuko Katoh, b Kiyomi Abe, b Akio Miyao, b Hirohiko Hirochika, b Tsukaho Hattori, a and Shin Takeda a,b,1 a Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan b National Institute of Agrobiological Sciences, Kannondai, Tsukuba 305-8602, Japan c Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8562, Japan Plasticity of root growth in response to environmental cues and stresses is a fundamental characteristic of land plants. However, the molecular basis underlying the regulation of root growth under stressful conditions is poorly understood. Here, we report that a rice nuclear factor, RICE SALT SENSITIVE3 (RSS3), regulates root cell elongation during adaptation to salinity. Loss of function of RSS3 only moderately inhibits cell elongation under normal conditions, but it provokes spontaneous root cell swelling, accompanied by severe root growth inhibition, under saline conditions. RSS3 is preferentially expressed in the root tip and forms a ternary complex with class-C basic helix-loop-helix (bHLH) transcription factors and JASMONATE ZIM-DOMAIN proteins, the latter of which are the key regulators of jasmonate (JA) signaling. The mutated protein arising from the rss3 allele fails to interact with bHLH factors, and the expression of a signicant portion of JA- responsive genes is upregulated in rss3. These results, together with the known roles of JAs in root growth regulation, suggest that RSS3 modulates the expression of JA-responsive genes and plays a crucial role in a mechanism that sustains root cell elongation at appropriate rates under stressful conditions. INTRODUCTION In land plants, roots absorb water and soluble nutrients, in ad- dition to anchoring the shoot system. The spatial distribution of roots, including primary and lateral roots, largely depends on environmental signals, such as water availability, gravity, and light, which are perceived by the root apex (Kiss et al., 2002, 2003; Kobayashi et al. 2007) and modulate regional cell elon- gation rates via mechanisms that involve auxins (Swarup et al., 2005; Kaneyasu et al., 2007). Postembryonic root growth is sustained by continuous cell proliferation and subsequent cell differentiation. Cell division is maintained in the root apical meristem, or meristematic zone (MZ), which provides cells for elongation in the basal region, called the elongation zone (EZ). After elongation, cells undergo further differentiation or maturation in the maturation zone (MTZ), where root hairs and thickened secondary cell walls are formed. In contrast with root orientation, the rate of root growth is elaborately regulated in response to environmental changes at the level of both cell division and cell elongation. For example, when a plant grows under conditions of low phosphate avail- ability, primary root growth stalls and the number and length of lateral roots increases (Svistoonoff et al., 2007). In the primary root, the phosphate deciency is perceived in the apical region, resulting in the arrest of cell division and subsequent inhibition of cell elongation. This mode of regulation is mediated by LOW PHOSPHATE ROOT1 (LPR1), which encodes a multicopper oxidase (Svistoonoff et al., 2007). LPR1 has been proposed to modify a growth regulator in response to phosphate depletion in the apical region of the primary root. This in turn leads to the expression of genes, including those responsive to reactive oxygen species and to jasmonate (JA) and its derivatives (Chacón-López et al., 2011). JAs are a class of plant hormone involved in defense-related responses caused by wounding and pathogens and in the regulation of developmental processes, including fruit ripening, pollen production, root growth, tendril coiling, and senescence (Creelman and Mullet, 1997; Wasternack, 2007). JAs are also involved in the responses to a variety of abiotic stresses, such as salinity and drought stress (Wasternack, 2007; Takeuchi et al. 2011). In cultured Arabidopsis thaliana cells, JAs mediate the transcriptional reprogramming of genes, as reected in the repression of the cell cycle and the biosynthesis of phenyl- propanoids, including lignin precursors (Pauwels et al., 2008). In JA signaling, JASMONATE ZIM-domain proteins (JAZs) have an important role in gene regulation by physically interacting with members of the R/B-like (or MYC-type) basic helix-loop-helix (bHLH) transcription factors, such as Arabidopsis homolog of myelocytomatosis oncogene MYC2. In the presence of 1 Address correspondence to [email protected]. The author responsible for distribution of materials integral to the ndings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Shin Takeda (takeda@agr. nagoya-u.ac.jp). C Some gures in this article are displayed in color online but in black and white in the print edition. W Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.113.112052 This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online reduces the time to publication by several weeks. The Plant Cell Preview, www.aspb.org ã 2013 American Society of Plant Biologists. All rights reserved. 1 of 17

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RICE SALT SENSITIVE3 Forms a Ternary Complex with JAZand Class-C bHLH Factors and Regulates Jasmonate-InducedGene Expression and Root Cell ElongationC W

Yosuke Toda,a Maiko Tanaka,a Daisuke Ogawa,a Kyo Kurata,a Ken-ichi Kurotani,a Yoshiki Habu,b Tsuyu Ando,b

Kazuhiko Sugimoto,b Nobutaka Mitsuda,c Etsuko Katoh,b Kiyomi Abe,b Akio Miyao,b Hirohiko Hirochika,b

Tsukaho Hattori,a and Shin Takedaa,b,1

a Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, JapanbNational Institute of Agrobiological Sciences, Kannondai, Tsukuba 305-8602, JapancBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8562, Japan

Plasticity of root growth in response to environmental cues and stresses is a fundamental characteristic of land plants.However, the molecular basis underlying the regulation of root growth under stressful conditions is poorly understood. Here,we report that a rice nuclear factor, RICE SALT SENSITIVE3 (RSS3), regulates root cell elongation during adaptation tosalinity. Loss of function of RSS3 only moderately inhibits cell elongation under normal conditions, but it provokesspontaneous root cell swelling, accompanied by severe root growth inhibition, under saline conditions. RSS3 is preferentiallyexpressed in the root tip and forms a ternary complex with class-C basic helix-loop-helix (bHLH) transcription factors andJASMONATE ZIM-DOMAIN proteins, the latter of which are the key regulators of jasmonate (JA) signaling. The mutatedprotein arising from the rss3 allele fails to interact with bHLH factors, and the expression of a significant portion of JA-responsive genes is upregulated in rss3. These results, together with the known roles of JAs in root growth regulation,suggest that RSS3 modulates the expression of JA-responsive genes and plays a crucial role in a mechanism that sustainsroot cell elongation at appropriate rates under stressful conditions.

INTRODUCTION

In land plants, roots absorb water and soluble nutrients, in ad-dition to anchoring the shoot system. The spatial distribution ofroots, including primary and lateral roots, largely depends onenvironmental signals, such as water availability, gravity, andlight, which are perceived by the root apex (Kiss et al., 2002,2003; Kobayashi et al. 2007) and modulate regional cell elon-gation rates via mechanisms that involve auxins (Swarup et al.,2005; Kaneyasu et al., 2007).

Postembryonic root growth is sustained by continuous cellproliferation and subsequent cell differentiation. Cell division ismaintained in the root apical meristem, or meristematic zone(MZ), which provides cells for elongation in the basal region,called the elongation zone (EZ). After elongation, cells undergofurther differentiation or maturation in the maturation zone(MTZ), where root hairs and thickened secondary cell walls areformed. In contrast with root orientation, the rate of root growthis elaborately regulated in response to environmental changes atthe level of both cell division and cell elongation. For example,

when a plant grows under conditions of low phosphate avail-ability, primary root growth stalls and the number and length oflateral roots increases (Svistoonoff et al., 2007). In the primaryroot, the phosphate deficiency is perceived in the apical region,resulting in the arrest of cell division and subsequent inhibition ofcell elongation. This mode of regulation is mediated by LOWPHOSPHATE ROOT1 (LPR1), which encodes a multicopperoxidase (Svistoonoff et al., 2007). LPR1 has been proposed tomodify a growth regulator in response to phosphate depletion inthe apical region of the primary root. This in turn leads to theexpression of genes, including those responsive to reactiveoxygen species and to jasmonate (JA) and its derivatives(Chacón-López et al., 2011).JAs are a class of plant hormone involved in defense-related

responses caused by wounding and pathogens and in theregulation of developmental processes, including fruit ripening,pollen production, root growth, tendril coiling, and senescence(Creelman and Mullet, 1997; Wasternack, 2007). JAs are alsoinvolved in the responses to a variety of abiotic stresses, suchas salinity and drought stress (Wasternack, 2007; Takeuchi et al.2011). In cultured Arabidopsis thaliana cells, JAs mediate thetranscriptional reprogramming of genes, as reflected in therepression of the cell cycle and the biosynthesis of phenyl-propanoids, including lignin precursors (Pauwels et al., 2008). InJA signaling, JASMONATE ZIM-domain proteins (JAZs) have animportant role in gene regulation by physically interacting withmembers of the R/B-like (or MYC-type) basic helix-loop-helix(bHLH) transcription factors, such as Arabidopsis homologof myelocytomatosis oncogene MYC2. In the presence of

1 Address correspondence to [email protected] author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Shin Takeda ([email protected]).C Some figures in this article are displayed in color online but in black andwhite in the print edition.W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.113.112052

This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been

edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online

reduces the time to publication by several weeks.

The Plant Cell Preview, www.aspb.org ã 2013 American Society of Plant Biologists. All rights reserved. 1 of 17

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Ile-conjugated JA, the F-box protein CORONATINE INSENSITIVE1(COI1) binds to JAZs, resulting in their polyubiquitination andsubsequent degradation by 26S proteasome (Chini et al., 2007;Thines et al., 2007). This releases the repression of R/B-likebHLH transcription factors from JAZs, leading to the transcrip-tional regulation of downstream target genes. In rice (Oryzasativa), overexpression of JAZ9, also called TIFY11a, usinga stress-inducible promoter was reported to alleviate the growthinhibition resulting from salt and dehydration stress (Ye et al.,2009), although the downstream factors involved in the processwere not identified.

In Arabidopsis, exogenously supplied JAs inhibit root growth,in terms of both cell division and cell elongation (Chen et al.,2011). This inhibition is completely suppressed in coi1 (Lorenzoet al., 2004; Chen et al., 2011). By contrast, loss-of-functionmutations of MYC2 (jasmonate-insensitive1) exhibit weakersuppression of JA-induced root growth inhibition than coi1,suggesting that activation of MYC2 does not account for all ofthe JA-mediated inhibition of root growth (Lorenzo et al., 2004).Consistent with this, MYC2 was found to influence the expres-sion of only a subset of JA-responsive genes (Dombrecht et al.,2007). Moreover, myc2 combined with loss-of-function muta-tions of MYC3 and MYC4, the R/B-like bHLHs closely relatedto MYC2, further suppressed the JA-induced root growth in-hibition, but the myc2 myc3 myc4 triple mutant was still rela-tively sensitive to JA than coi1 (Fernández-Calvo et al., 2011).Recently, MYC2 was suggested to reduce root meristem activityby repressing PLETHORA expression upon the activation of JAsignaling (Chen et al., 2011). However, the transcriptional reg-ulatory mechanisms that inhibit root cell elongation after JAperception remained unclear.

Salinity represents a widespread problem in agriculture. De-spite extensive studies of the salt stress response and toleranceusing a model dicot, Arabidopsis (Zhu, 2001; Yamaguchi andBlumwald, 2005; Takeda and Matsuoka, 2008; Qin et al., 2011),the mechanism underlying the control of root growth undersalinity is still not well understood, especially in monocots. Wepreviously showed that a rice protein RICE SALT SENSITIVE1(RSS1), whose stability is controlled in a cell cycle phase–dependent manner, is required for the maintenance of meristemfunctions in the shoot and root under stressful conditions(Ogawa et al., 2011). In rss1 roots, cell proliferation activity isreduced upon salinity stress, resulting in a decrease in thenumber of cells in both the MZ and EZ. This finding illustratesthe importance of the coordinated regulation of cell division andelongation in response to environmental changes.

In this study, we show that a rice nuclear factor, RSS3, hasa regulatory role in root cell elongation. RSS3 interacts not onlywith JAZs, but also with non-R/B-like bHLH transcription factorsand forms an RSS3-JAZ-bHLH ternary complex in the nucleus.Loss of function of RSS3 activates the expression of a subset ofJA-induced genes in the root apex and restricts cell elongationbut does not primarily affect cell division activity. Under high-salinity conditions, rss3 mutants exhibit severely inhibited rootgrowth, concomitant with root cell swelling. These results reveala molecular mechanism for the modulation of JA-responsivegene regulation, which is especially crucial for the control of rootelongation in response to stressful conditions.

RESULTS

The rss3 Mutant Has a Defect in Root Cell Elongation

In the course of a genetic screen of rice to identify loci responsiblefor salt tolerance (Ogawa et al., 2011), we identified a recessivemutant designated rss3. When grown under high-salinity con-ditions, rss3 exhibited severely impaired root growth comparedwith the wild type (Figures 1A and 1B; see Supplemental Figure 1Aonline). Root growth in the absence of salinity stress was also in-hibited in rss3, but only moderately. The length of cells in the MTZof roots of rss3 plants grown under unstressed conditions wasmarkedly shorter than those of the wild type (see SupplementalFigure 1B online). The inhibition of root growth in rss3 appeared tobe primarily due to reduced cell elongation because the size of theEZ, but not of the MZ, was reduced in the mutant, and the numberof cells in both the EZ and MZ was not affected in the mutant(Figures 1C and 1D). The exaggerated inhibition of root growthunder salinity conditions also appeared to be mainly due to com-promised cell elongation but was accompanied by aberrant cellulararrangement and the formation of oblique cell plates in the MZ,swelling of the cells in the EZ (see Supplemental Figures 2A and2B online), and a waviness of the root surface at the MTZ (Figure1E). Moreover, rss3 roots show impaired flexibility, in a salinity-dependent manner. When extracted from the medium, the wild-type roots exhibited a paintbrush-like shape, but rss3 roots did not,due to stiffness (Figure 1A). These rss3 phenotypes were observedboth in seminal and adventitious crown roots, although rss3showed a slight decrease in the number of crown roots, regardlessof the salinity conditions (see Supplemental Figure 1C online).Collectively, these results suggest that RSS3 has an importantrole in root cell elongation and has additional functions inregulating root elongation under high-salinity conditions.When seedlings were grown under continuous light, root coiling

was frequently observed in the wild type under nonstressed con-ditions but not under salinity stress. By contrast, root coiling wasnot observed in rss3, even under nonstressed conditions (seeSupplemental Figures 3A and 3B online). This is not due to a de-fect in the gravity-sensing machinery since root gravitropism is notimpaired in rss3 (see Supplemental Figure 3C online). These re-sults also suggest that the flexibility of rss3 roots is impaired.

RSS3 Encodes a Nuclear Protein Homologous to theRegulatory Domain of the R/B-Like bHLHTranscription Factors

The causative gene of the rss3 mutant was identified by map-based cloning as Os11g0446000 (Figure 2A). Among four geneslocated in the mapped region, we found a deletion inOs11g0446000 in rss3. Transformation with a genomic fragmentof the wild-type allele of Os11g0446000 complemented the re-cessive mutant phenotype of rss3 (see Supplemental Figure 4Aonline). RSS3 encodes a protein consisting of 458 amino acids.RSS3 contains a region homologous to the postulated regula-tory domain conserved among the R/B-like bHLH transcriptionfactors (Li et al., 2006), which contains four conserved regions(I to IV) (Figures 2B and 2C). Region II is predicted to constitutethe central sequence of the JAZ interacting domain (Cheng et al.,

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2011; Fernández-Calvo et al., 2011; Niu et al., 2011). However,RSS3 does not contain a bHLH domain or another known DNAbinding motif. rss3 contains a 47-bp deletion at the junctionbetween the 6th intron and 7th exon (Figure 2A), which results inan altered splicing pattern (see Supplemental Figure 4B online)and the production of a mutated protein that lacks 15 aminoacids in region IV and six amino acids in the subsequentC-terminal nonconserved region (Figure 2C).

In seedlings, RSS3 was expressed predominantly in the roots,particularly in the root tip (see Supplemental Figure 5 online). Atruncated form of RSS3 transcript was expressed in the mutantroots, at levels that were apparently higher than those of thenondeleted RSS3 transcript in the wild type (see SupplementalFigure 4C online). When enhanced green fluorescent protein(EGFP)–tagged RSS3 was expressed under the control of theRSS3 promoter, EGFP signals were detected at high levels inthe MZ and at low levels in the EZ of the root tip (Figure 2D, left).The fusion protein was localized almost exclusively in the nucleiof the MZ cells (Figure 2D, top right). By contrast, nonfusedEGFP expressed under the control of the constitutive actin genepromoter was localized in both nuclei and the cytoplasm of MZcells. In the EZ region bordering the MZ, RSS3-EGFP wasdetected in both the nucleus and the cytoplasm (Figure 2D,bottom right). These data suggest that RSS3 functions as

a nuclear protein in the root tip and are consistent with themutant phenotypes.

RSS3 Interacts with the Transcription Factors bHLH089and bHLH094

We then used yeast two-hybrid (Y2H) screening to search fora protein that interacts with RSS3. Because the full-length RSS3caused a self-activation in the Y2H system, we used a version ofRSS3 that lacked the C-terminal 140 amino acids (RSS3N318) asbait and identified a non-MYC-type bHLH transcription factor(bHLH094) as an RSS3 binding protein. The cDNA for bHLH094expressed in the screening was truncated and encoded only theC-terminal part (amino acids 143 to 256) of this protein (Figure3A). An analysis using further deletions of the truncatedbHLH094 revealed that a region (amino acids 143 to 200) cor-responding to the bHLH and subsequent C-terminal regionswas necessary and sufficient for the interaction with RSS3(Figure 3B). We also examined the ability of a deletion series ofthe RSS3 bait to interact with bHLH094 and identified a region ofRSS3 (amino acids 1 to 203) as being responsible for the in-teraction (Figure 3C). Importantly, the 21–amino acid sequencemissing from RSS3 in the rss3 mutant was required for the in-teraction with the bHLH factor.

Figure 1. Impaired Root Growth of rss3 under Salinity Conditions.

(A) and (B) Wild-type (WT) and rss3 seedlings were grown with (+) or without (2) 100 mM NaCl for 7 d.(A) Seedlings.(B) Roots floating on water.(C) Propidium iodide staining of the primary roots of 3-d-old wild-type and rss3 seedlings, grown in the presence or absence of 100 mM NaCl. Red andwhite arrowheads indicate the border of the MZ and EZ, and of the EZ and MTZ, respectively. Images were generated by concatenating two or morepictures.(D) Length (left) and number of cells (right) in the MZ and EZ of the root tips of 3-d-old wild-type and rss3 seedlings, grown with or without 100 mM NaCl.Mean 6 SD, n $ 3. Asterisks indicate a Student’s t test P value < 0.05.(E) The MTZs of 7-d-old roots of wild-type and rss3 seedlings grown in the presence or absence of NaCl were observed by differential interferencecontrast microscopy.Bars = 5 cm in (A), 1 cm in (B), 400 µm in (C), and 100 µm in (E).

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In vivo interaction of RSS3 and bHLH094 was examined bybimolecular fluorescence complementation (BiFC) and acceptorphotobleaching Förster resonant energy transfer (apFRET)analyses (Figure 4). When expressed in onion (Allium cepa)epidermal cells, yellow fluorescent protein (YFP)–fused RSS3localized to both the nucleus and cytoplasm, whereas cyanfluorescent protein (CFP)–fused bHLH094 (bHLH094-CFP) lo-calized exclusively to the nucleus (Figure 4A). For BiFC assay,the N-terminal region of RSS3 fused to the N-terminal half ofYFP (YFPN-RSS3N203) was coexpressed with the full-lengthbHLH094 fused to the C-terminal half of YFP (bHLH094-YFPC).The complemented YFP signals were predominantly detected in

the nucleus (Figure 4B), indicating the in vivo interaction of thetwo proteins. In the apFRET assay, bHLH094-CFP was ex-pressed together with YFP-RSS3N203 or YFP-RSS3full-length inonion epidermal cells. We measured the strength of the CFPsignal before and after photobleaching of the YFP and calcu-lated the efficiency of FRET (see Methods for details). Afterphotobleaching of YFP-RSS3N203 or YFP-RSS3full-length in thenucleus, an increase in bHLH94-CFP signal was observed(Figures 4C to 4E), reflecting the interaction of the YFP and CFPfusion proteins. By contrast, energy transfer was not detectedwhen nonfused YFP was expressed as a control, instead of YFPfused to RSS3N203 or RSS3full-length (Figures 4C to 4E). These

Figure 2. RSS3 Encodes a Nuclear Protein Homologous to the Regulatory Domains of R/B-Like bHLH Proteins.

(A) Map-based cloning of RSS3 (top) and the schematic representation of the RSS3 gene structure (bottom). The rss3 locus was delimited between theindicated markers. The chromosomal position of the markers and the number of F2 lines exhibiting the rss3 phenotype among 1501 lines examined areprovided in brackets and parentheses, respectively. Exons (closed boxes), 59- or 39-untranslated regions (open boxes), and the position of the firstcodon (ATG), stop codon (TAG), and 47-bp deletion in rss3 are indicated.(B) Schematic structure of RSS3 and an R/B-like bHLH protein, MYC2, of Arabidopsis. The regions conserved between RSS3 and At-MYC2 (I to IV) andthe position of the 21–amino acid (a.a.) deletion in rss3 are shown. bHLH represents the basic helix-loop-helix DNA binding domain.(C) The amino acid sequences of the N-terminal region of RSS3, RSS3-related proteins, and R/B-like bHLH factors were aligned using the MAFFTprogram. The red dotted line represents the sequence lacking in rss3. The conserved regions (I to IV) and JAZ interacting domain of At-MYC2 and At-MYC3 (Fernández-Calvo et al., 2011) are indicated.(D) GFP signals of rice roots carrying RSS3-EGFP driven by the RSS3 promoter or EGFP driven by the Actin promoter. The red arrowhead indicates theborder between the MZ and EZ. Magnified views of the MZ (top) and EZ (bottom) are shown in the right panels. Bars = 200 mm (left) and 10 mm (rightpanels).

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results strongly suggest that RSS3 and bHLH094 can interact inplant cells.

We also examined which types of bHLH factors bind to RSS3.Among more than 150 bHLH factors in rice, bHLH094 belongsto the class-C group (Li et al., 2006). The distinguishing featureof class-C members is the conservation of bHLH sequences andthe subsequent C-terminal regions (see Supplemental Figure6 online). To examine whether RSS3 interacts with other riceclass-C bHLH factors, we selected bHLH089, which is classifiedin the same minor clade that bHLH094 belongs to, and threerepresentative bHLH transcription factors from other clades of

Figure 3. The Interaction between RSS3 and bHLH094 in Yeast.

(A) Schematic representation of the full-length rice bHLH094 protein andits derivative (Y2H prey) obtained by Y2H screening. The bHLH domain isrepresented in blue, and the region that is conserved specifically in theclass-C bHLH subfamily is shown in orange. AD, activation domain ofGAL4.(B) Analysis of the domains of bHLH094 required for binding to the baitRSS3N318 as determined by Y2H assay. Numbers indicate terminalamino acid residues of bHLH094 fused to AD.(C) bHLH094 interacting domains of RSS3 analyzed by a Y2H assay.Numbers indicate the terminal amino acid residues of RSS3 fused toGAL4 DNA binding domain (DBD). rss3 indicates the truncated proteintranslated in rss3 with a 21–amino acid deletion. Plasmids carrying ADalone (B) or DBD alone (C) were used as controls. Yeast transformantswere spotted onto control medium [-(L,W), lacking Leu and Trp] or se-lective medium [-(A,H,L,W), lacking Ade, His, Leu, and Trp], and theactivation of ADE2 and HIS3 reporter genes was monitored.

Figure 4. The in Vivo Interaction between RSS3 and bHLH094.

(A) The subcellular localization of rice bHLH094-CFP and YFP-RSS3N203

in onion epidermal cells. bHLH094 localized exclusively to the nucleus,whereas RSS3 localized to both the nucleus and cytoplasm. c, cyto-plasm; n, nucleus.(B) BiFC assay to verify interaction between RSS3 with bHLH094. YFPN-RSS3N203 and bHLH094-YFPC or YFPC were transiently expressed withmRFP1 in onion epidermal cells.(C) Representative images of the apFRET assay using bHLH094-CFP andYFP-RSS3N203 (left four) or nonfused YFP (right four). Signal intensities of theacceptor and donor in onion cells before and after acceptor photobleachingwere analyzed. Pseudocolor images represent fluorescence intensities.(D) Evaluation of the interaction between YFP-RSS3N203 and the class-CbHLH proteins fused to CFP by an apFRET assay. FRET efficiency wasshown with mean values 6 SD; n = 3 to 8.(E) apFRET analysis of full-length RSS3 fused to YFP and class-C bHLHproteins fused to CFP in the nucleus of onion cells. Mean6 SD; n = 3 to 5.The names of rice bHLHs are as reported (Li et al., 2006). The controlindicates the combination of YFP and bHLH094-CFP. Asterisks indicateP value < 0.05 (Student’s t test).Bars = 200 µm in (A) and (B) and 20 µm in (C).

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class-C (bHLH092, bHLH079, and bHLH096). The in vivo in-teraction between these bHLH factors and RSS3 was examinedby the apFRET assay. By the combinatorial expression of therespective bHLH-CFPs with YFP-RSS3N203, FRET efficiencythat reflects the physical interaction was positive with bHLH089but negative with bHLH092, bHLH079, and bHLH096 (Figure4D). Similarly, the interaction of RSS3full-length with bHLH094 andbHLH089, but not with bHLH092, was demonstrated by theapFRET assay (Figure 4E). These results indicate that RSS3 canspecifically interact with bHLH094 and bHLH089. Interestingly,both full-length bHLH094 and bHLH089 lack the N-terminalregulatory domains that are conserved among R/B-like bHLHfactors and RSS3 (Li et al., 2006). The genes encoding bHLH094and bHLH089 were expressed in the root tip, although highlevels of expression were also observed in the basal tissues ofthe shoot (see Supplemental Figure 5 online). Taken together,these results suggest that RSS3 acts as a regulatory module forbHLH094 and bHLH089 and thus is directly involved in tran-scriptional regulation in the root tip.

RSS3 Interacts with JAZ Proteins

As described above, RSS3, but not bHLH094 or bHLH089, con-tains the regulatory domain conserved among R/B-like bHLHtranscription factors. Considering that region II of the regulatorydomain is a part of the JAZ interacting domain, we speculated thatRSS3 is capable of binding to JAZs. To test this possibility, thebinding abilities of 12 of the 15 rice JAZs (Ye et al., 2009) to RSS3were examined by the Y2H assay. This analysis showed that JAZ8,JAZ9, and JAZ11 interact specifically with RSS3 (Figure 5A).Binding of RSS3 to JAZ9 and JAZ11, but not to JAZ8, was furtherconfirmed in vivo by the apFRET assay (Figures 5B and 5C).JAZ9 and JAZ11 were preferentially expressed in the apical

and distal regions of the root tip, respectively (see SupplementalFigure 5 online). A comparison of Arabidopsis and rice JAZproteins revealed that Os-JAZ9 and Os-JAZ11 belong to a rice-specific clade (Ye et al., 2009; see Supplemental Figure 7online). Both Os-JAZ9 and Os-JAZ11 contain a canonical ZIMdomain and a Jas domain, which are highly conserved amongJAZ proteins (Figure 5D; see Supplemental Figure 7 online). TheJas domain contains a JAZ degron, which contains a critical rolefor JA-Ile–dependent binding to COI1 (Chini et al., 2007; Melottoet al., 2008; Sheard et al., 2010). In several Arabidopsis JAZproteins, the Jas domain and its surrounding region is involvedin the interaction with MYC-type bHLH transcription factors (Chiniet al., 2007; Shyu et al., 2012). Several Os-JAZs were demon-strated to interact with COI1 (Seo et al., 2011), suggesting that the

Figure 5. The Interaction between RSS3 and Rice JAZ Proteins.

(A) Characterization of the interaction between RSS3N318 and riceJAZ proteins using the Y2H assay. Yeast transformants were spottedon control medium [-(L,W)] or selective medium [-(H,L,W) + 3-AT,or –(A,H,L,W)] to monitor ADE2 and HIS3 reporter activity. 3-AT,3-amino-1,2,4-triazole (5 mM). The vector plasmid expressing onlythe DNA binding domain was used as a control (empty). Among the15 rice JAZs proteins reported by Ye et al. (2009), 12 JAZs wereexamined.(B) and (C) Evaluation of the interaction between RSS3N203 and JAZproteins by apFRET analysis. Representative images of apFRET in onioncells using YFP-RSS3N203 and ECFP-JAZ9 (left four) or nonfused ECFP(right four) are shown in (B). Pseudocolor images represent fluorescenceintensities. Bar = 10 µm.

(C) FRET efficiencies between YFP-RSS3N203 and the respective JAZproteins fused to ECFP or nonfused ECFP are shown (mean 6 SD; n = 3to 4). Asterisks indicate P value < 0.05 (Student’s t test). The respectiveJAZ proteins fused to ECFP were coexpressed with YFP fused toRSS3N203 in onion cells.(D) Schematic structure of Os-JAZ9, Os-JAZ11, and At-JAZ1. Numbersindicate the terminal amino acid residues. The position of the conserveddomains is shown. The names of rice JAZs are as reported (Ye et al.,2009).

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rice JAZ homologs are able to function with COI1 in JA percep-tion. Os-JAZ9 and Os-JAZ11 also contain the consensus se-quence of the JAZ degron, including the two basic amino acidresidues (6th and 7th relative to the first amino acid of the JAZdegron) and the strictly conserved Phe residue that are requiredfor the interaction with COI1 and JA-Ile (Melotto et al., 2008;Sheard et al., 2010) (see Supplemental Figure 7B online). In ad-dition, most of the amino acids that are involved in hydrogen bondformation in the complex that forms between JA-Ile, COI1, andthe JAZ peptide are conserved in Os-JAZ9 and Os-JAZ11 and thetwo rice COI1 proteins (see Supplemental Figure 8 online).Moreover, both JAZ9 and JAZ11 was shown to bind to the riceCOI1 homologs at least by Y2H assay in the presence of coro-natine, a structural analog of JA-Ile (Lee et al., 2013). Therefore,Os-JAZ9 and Os-JAZ11 may also function through their bindingto JA-lle.

RSS3 Forms a Ternary Complex with Rice bHLH094and JAZ9

Based on the finding that RSS3 has two different binding part-ners, we examined whether RSS3 is capable of forming a ternarycomplex with the bHLH factor and JAZ by yeast three-hybridassay (Figure 6A). Positive interaction was observed whenRSS3N318 was coexpressed with bHLH094 and JAZ9. Moreover,when the 21–amino acid deletion mimicking the rss3 mutationwas introduced into RSS3N318 (RSS3N318_D21), the interactionwas disrupted. In vivo ternary complex formation was furtherexamined using a combination of the BiFC and apFRET assays(BiFC-apFRET) (Figures 6B and 6C). YFPN-RSS3N203 andOs-bHLH094-YFPC were coexpressed in onion epidermal cells,together with ECFP-fused Os-JAZ9 (ECFP-OsJAZ9). After photo-bleaching, the YFP fluorophore reconstituted from YFPN-RSS3N203

and OsbHLH094-YFPC, an increase in ECFP-OsJAZ9 signal wasobserved in the nucleus (Figures 6B and 6C). By contrast, no signalincrease was detected when YFPN-RSS3N203 and OsbHLH094-YFPC were coexpressed with nonfused ECFP. These resultssuggest that RSS3, Os-JAZ9, and Os-bHLH094 form a ternarycomplex in the nucleus.

RSS3 and JAZ together Repress bHLH094-MediatedTranscriptional Activation

bHLH factors are known to bind to specific DNA sequences witha minimal consensus motif, called the E-box (CANNTG, where Ncorresponds to any nucleotide) (Atchley and Fitch, 1997). Thestrength of the interaction varies with the identity of N and differsbetween different bHLH transcription factors. For example,Arabidopsis MYC2 has high binding affinities for CACGTG (alsocalled the G-box) and CATATG, but not for CAATTG andCAGCTG sequences (Godoy et al., 2011). We examined whetherOs-bHLH094 acts on the same DNA sequences as ArabidopsisMYC2. We constructed LUC reporter genes carrying a triplicaterepeat of the respective E-box sequences fused to the upstreamregion of a cauliflower mosaic virus (CaMV) 35S minimal pro-moter and cotransformed rice cultured cell protoplasts with theseconstructs and bHLH094. As shown in Figure 7A, bHLH094 en-hanced the expression of reporters carrying (CACGTG)x3, (CAGCTG)

Figure 6. RSS3, bHLH, and JAZ Form a Ternary Complex.

(A) Yeast three-hybrid assay to verify interaction of RSS3N318, bHLH094,and JAZ9. Representative results of more than six independent trans-formants are shown for each combination. D21 represents the 21–aminoacid deletion mimicking rss3 mutation. The control and selective mediumwere as described in Figure 5. Media lacking Met (-M) was used for in-duction of the gene encoding the 3rd protein by MET25 promoter fortesting interaction between the three proteins expressed. “Empty”means the vector control. 3AT, 3-amino-1,2,4-triazole(B) and (C) Interaction among JAZ9, RSS3N203, and bHLH094, as ana-lyzed by BiFC-apFRET. Representative images (B) and FRET efficiencies(C) of apFRET in onion cells using YFPN-RSS3N203, bHLH094-YFPc, andECFP-JAZ9 or nonfused ECFP are shown. Pseudocolor images repre-sent fluorescence intensities. Bar = 10 µm. Mean 6 SD; n = 3 (ECFP) and6 (ECFP-JAZ9). Asterisks indicate P value < 0.05 (Student’s t test).

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x3, or (CATATG)x3 but not of those carrying (CAATTG)x3. These re-sults indicate that bHLH094 can activate transcription throughspecific E-box sequences. Activation of the promoter carrying(CAGCTG)x3 may indicate that the binding specificity of Os-bHLH094 is different from that of At-MYC2.

To examine the possibility that RSS3 functions as an effectorof bHLH094-dependent transcriptional regulation, RSS3 (drivenby the CaMV 35S promoter) was additionally coexpressed withbHLH094. However, no significant effect on reporter expressionwas observed (Figure 7A). When RSS3 and JAZ9 were coex-pressed, bHLH094-dependent activation of the reporter geneswas significantly repressed (Figure 7B). By contrast, this re-pression was not observed by the expression of JAZ9 alone orwhen RSS3 was replaced with RSS3_D21, which encodes theRSS3 variant with the 21–amino acid deletion as in rss3 (Figure7B). Together with the finding that RSS3 forms a complex withJAZ9 and bHLH094, these results indicate that the combinedRSS3-JAZ9 represses the bHLH094-mediated transcriptionalactivation, and this regulation is impaired in rss3.

Expression of Salt-Responsive Genes Is Largely Alteredin rss3

To gain further insight into the function of RSS3, we performeda transcriptomic analysis of the root tip, using a rice 44K micro-array. First, we analyzed the relationship between the effects of themutant genotype (rss3 mutation) and those of salinity (100 mMNaCl treatment) using two-way analysis of variance (ANOVA). Asillustrated in the Venn diagram (Figure 8A), nearly half (49.5%; 3366/6804) of the genes affected by salinity stress were also affected byrss3 (bold numbers). These 3366 genes also accounted for half(48.9%) of the 6884 genes affected by rss3. For most of these 3366genes, the altered expression in rss3 was observed under eithernonstress or salinity stress conditions, and the salt-responsivenessof these genes seemed not to be changed between the wild typeand rss3 (Figure 8B). This is reflected by the small proportion ofthe interactively affected genes (genotype 3 salinity), the salt-responsiveness of which was modified in rss3 (183/3366; Figure8A). The proportion of the 183 genes interdependently affected bysalinity and rss3 was also small among the total number of affectedgenes (10372; Figure 8A). Similar tendencies were observed whenthe differentially expressed genes were further filtered with morestringent fold-change criteria (>2 or <0.5) (Figure 8A, numbers ingray, parentheses). These results suggest that RSS3 has globaleffects on the salt stress response pathways but does not con-stitute stress response signaling mechanisms.

A Substantial Proportion of JA-Responsive Genes AreActivated in rss3

Prompted by the finding that RSS3 forms complexes with JAZand bHLH proteins, we explored whether the expression ofJA-responsive genes was affected by rss3 (Figure 8C; seeSupplemental Data Sets 1A and 1B online). For this analysis,genes that were altered in expression by rss3were further selectedwith a cutoff of 1.5-fold change and with significance evaluated byrank product (P < 0.01, percentage of false positive [pfp] < 0.05).JA-responsive genes were selected in the same manner using the

data deposited by Miyamoto et al. (2012) in the Gene ExpressionOmnibus of the National Center for Biotechnology Information(GSE32633). Notably, 20% of 112 JA-inducible genes were up-regulated in rss3 under nonstressed conditions (Figure 8C). Simi-larly, 28% of the JA-inducible genes were upregulated in rss3under salinity stress. These correspond to approximately aneightfold (P = 4.5e214, hypergeometric distribution) and 11-fold(P = 2.1e223, hypergeometric distribution) enrichment, as thepercentage of genes upregulated in rss3 out of the total number ofgenes examined in the array was 2.5 and 2.6% under nonstressedand salinity conditions, respectively. Only a small portion of JA-induced genes was downregulated in rss3 in either nonstressed orsalinity conditions (5 and 2%, respectively). The expression ofgenes that are repressed by JA was less significantly affected byrss3. These results suggest that RSS3 has a role in repressinga portion but not all of the JA-inducible genes. Among the JA-responsive genes, the expression of which was altered in rss3, wefound no significant biases to certain gene categories with respectto cellular metabolism and/or molecular function (see SupplementalData Set 1C online).To further evaluate the role of RSS3 in the regulation of JA-

inducible genes, we examined the expression pattern of severalJA-responsive genes. Wild-type and rss3 seedlings were grownin a hypotonic culture medium and transferred into the mediumin the presence or absence of 100 mM JA. In the wild-type roottips, the expression of JAZ7, JAZ8, and SECOLOGANIN SYN-THASE (SLS) was induced by JA within 6 to 16 h (Figure 8D).The expression levels of these genes after JA treatment werefurther increased in rss3 (Figure 8D), indicating that RSS3 hasa role in mediating negative regulation of JA-induced geneexpression. Taken together, our results suggest that the JAZ-RSS3-bHLH ternary complex regulates the expression of JA-responsive genes in root tips. It is likely that the repressive functionof this complex is due to the JAZ component, as is the case for thewell-known JAZ-MYC2 complex in Arabidopsis (Chini et al., 2007;Thines et al., 2007).It is noteworthy that, in the absence of exogenously supplied

JA, the JA-induced genes are upregulated in rss3 in the con-ditions used for microarray analysis (Figure 8C; see SupplementalFigure 9A online) but not significantly in the hypotonic cultureconditions (Figure 8D). This suggests that difference in physio-logical state in the root tip between the experimental setups af-fects the expression of JA-induced genes. We confirmed that, inthe growth conditions in which root elongation was reducedin rss3 (Figure 1), the expression levels of the JA-induced genes inrss3 was considerably higher than those in the wild type grown inthe absence of exogenous JA (see Supplemental Figure 9B on-line).

Low Levels of JA Inhibit Cell Elongation but Not Cell Divisionin the Root Tip

JAs inhibit cell division in the Arabidopsis root tip (Chen et al.,2011). In Arabidopsis cultured cells, cell cycle arrest at G2 hasbeen reported for treatment with 50 mM JA (Pauwels et al.,2008). By contrast, rss3 resulted in reduced cell elongation butdid not affect meristematic activity in the absence of exogenousJA (Figure 1). To verify the possibility that this reflects the

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derepression of JA-responsive genes in rss3, we examined iftreatment of the wild type with low concentrations of JA wouldphenocopy the reduced root cell elongation phenotype of rss3.Even though JA response is provoked in rss3 without JA treat-ment, it was less than those in wild type grown in the presenceof 1 or 5 mM JA (see Supplemental Figure 9B online). Wetherefore expected that these concentrations of JA would inhibitroot cell elongation specifically in EZ in the wild type. In fact,5 mM JA caused a decrease in the size of the EZ but not of theMZ in the wild-type root tip, whereas both the MZ and EZ werereduced in size by 10 mM JA. In the MTZ, a reduction in cell sizewas observed even with 5 mM JA (see Supplemental Figures10A and 10B online).To explore these concentration-dependent differential effects

of JAs on root tip cells, we monitored the expression levels ofa S-phase marker gene, PCNA, and a M-phase cyclin, CycB2;1.The expression of CycB2;1 was repressed by 10 mM JA, but notby 5 mM, while the expression of PCNA was not significantlychanged by JA at either concentration (see Supplemental Figure10C online). These results further support the above idea thatthe effect of rss3 is mimicked by low concentrations of JA.

DISCUSSION

JAs are known to regulate root elongation in a wide variety ofplant species (Corbineau et al., 1988; Creelman and Mullet,1997; Ulloa et al., 2002; Uppalapati et al., 2005). In this article,we have shown that RSS3 is a novel regulatory factor of tran-scription, particularly in the regulation of JA-induced genes, thatcontrols root cell elongation. RSS3 is expressed predominantlyin the root tip, particularly in the MZ and in the EZ region bor-dering MZ. The function of RSS3 in transcriptional regulationwas mainly evidenced by the interaction of RSS3 with bHLH094and bHLH089 in the nucleus. We also demonstrated that RSS3binds to JAZ9 and JAZ11 and thereby forms a ternary complexwith bHLH094 and JAZ9. It is conceivable that bHLH089 andJAZ11 also form a similar complex with RSS3, based on theirstructural similarities to bHLH094 and JAZ9, respectively. RSS3is likely to mediate the repression of bHLH factors by JAZ pro-teins, some of which are proposed to mediate active repressionthrough NOVEL INTERACTOR OF JAZ (NINJA) and TOPLESS(TPL) (Pauwels et al., 2010).We propose that RSS3 represses the expression of JA-

induced genes because a portion of JA-responsive genes wasupregulated in the root tip of rss3, and the levels of expression of

Figure 7. RSS3 and JAZ9 together Suppress bHLH094-Mediated GeneActivation.

(A) bHLH094 enhances the activity of the gene promoter containingE-box (CANNTG).(B) A combinational expression of RSS3 and JAZ9 suppresses thebHLH094-mediated gene activation driven by the promoter containingG-box (CACGTG). Effector plasmids containing bHLH094, RSS3,

RSS3_D21, and JAZ9 driven by the CaMV 35S promoter were in-troduced into rice protoplasts, concomitant with a reporter plasmidcontaining a triplicate E-box (A) or G-box (B) fused to the basal pro-moter-fLUC (reporter). fLUC activity was measured and normalized withthat of rLUC. Empty vector was added to equalize the amount of plas-mids transfected in each combination. Mean 6 SD; n = 3 to 8. Asterisksindicate a Student’s t test P value < 0.05 in (A). Statistical differenceswere determined by one-way ANOVA followed by Tukey posthoc anal-ysis, and different letters indicate significant differences (P value < 0.05)in (B). fLUC, firefly luciferase; rLUC, renilla luciferase.[See online article for color version of this figure.]

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Figure 8. The Expression of Salt- and JA-Responsive Genes Is Altered in rss3.

(A) Venn diagram summarizing the gene expression patterns examined using rice 44K microarrays. Differentially expressed genes based on threeparameters, genotype (wild type versus rss3), salinity (with or without 100 mM NaCl), and interaction (genotype3 interaction), were categorized by two-way ANOVA (P < 0.01) (bold). The differentially expressed genes were further filtered by fold change criteria ($2 or #0.5) (gray, parentheses). Thenumbers of nonredundant genes in each category are indicated. The interaction between genotype and salinity, genes that were interdependentlyaffected by differences in the genotype and growth conditions.(B) Heat map views of 3366 nonredundant genes whose expression was altered in rss3 and by salinity in (A). WT, the wild type.(C) Proportion of JA-responsive genes among upregulated (red) and downregulated (blue) genes in rss3 under nonstressed and high-salinity conditions.The up- and downregulated genes in rss3 were categorized as described in Methods. The JA-responsive genes were selected using data deposited byMiyamoto et al. (2012) into the Gene Expression Omnibus of the National Center for Biotechnology Information (GSE32633) with a cutoff of 1.5-foldupregulation and rank product (P < 0.01, pfp < 0.05). Numbers indicate the amount of nonredundant genes in each category. P values indicate thehypergeometric distribution significance, evaluating the enrichment of JA-induced genes.(D) The expression of JAZ7 (top), JAZ8 (middle), and SLS (bottom) after treatment with 100 µM JA or mock treatment, quantified by real-time RT-PCRanalysis. Root tips of 5-d-old seedlings grown in the water were used. Expression levels were normalized by the levels of eEF-1a. The expression levelsrelative to those in the wild type before treatment with JA are shown. Statistical differences were determined by one-way ANOVA followed by Tukeyposthoc analysis, and different letters indicate significant differences (P value < 0.05). Mean 6 SD; n = 4. SLS, secologanin synthase.

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JA-responsive genes were significantly increased in rss3 afterJA treatment. bHLH094 enhanced the transcriptional activationof promoters carrying specific E-box sequences, but this wassuppressed by the combined expression of RSS3 and JAZ9.Based on these data, we propose that the bHLH factors andRSS3 combine with JAZs to negatively regulate JA-responsivegenes (Figure 9). This is reminiscent of the function of Arabi-dopsis MYC2, a transcriptional activator of JA-induced genes(Dombrecht et al., 2007). The activity of At-MYC2 is blocked bythe binding of JAZs. Upon JA-Ile perception by COI1, the JAZ isdegraded, which leads to the recovery of the activation functionof At-MYC2 and thereby results in JA-responsive gene induction(Chini et al., 2007; Thines et al., 2007). Similarly, the bindingof JAZ9 to RSS3-bHLH094 might repress bHLH094-mediatedtranscriptional activation. In this scenario, defects in the bindingof RSS3 to bHLH094 in rss3 should release the repression ofbHLH094-mediated gene expression as illustrated in Figure 9.

However, in the rss3 mutant, the JA-induced genes were notfully activated and were still responsive to exogenous JA (Figure9, bottom). Moreover, gene induction level by bHLH094 alone inthe reporter assay was moderate. Therefore, it is suggested that,in conjunction with bHLH089 and bHLH094, other positive regu-latory factors are involved in the activation of the JA-responsivegenes (Figure 9, depicted as factor X). Probably, such factors arethemselves activated by JA but regulated independently of RSS3and its binding JAZs. The action of the factor X may be dependenton the downstream gene promoter. A possible candidate for suchregulators is a MYB transcription factor. In the case of PAL, forexample, the promoter region contains potential cis-regulatorysequences for bHLH094 (CAGCTG) and R2R3 MYB factors(CACCAACC) (Takeda et al., 1999; Sugimoto et al., 2000), re-spectively. A JA-induced MYB exists in rice (Os11g0684000) (Leeet al., 2001). Regulation of this MYB seems to be independent ofRSS3 because it was not significantly activated in rss3 in ourmicroarray data (GSE41442). It is also possible that the activity ofMYB factor(s) in rice is regulated by interaction with several JAZproteins, as recently reported in Arabidopsis (Qi et al., 2011).Another candidate for the factor X can be a different type of bHLHthat is directly repressed by a JAZ factor independently of RSS3.

Importantly, in rss3, JA induces further activation of genes(Figure 8D). Therefore, RSS3 may have an additional role in re-pression of the bHLH094/089-mediated gene expression, throughunknown mechanism that is affected by the interaction betweenRSS3 and the bHLH factors (Figure 9, bottom right). The elevatedlevels of gene expression in rss3 suggest that in the wild type,RSS3 restricts JA responses in the root tip to a certain extent. Ingeneral, activation of JA signaling in response to unfavorableconditions causes root growth inhibition (Chacón-López et al.,2011). Under weak stress conditions, however, there should bea control to sustain root growth by preventing excessive JA re-sponse. RSS3 may function in such regulation in the root tip. Bycontrast, JA-induced expression might be fully activated in tissuesother than the root tip, where RSS3 is absent.

Accumulating evidence suggests that JAZ factors, togetherwith the F-box protein COI1, play central roles in JA signaling(Pauwels and Goossens, 2011; Kazan and Manners, 2012; Wagerand Browse, 2012). JAs are converted to JA-Ile, which in turnbinds to the COI1-JAZ complex in conjunction with ionositol

pentakiphosphate, which functions as a cofactor (Chini et al.,2007; Sheard et al., 2010). In JAZ factors, the C-terminal Jasdomain has a crucial role in JA-Ile–dependent binding to COI1and the subsequent degradation of JAZ by the 26S proteasome(Chini et al., 2007; Melotto et al., 2008; Sheard et al., 2010). Thestrength of association with COI1 differs among JAZs, whichcorrelates with sequence variation in the Jas domain (Shyu et al.,2012). The differential association of JAZs with COI1 may enableplants to respond dynamically to a wide range of JA concen-trations. Rice JAZ9 and JAZ11, which interact with RSS3 (Figure5) and with COI1 in the presence of coronatine (Lee et al., 2013),also contain the JAZ degron, the core sequence of the Jas do-main, but an incomplete PY consensus at the C terminus (seeSupplemental Figure 7B online). The Jas domain of JAZ9 andJAZ11 likely binds to COI1 and JA-Ile because the amino acidsresponsible for complex formation are well conserved in theseJAZ proteins (see Supplemental Figure 8 online). The PY se-quence has only a limited effect on the binding to COI1 in vitro

Figure 9. Possible Functions of RSS3 and Its Binding Proteins in JA-Inducible Gene Expression.

RSS3 is proposed to have a role in mediating JAZ-dependent in-activation of genes. Some JAZ factors have been reported to mediateactive repression through NINJA and TPL (Pauwels et al., 2010). It isunclear whether rice JAZ9 and JAZ11 are involved in active repression orpassive repression. Top left: In the absence of JA (-JA), bHLH094 (orbHLH089)-mediated transcription is repressed by a combination ofRSS3 and JAZ9 (or JAZ11) proteins that form a trimeric complex with thebHLH factor. Bottom left: In rss3, this repression was abolished becauseRSS3 with the 21–amino acid deletion (RSS3D21) cannot bind to thebHLH factor. bHLH094 is a weak activator by itself, but the bHLH-mediated transcription is readily activated depending on other positiveregulators. Top right: In the presence of JA (+JA), JAZ is possibly de-graded by 26S proteasome by the proposed COI1-dependent pathway(Chini et al., 2007; Thines et al., 2007), leading to derepression of the bHLH-mediated transcription. Since RSS3 alone cannot activate bHLH094, an-other positive regulation (depicted as factor X) is likely involved in the geneactivation by JA. The action of factor X might be dependent on respectivedownstream gene promoters. In the wild type (WT), the JA-responsivegenes are active but incompletely (“restricted”). Bottom right: In rss3, JAinduces full activation of genes. This might be achieved by release fromrepression through the JAZ, the positive regulation with the factor X, andadditional unknown mechanism that is interfered by interaction of RSS3with the bHLH factors.[See online article for color version of this figure.]

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(Chini et al., 2007; Chung et al., 2010; Sheard et al., 2010). Insome cases, however, the lack of a sequence including PYcauses changes in the stability of JAZ and affects JA sensitivity inplants (Chung and Howe, 2009; Chung et al., 2010). Therefore, wecannot rule out the possibility that JAZ9 and JAZ11 have uniquefeatures that do not occur in canonical JAZ proteins. The complexthat forms between RSS3 and JAZs remains to be characterizedin terms of its affinity for COI1 and its stability in the presence ofJAs.

Previous studies reported that the N terminus of R/B-likebHLHs functions as a JAZ interacting domain (JID) (Chini et al.,2007; Fernández-Calvo et al., 2011). Within the JID, the region IIis highly conserved both in RSS3 and in R/B-like bHLHs (Figure2C). Moreover, a single amino acid substitution (D94N) of ATR2(Arabidopsis MYC3) in the region II [M/I/V-L(G)W(G)DG-F/Y] re-sulted in a dominant mutation causing activation of Trp-relatedgenes and stress-related genes (Smolen et al., 2002). It wasspeculated that this is due to a defect of its interaction with JAZproteins, possibly leading to release of repression mediated byJAZs (Fernández-Calvo et al., 2011). Therefore, the region II islikely the central part of JID. It was also suggested that the firstGly of the LGWGDG sequence in the region II is important forbinding to JAZ (Fernández-Calvo et al., 2011). This residue isconserved between JAZ binding MYC2, MYC3, and MYC4 ofArabidopsis but not its close homolog bHLH028 (At5g46830)that seems not to bind to JAZ factors (Fernández-Calvo et al.,2011). Interestingly, RSS3 has Met instead of Gly at this posi-tion, but nevertheless is able to bind to JAZ9 and JAZ11. Thus,variations within its amino acid sequence in the region II as wellas their surrounding sequence may be responsible for thespecificity of binding to respective JAZs.

The rice genome encodes two proteins homologous to RSS3,RSS3-LIKE1 and RSS3-LIKE2 (see Supplemental Figure 11Aonline). When RSS3-LIKE1 was constitutively expressed undera rice actin promoter in rss3, salt-dependent root growth inhibitionand cell swelling were suppressed (see Supplemental Figures11C and 11D online). Thus, RSS3 and its homologs possiblyregulate overlapping target genes through members of the class-C bHLH factors, including bHLH089 and bHLH094. RSS3-LIKE1and RSS3-LIKE2 are expressed not only in roots but also in otherorgans (see Supplemental Figure 11B online), as are bHLH089and bHLH094. Therefore, similar regulatory mechanisms mayalso operate in tissues other than roots. Conceivably, the same istrue for other plant species because genes homologous to RSS3and bHLH089, such as Arabidopsis At5G53900 and At1G59640(BPEp; Szécsi et al., 2006), respectively, are found in the genomesof other plants. Interestingly, it has been reported that BPEp alsoacts downstream of the JA signal and functions as a negativeregulator of cell elongation in petals (Brioudes et al., 2009).

It remains to be clarified which factors downstream of theRSS3-JAZ-bHLH complex mediate the control of root growth.To classify the genes whose expression was altered in rss3, weapplied our transcriptomic data for gene set enrichment analysisusing MapMan (Thimm et al., 2004). Among the downregulatedgenes in rss3, highly enriched gene categories are related toabscisic acid–induced and late embryogenesis abundant genes(see Supplemental Figure 9C online). By contrast, genes upre-gulated in rss3 were classified into a wide variety of categories

(see Supplemental Figure 9C online). Notably, many of themwere related to cell wall metabolism (categories such as “cell wallmodification,” “b-1,3 glucosidase,” “lignin biosynthesis,” “phenyl-propanoids,” and “simple phenols”) (see Supplemental Figure 9Conline). It is plausible that RSS3 regulates the expression of genesinvolved in cell wall metabolism, which are required for proper cellelongation and maturation. In fact, genes encoding enzymes in-volved in lignin biosynthesis and/or the metabolism of other cellwall components, such as PAL, COMT, Cellulase, and Peroxi-dase, are upregulated in rss3 (microarray data: GSE41442) (seeSupplemental Data Set 1D online), which may result in changes incell wall properties. This may in turn affect the elongation of rss3root cells under nonstress conditions and cause further defectsunder salinity conditions. In tobacco (Nicotiana tabacum) cells, thephysical and chemical structures of the primary cell wall are alteredwhen the plants are cultured under high-salinity conditions; spe-cifically, the wall exhibits weakened tensile strength, concomitantwith a decrease in cellulose content, compositional changes of thecell wall–associated proteins, and reorganization of pectin (Irakiet al., 1989). It is reasonable to assume that RSS3 is involved in theregulation of genes involved in such processes. This may explainthe impaired flexibility in rss3 roots. Experiments to examine howrss3 affects cell wall properties are underway.We demonstrated that RSS3 globally modulates the expres-

sion of salinity stress–responsive genes. The link between thismodulation and the RSS3-mediated regulation of JA-responsivegenes remains unclear, although JAs are known to be involvedin various stress responses (Wasternack, 2007; Takeuchi et al.,2011). Notably, Arabidopsis MYC2 was originally identified asa transcriptional activator of stress-responsive genes (Abe et al.,1997). The global effects of rss3 on the stress-responsive genesmay be accounted for by assuming that the target cis-elementsof Arabidopsis MYC2- or rice bHLH89/94-related Transcriptionfactors prevail in primary stress-responsive genes, including theupstream transcription factors, and that they variously modulatethe expression levels of the stress-responsive genes. SuchbHLH-mediated controls would contribute to the adaptation ofroot growth in response to stress conditions.In conclusion, we have shown that RSS3 forms a ternary

complex with bHLH and JAZ factors. Moreover, our results in-dicate that RSS3, possibly through this complex, has an importantrole in stress-responsive gene regulation and the control of cellelongation, providing a novel molecular mechanism by which JA-responsive genes and plant growth are regulated. The RSS3-mediated regulation of cell elongation would allow the elaboratecontrol of root growth in response to environmental stresses andalso be linked to the regulation of cell division in the root tip. Afurther understanding of the mechanisms that sustain root growthunder stressful conditions will enhance our understanding of howplants adapt to environmental changes.

METHODS

Plant Materials and Growth Conditions

The rss3 mutant was originally identified in a screen for salt-dependentphenotypes (Ogawa et al., 2011) in the rice (Oryza sativa) mutant pop-ulation prepared by Miyao et al. (2003) (line ND2084). Wild-type and rss3

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seeds (O. sativa cv Nipponbare) were surface sterilized and germinated onsolid MS-based medium (Murashige and Skoog basal medium, 1% Suc,0.25% gellan gum, and 0.05% MES-KOH, pH 5.8) in 13-cm-high cappedbottles. Seedlings were grown at 25°C under a photoperiod of 14 h light(4000 lux, white light) and 10 h dark for 3 to 7 d. For the salinity stress test,100 to 150 mM NaCl was applied to the medium. JA [(6)-JA; Wako] wasdissolved in DMSO (Wako) to produce a 100 mM stock solution andstored at 220°C. For JA treatment of rice seedlings, JA or mock solutionwas applied to the solid MS-based medium, except that the short-termtreatment with JA was performed in water.

Microscopy Observation of Root Cells

Root samples were treated with chloral hydrate solution (8 g chloralhydrate, 1 mL glycerol, and 2 mL water) or stained by the modifiedpseudo-Schiff propidium iodide technique (Truernit et al., 2008; Ogawaet al., 2011). The tissues were observed under a fluorescence microscope(Axio Imager.MI; Carl Zeiss) or under a confocal laser scanning micro-scope (FV-1000D; Olympus).

Positional Cloning of the RSS3 Gene

Genetic mapping of rss3was performed with F2 populations derived fromcrosses between rss3 and an indica cultivar, Kasalath. DNA extractedfrom 1501 F2 seedlings exhibiting the rss3 phenotype was used to typesubspecies-specific polymorphisms for simple sequence repeat andcleaved amplified polymorphic sequence marker design (InternationalRice Genome Sequence Project, 2005; see Supplemental Table 1 online).Transcripts of four genes located in the mapped regions, i.e.,Os11g0446000, Os11g0446500, Os11g0446700, and LOC_Os11g25970(the first three genes were annotated in the Rice Annotation Project Da-tabase and the last in Michigan State University/The Institute for GenomicResearch), were analyzed by RT-PCR. A truncation of Os11g0446000mRNA was detected in rss3. The causative RSS3 gene was confirmed bycomplementation of rss3 using a genomic fragment (7.4 kb) of the wild-typeallele of Os11g0446000. We cloned the RSS3 cDNA fragments by 59- and39-rapid amplification of cDNA ends using GeneRacer (Invitrogen) andamplified the cDNA encoding the full-length RSS3 amino acid sequence.

Sequence Alignment and Phylogenic Analysis

Alignment of the amino acid sequences was performed using MAFFTsoftware (http://mafft.cbrc.jp/alignment/software/) with the G-INS-I strategyand/or the MUSCLE algorithm from MEGA5.0 (http://www.megasoftware.net/). To generate the graphical output, the sequences listed inSupplementalData Set 2, TreeView (Page, 1996) and Genedoc (http://www.nrbsc.org/gfx/genedoc/) were used.

Plasmid Construction

A 7.4-kb genomic fragment of RSS3 (4.0 kb of coding region, 2.5 kb of theupstream promoter region, and 0.9 kb of the downstream region) wasamplified by PCR with the specific primers 59-TCGAGGCTGAC-GAATGCAGCCG-39 and 59-AGGCTTAAAGATTCGTCTCGCAG-39 andinserted into pCR-Blunt II-TOPO (Invitrogen). The fragment containingProRSS3:RSS3 was excised with XbaI and KpnI, inserted between XbaIand KpnI sites of the binary vector pCAMBIA1301 (CAMBIA), and thenused for transformation of rss3 for the complementation assay. Fur-thermore, the plasmid was modified to construct ProRSS3:RSS3-EGFP.Briefly, the stop codon (TAG) of RSS3was substituted into a codon (TCG)followed by an EcoRI-BamHI linker sequence. The EGFP gene fragment,derived from ProAct:EGFP (Ogawa et al., 2011), was then inserted be-tween the EcoRI and BamHI sites. The resulting ProRSS3:RSS3-EGFP

fusion gene, containing an amino acid junction sequence (Ser-Glu-Phe-Ala-Ala-Ala-Ser-Thr) between RSS3 and EGFP, was inserted betweenXbaI and KpnI sites of the binary vector pPZP2H-lac (Fuse et al., 2001).The binary plasmid constructs were used for Agrobacterium tumefaciens–mediated transformation. Introduction of the ProRSS3:RSS3-EGFP fu-sion gene into the rss3 mutant complemented the severely impaired rootgrowth phenotype under salinity conditions.

For transient assays using rice protoplasts, cDNAs encoding RSS3,bHLH094, and JAZ9 were amplified by PCR with specific primer sets (seeSupplemental Table 1 online) and subcloned into the pENTR/D-TOPOvector. These genes were then transferred using the LR recombinationreaction (Gateway technology; Invitrogen) into a vector containing 35S:ShD:GW:stop (courtesy of Hirokazu Kato, Nagoya University), whichconsists of the CaMV 35S promoter, the first intron of maize (Zea mays)Sh1 with an internal deletion, the Gateway cassette, stop codons in threeframes, and a nopaline synthase terminator. For constructing the reporterplasmid containing E-box:TATA:fLUC (E-box repeats fused to the CaMV35Sminimal promoter and firefly LUC ), the GAL4 binding sequence (UAS)of the UAS:TATA:fLUC plasmid (courtesy of Yutaka Sato, Nagoya University)was replacedwith an artificial enhancer sequence that contains the triplicate ofthe E-box regulatory sequence (underlined), 59-GGATCCTGGCAAGCANNTG-AAGCANNTGAAGCANNTGAAGGGATCC-39.

For construction of YFPN-RSS3 and Os-bHLH094-YFPC driven by theCaMV 35S promoter, cDNAs for RSS3 and bHLH094 subcloned in thepENTR/D-TOPO vector were transferred to Gateway expression vectors(nYFP/pUGW0 and cYFP/pUGW2) by LR recombination. The pDH51-mRFP1 plasmid (Zhong et al., 2008) was used as a control for the BiFCassay. For fluorescence observation and the apFRET assay, cDNAs en-coding RSS3, class-C bHLHs, and JAZs were subcloned into the pENTR/D-TOPOvector and transferred toGateway expression vectors (YFP/pUGW0,pDH51-GW-CFP, and ECFP/pUGW0) (Nakagawa et al., 2007; Zhong et al.,2008). Primer sets used for cDNA amplification are listed in SupplementalTable 1 online.

Y2H and Three-Hybrid Assay

Transformation of yeast (AH109) and the interaction assays were per-formed using Matchmaker Library Construction and Screening Kits(Clontech) according to the manufacturer’s instructions. Summary ofscreening of RSS3 binding proteins are described in Supplemental Table2 online. Fragments of cDNA encoding RSS3, bHLH094, and JAZs wereamplified by PCR with the primer sets listed in Supplemental Table 1online. These cDNAs were derived from mRNA expressed in the root tip,except for JAZ12, which was from whole seedlings. Among the 15 JAZgenes reported by Ye et al. (2009), cDNAs for 12 JAZ genes other thanJAZ5, JAZ13, and JAZ14were amplified. Amplicons were either cloned intothe NdeI and EcoRI site of pGBKT7 or introduced by homologous re-combination into pGADT7-Rec. For the yeast three-hybrid assay, RSS3 andbHLH094 were amplified by PCR and cloned into theNotI site or EcoRI andBamHI sites of pBRIDGE (Clontech).

For screening of RSS3 interactors, rice cDNA libraries derived fromroot tissues of 7-d-old seedlings were amplified and cloned into the preyvector pGADT7-Rec in yeast. Using the N-terminal 318 amino acids ofRSS3 as bait, a total of 7.2 3 106 clones were screened based on theactivation of the HIS3 reporter gene. The 336 candidate clones werefurther selected by activation of the ADE2 and MEL1 reporter genes,resulting in 333 clones. Within the 40 high MEL1 expressing clones, ninecandidate clones were selected by their reading frame and repeatedreporter assay after retransformation of the respective plasmids (listed inSupplemental Table 2 online). For four candidate clones that carriedtranscription factors genes, the dependency of the bait plasmid wasexamined. As a result, only one clone was selected, which had a genefragment for a DNA binding protein (Os07g0193800; bHLH094). Byscreening another cDNA library, derived from the root tips of rice

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seedlings grown under salinity conditions (9.9 3 105 clones), using theN-terminal 203 amino acids of RSS3 as bait, no positive clones wereobtained after further selection.

Expression Analysis

Total RNA was extracted and purified using the RNeasy Mini Kit or RNeasyMicro Kit (Qiagen). Reverse transcription was performed using the Quanti-Tect Rev transcription kit (Qiagen). First-strand cDNAwas amplified by PCRPrimeStar HS DNA polymerase (Takara) using the GeneAmp PCR System9700 (Applied Biosystems) or by real-time PCR with Power SYBR GreenPCR master mix (Applied Biosystems) using Mx3000P (Agilent). The primersets are listed in Supplemental Table 1 online. The quantified expressionlevels of the tested genes were normalized against those ofUBQ, 25S rRNA,or eEF1-a (Jain et al., 2006).

Transient Expression Assay Using Rice Protoplasts

Electroporation of rice cultured cell–derived protoplasts was performedas described previously (Hattori et al., 1995). Ten micrograms of eachplasmid was used for each electroporation, with 5 mg of a referenceplasmid encoding renilla LUC driven under a CaMV 35S promoter used fornormalization. The activity of firefly and renilla luciferase was monitoredby a luminometer (Lumat LB 9507; Berthold Technologies) using thePicaGene Dual Sea Pansy Luminescence Kit (Toyo-Ink). Each experi-ment was repeated at least three times using a single preparation ofprotoplasts.

BiFC, apFRET, and BiFC-apFRET Analysis

To evaluate the protein–protein interaction by BiFC, apFRET, or BiFC-apFRET analysis, transient assays were performed by particlebombardment using the PDS-1000/He biolistic particle delivery system(Bio-Rad). Plasmids encoding the protein of interest were adsorbed ontogold particles (1.6 mm in diameter) according to the manufacturer’s in-structions (Bio-Rad). Gold particles were then bombarded into onionpieces at 7584 kPa under a vacuum of 94.82 kPa. The onion pieces werethen incubated in a Petri dish for 12 h at 30°C in the dark. Epidermal cellswere observed by fluorescence microscopy (Axio Imager.M1; Carl Zeiss).

The nuclei of onion epidermal cells expressing the proteins of interestwere subjected to apFRET and BiFC-apFRET analyses using a confocallaser scanning microscope (LSM700; Carl Zeiss). ECFP or CFP was usedas the FRET donor, and YFP or BiFC-derived YFP was used as the FRETacceptor. After bleaching the acceptor, FRET was monitored as an in-crease of donor fluorescence. Calculation of the FRET efficiency used theformula (donor intensitypostbleach 2 donor intensityprebleach) 3 100/donorintensitypostbleach.

Microarray and Statistical Analysis

For transcriptomic analysis, total RNA was extracted using the RNeasyMini Kit (Qiagen) from root tips (1 mm long) excised from wild-type andrss3 seedlings that had grown for 3 d on the solid MS medium in thepresence (+NaCl) or absence (2NaCl) of 100 mM NaCl. The excised roottips include the MZ and EZ, but not the MTZ, even in rss3 plants grownunder salinity stress. Three biological replicates, each of which consistedof at least 15 individual root tips, were prepared. The quality and quantityof RNAs were evaluated using a NanoDrop ND-1000 UV-VIS spectro-photometer (NanoDrop) and Agilent 2100 bioanalyzer (Agilent Technol-ogy). The RNAs were labeled with a LOW RNA input linear amplification/labeling kit (Agilent Technology). Four different aliquots (WT[2NaCl], WT[+NaCl], rss3[2NaCl], and rss3[+NaCl]) of Cy5-labeled cRNA (825 ng) anda control aliquot (WT[2NaCl]) of Cy3-labeled cRNA (825 ng) were pre-pared and used for hybridization using the Agilent-015241 rice gene

expression 4X44K microarray (Agilent Technology). Scanned imageswere analyzed with Feature Extraction Software v10.5.1.1 (Agilent). Mi-croarray data were normalized using the variance stabilization normali-zation method (Huber et al., 2002) packaged in R software (http://www.r-project.org/). After normalization, the relative log2 expression values ofthe genes in each sample were calculated by subtracting the Cy-3 valuefrom the Cy-5 value. Differentially expressed genes were categorizedusing two-way ANOVA (P < 0.01) by genotype (the wild type versus rss3)and stress treatment (2NaCl versus +NaCl) with TIGR Multi-ExperimentViewer (http://www.tm4.org/mev/), where the P value was adjusted withmultiple testing corrections (Benjamini and Hochberg, 1995). Alterna-tively, differentially expressed genes were selected by a cutoff of 1.5-foldchange and by rank product (P < 0.01, pfp < 0.05) (Breitling et al., 2004) ofR, to examine the effect of rss3 on the JA-responsive genes. Relative log2

expressions were Z-scaled and visualized with R and Multi-ExperimentViewer, respectively. Functional categorization of genes was performed,based on the classification available in the MapMan program (http://mapman.gabipd.org/) (Thimm et al., 2004). The raw and processed mi-croarray data are available at the Gene Expression Omnibus with re-pository number GSE41442.

For extraction of the JA-induced or JA-repressed genes, the micro-array data by Miyamoto et al. (2012) were also normalized using thevariance stabilization normalization method and filtered by a cutoff of 1.5-fold change and by rank product (P < 0.01, pfp < 0.05).

Accession Numbers

Sequence data from this article can be found in GenBank/EMBL databasesunder the accession numbers listed in Supplemental Table 3 online. Thenewly obtained cDNA sequence of Os11g0446000 encoding full-lengthRSS3 was deposited to GenBank under accession number AB753860.

Supplemental Data

Supplemental Figure 1. rss3 Displays Defects in Root Growth.

Supplemental Figure 2. rss3 Displays Aberrant Cellular Arrangementin the MZ.

Supplemental Figure 3. Responsiveness of rss3 to External Stimuli.

Supplemental Figure 4. Complementation Assay of the rss3 Mutant,Diagrammatic Representation of the rss3 Mutation, and ExpressionLevels of RSS3 in rss3.

Supplemental Figure 5. Expression Pattern of RSS3, bHLH089,bHLH094, JAZ9, and JAZ11 in Wild-Type Plants.

Supplemental Figure 6. Sequence Alignment of the RSS3 InteractionDomain of bHLH094, bHLH089, and Class-C bHLH Proteins in Rice.

Supplemental Figure 7. Comparison of the ZIM and Jas Domains ofRice and Arabidopsis JAZ Proteins.

Supplemental Figure 8. Comparison of the Amino Acid Residues ofJAZ Proteins Required for the Interaction with COI1 and JA-Ile.

Supplemental Figure 9. Altered Expression of Genes in rss3.

Supplemental Figure 10. Dose-Dependent Effects of JA on CellDivision and Cell Elongation in the Root Tip of Rice.

Supplemental Figure 11. Characterization of RSS3 Homologs ofRice.

Supplemental Table 1. Oligonucleotides Used in This Study.

Supplemental Table 2. Summary of the Yeast Two-Hybrid Screen.

Supplemental Table 3. Accession Numbers of Genes and Proteins.

Supplemental Data Set 1A. Expression Analysis of JA-InducedGenes in rss3.

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Supplemental Data Set 1B. Expression Analysis of JA-RepressedGenes in rss3.

Supplemental Data Set 1C. Functional Characterization of GenesWhose Expression Was Altered by JA and in rss3.

Supplemental Data Set 1D. Lists of the Categorized Genes WhoseExpression Was Altered in rss3.

Supplemental Data Set 2. Sequence Alignments Used for Phyloge-netic Analysis in This Study.

ACKNOWLEDGMENTS

We thank Tokunori Hobo, Yuka Kitomi, and Yoshiaki Inukai for experi-mental suggestions, Taisuke Nishimura, Chiharu Ueguchi, Moez Hanin,and Hironaka Tsukagoshi for helpful discussions, Masahiro Yano forgenerous support on genetic mapping, Ritsuko Motoyama and YoshiakiNagamura for help with the microarray analyses, Miyuki Hattori andShukushin Endo for technical assistance, and Tomoko Tsuchida-Mayama,Masaru Ohme-Takagi, and Hiroaki Ichikawa for helpful comments on thearticle. We thank Miyako Ueguchi-Tanaka for guidance with the particlebombardment experiments. We also thank Tsuyoshi Nakagawa and ShyojiMano for providing nYFP or cYFP inserted into pUGW0 or pUGW2 vectors,Yutaka Sato for 35S:rLUC and UAS:TATA:fLUC, H. Kato for 35S:ShD:GW:stop, and the Nottingham Arabidopsis Stock Centre for pDH51-mRFP1and pDH-GW-CFP. This work was supported in part by grants from theMinistry of Agriculture, Forestry, and Fisheries of Japan (integratedresearch project for plants, insects, and animals using genome technologyMP-2146 and IP-5002; Genomics for Agricultural Innovation AMR0002 andAMR0004) and from the Bio-oriented Technology Research AdvancementInstitute of Japan and by Japan Society for the Promotion of ScienceKAKENHI (24580493 and 21.5771). D.O. was supported by a fellowship ofthe Japan Society for the Promotion of Science.

AUTHOR CONTRIBUTIONS

H.H., T.H., and S.T. designed the research. Y.T., M.T., D.O., K.K., K.-I.K.,Y.H., T.A., K.S., N.M., E.K., K.A., and A.M. performed the research. Y.T.,T.H., and S.T. wrote the article.

Received March 28, 2013; revised April 20, 2013; accepted April 28,2013; published May 28, 2013.

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DOI 10.1105/tpc.113.112052; originally published online May 28, 2013;Plant Cell

Hirochika, Tsukaho Hattori and Shin TakedaAndo, Kazuhiko Sugimoto, Nobutaka Mitsuda, Etsuko Katoh, Kiyomi Abe, Akio Miyao, Hirohiko

Yosuke Toda, Maiko Tanaka, Daisuke Ogawa, Kyo Kurata, Ken-ichi Kurotani, Yoshiki Habu, TsuyuRegulates Jasmonate-Induced Gene Expression and Root Cell Elongation

RICE SALT SENSITIVE3 Forms a Ternary Complex with JAZ and Class-C bHLH Factors and

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