DELLAs Modulate Jasmonate Signaling via Competitive Binding to JAZs

Gibberellins (GAs) modulate jasmonate (JA) signaling, which is essential for stress response and development in plants. However, the molecular details of such phytohormone interaction remain largely unknown. Here, we show that the JA ZIM-domain 1 (JAZ1) protein, a key repressor of JA signaling, interacts in vivo with DELLA proteins, repressors of the GA pathway. DELLAs prevent inhibitory JAZ1 interaction with a key transcriptional activator of JA responses, MYC2, and, thus, enhance the ability of MYC2 to regulate its target genes. Conversely, GA triggers degradation of DELLAs, which allows JAZ1 to bind MYC2 and suppress MYC2-dependent JA-signaling outputs. Therefore, our results reveal one means by which GAs suppress cellular competence to respond to JA. Because DELLAs serve as central regulators that mediate the crosstalk of various phytohormones, our model also suggests a candidate mechanism by which JA signaling may be fine-tuned by other signaling pathways through DELLAs. Gibberellins (GAs) modulate jasmonate (JA) signaling, which is essential for stress response and development in plants. However, the molecular details of such phytohormone interaction remain largely unknown. Here, we show that the JA ZIM-domain 1 (JAZ1) protein, a key repressor of JA signaling, interacts in vivo with DELLA proteins, repressors of the GA pathway. DELLAs prevent inhibitory JAZ1 interaction with a key transcriptional activator of JA responses, MYC2, and, thus, enhance the ability of MYC2 to regulate its target genes. Conversely, GA triggers degradation of DELLAs, which allows JAZ1 to bind MYC2 and suppress MYC2-dependent JA-signaling outputs. Therefore, our results reveal one means by which GAs suppress cellular competence to respond to JA. Because DELLAs serve as central regulators that mediate the crosstalk of various phytohormones, our model also suggests a candidate mechanism by which JA signaling may be fine-tuned by other signaling pathways through DELLAs. Gibberellins (GAs) inhibit DELLA-mediated upregulation of jasmonate (JA) target genes DELLAs compete the JA signaling repressor JAZ1 away from the JA effector MYC2 DELLA/JAZ binding derepresses MYC2's ability to regulate JA-responsive genes DELLAs promote cellular competence to respond to JA Seed plants have evolved a set of sophisticated mechanisms to respond to diverse exogenous and endogenous stimuli to promote their survival. Jasmonates (JAs) are lipid-derived small-molecule phytohormones that regulate multiple plant growth responses, including the defense against pathogens and insects, the adaptation to abiotic stresses such as wounding and drought, and the involvement in developmental processes such as root growth and stamen development (Browse and Howe, 2008Browse J. Howe G.A. New weapons and a rapid response against insect attack.Plant Physiol. 2008; 146: 832-838Crossref PubMed Scopus (169) Google Scholar, Farmer et al., 2003Farmer E.E. Almeras E. Krishnamurthy V. 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SCFCOI1 degrades JA ZIM-domain (JAZ) proteins, a family of key repressors of JA signaling, through the 26S proteasome in the presence of jasmonoyl-L-isoleucine (JA-Ile) or its mimic, coronatine (COR) (Chini et al., 2007Chini A. Fonseca S. Fernandez G. Adie B. Chico J.M. Lorenzo O. Garcia-Casado G. Lopez-Vidriero I. Lozano F.M. Ponce M.R. et al.The JAZ family of repressors is the missing link in jasmonate signalling.Nature. 2007; 448: 666-671Crossref PubMed Scopus (1412) Google Scholar, Katsir et al., 2008Katsir L. Schilmiller A.L. Staswick P.E. He S.Y. Howe G.A. COI1 is a critical component of a receptor for jasmonate and the bacterial virulence factor coronatine.Proc. Natl. Acad. Sci. USA. 2008; 105: 7100-7105Crossref PubMed Scopus (526) Google Scholar, Melotto et al., 2008Melotto M. Mecey C. Niu Y. Chung H.S. Katsir L. Yao J. Zeng W. Thines B. Staswick P. Browse J. et al.A critical role of two positively charged amino acids in the Jas motif of Arabidopsis JAZ proteins in mediating coronatine- and jasmonoyl isoleucine-dependent interactions with the COI1 F-box protein.Plant J. 2008; 55: 979-988Crossref PubMed Scopus (254) Google Scholar, Thines et al., 2007Thines B. Katsir L. Melotto M. Niu Y. Mandaokar A. Liu G. Nomura K. He S.Y. Howe G.A. Browse J. JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling.Nature. 2007; 448: 661-665Crossref PubMed Scopus (1494) Google Scholar). JAZ proteins serve as the major molecular link between the SCFCOI1 E3 ubiquitin ligase and MYC2, a key transcriptional activator that regulates JA-dependent transcriptional reprogramming (Boter et al., 2004Boter M. Ruiz-Rivero O. Abdeen A. Prat S. Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis.Genes Dev. 2004; 18: 1577-1591Crossref PubMed Scopus (423) Google Scholar, Chini et al., 2007Chini A. Fonseca S. Fernandez G. Adie B. Chico J.M. Lorenzo O. Garcia-Casado G. Lopez-Vidriero I. Lozano F.M. Ponce M.R. et al.The JAZ family of repressors is the missing link in jasmonate signalling.Nature. 2007; 448: 666-671Crossref PubMed Scopus (1412) Google Scholar). Upon degradation of JAZs, MYC2 is released to promote JA-induced gene expression. JA signaling is integrated in complex regulatory networks that include its crosstalk with other phytohormone signaling pathways (Grant and Jones, 2009Grant M.R. Jones J.D. Hormone (dis)harmony moulds plant health and disease.Science. 2009; 324: 750-752Crossref PubMed Scopus (309) Google Scholar, Lorenzo and Solano, 2005Lorenzo O. Solano R. Molecular players regulating the jasmonate signalling network.Curr. Opin. Plant Biol. 2005; 8: 532-540Crossref PubMed Scopus (338) Google Scholar). Recent studies have implicated an important function of gibberellins (GAs) in mediating JA signaling in both stress responses and plant development. DELLAs, known as plant growth repressors whose degradation is promoted by GA, confer plants elevated resistance to necrotrophs via potentiating JA signaling, whereas they attenuate salicylic acid (SA) signaling to make plants more vulnerable to biotrophs, suggesting a role of DELLAs in modulating the balance of JA and SA signaling in response to pathogen stress (Navarro et al., 2008Navarro L. Bari R. Achard P. Lison P. Nemri A. Harberd N.P. Jones J.D. DELLAs control plant immune responses by modulating the balance of jasmonic acid and salicylic acid signaling.Curr. Biol. 2008; 18: 650-655Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). In addition, GA has been found to promote JA biosynthesis through DELLAs to control the expression of MYB21, MYB24, and MYB57, which in turn promote stamen development (Cheng et al., 2009Cheng H. Song S. Xiao L. Soo H.M. Cheng Z. Xie D. Peng J. Gibberellin acts through jasmonate to control the expression of MYB21, MYB24, and MYB57 to promote stamen filament growth in Arabidopsis.PLoS Genet. 2009; 5: e1000440Crossref PubMed Scopus (241) Google Scholar). Although these observations have suggested the crosstalk between GA and JA signaling in pathogen interaction and plant development, the detailed molecular mechanisms by which DELLAs modulate JA signaling still remain elusive. Here, we report a “relief of repression” model in which DELLAs compete with MYC2 for binding to JAZ1 in Arabidopsis. Without GA, stabilized DELLA proteins bind to JAZ1 and release MYC2 to promote JA signaling. GA triggers degradation of DELLAs, which releases free JAZ1 to bind to MYC2 and, thus, attenuates JA signaling. Our results provide the mechanism of how DELLAs contribute to JA signaling through upregulating the expression of JA-responsive genes. Because DELLAs have been suggested to integrate plant responses to various hormonal and environmental signals (Achard et al., 2003Achard P. Vriezen W.H. Van Der Straeten D. Harberd N.P. Ethylene regulates Arabidopsis development via the modulation of DELLA protein growth repressor function.Plant Cell. 2003; 15: 2816-2825Crossref PubMed Scopus (309) Google Scholar, Achard et al., 2006Achard P. Cheng H. De Grauwe L. Decat J. Schoutteten H. Moritz T. Van Der Straeten D. Peng J. Harberd N.P. Integration of plant responses to environmentally activated phytohormonal signals.Science. 2006; 311: 91-94Crossref PubMed Scopus (966) Google Scholar, Achard et al., 2008Achard P. Renou J.P. Berthome R. Harberd N.P. Genschik P. 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Several studies have shown that GA and DELLA proteins affect the expression of JA-responsive genes (Cao et al., 2006Cao D. Cheng H. Wu W. Soo H.M. Peng J.R. Gibberellin mobilizes distinct DELLA-dependent transcriptomes to regulate seed germination and floral development in Arabidopsis.Plant Physiol. 2006; 142: 509-525Crossref PubMed Scopus (196) Google Scholar, Hou et al., 2008Hou X. Hu W.W. Shen L. Lee L.Y. Tao Z. Han J.H. Yu H. Global identification of DELLA target genes during Arabidopsis flower development.Plant Physiol. 2008; 147: 1126-1142Crossref PubMed Scopus (70) Google Scholar). In addition, DELLAs have been demonstrated to control JA-mediated plant immune responses (Navarro et al., 2008Navarro L. Bari R. Achard P. Lison P. Nemri A. Harberd N.P. Jones J.D. DELLAs control plant immune responses by modulating the balance of jasmonic acid and salicylic acid signaling.Curr. Biol. 2008; 18: 650-655Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). These observations suggest that GA and DELLA proteins modulate JA perception and/or signaling. To test how GA and DELLA proteins are involved in JA signaling, we measured the expression of several typical JA-responsive genes, including LOX2, TAT1, PDF1.2, and b-CHI (Lorenzo et al., 2004Lorenzo O. Chico J.M. Sanchez-Serrano J.J. Solano R. JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis.Plant Cell. 2004; 16: 1938-1950Crossref PubMed Scopus (878) Google Scholar), under JA and/or GA treatment. In the GA-deficient mutant ga1-3, these genes were upregulated in response to JA treatment, whereas such elevated expression was significantly attenuated under the combined treatment with JA and GA (Figure 1A ). GA treatment alone did not significantly affect the expression of these JA-responsive genes (Figure 1A). Expression analyses on several other primary JA-responsive genes, including LOX3, LOX4, JAZ1, and JAZ3 (Chung et al., 2008Chung H.S. Koo A.J. Gao X. Jayanty S. Thines B. Jones A.D. Howe G.A. Regulation and function of Arabidopsis JASMONATE ZIM-domain genes in response to wounding and herbivory.Plant Physiol. 2008; 146: 952-964Crossref PubMed Scopus (300) Google Scholar), revealed similar changes in their expression trends in ga1-3 under JA and/or GA treatment (see Figure S1A available online). These results suggest that GA suppresses cellular competence to respond to JA. This is further substantiated by the observation that increased concentrations of JA more significantly induced the expression of JA-responsive genes in ga1-3 than in wild-type seedlings (Figure S1A). In both wild-type and ga1-3 backgrounds, loss of function of DELLA proteins (RGA, GAI, RGL1, and RGL2) impaired the upregulation of JA-responsive genes by JA (Figure S1B). Moreover, upregulation of these genes in wild-type plants by JA was enhanced by the GA biosynthesis inhibitor, paclobutrazol (PAC), whereas their upregulation in ga1-3 was suppressed by GA (Figure S1B). Such changes in response to PAC or GA were compromised in either gai-t6 rga-t2 rgl1-1 rgl2-1 or ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1, respectively, except for PDF1.2 expression in penta mutants (Figure S1B). These results indicate that DELLA proteins degraded by GA are involved in upregulating these genes in response to JA. We then examined the expression of JA-responsive genes in response to RGA activity using a steroid-inducible RGA (RGA-GR) in ga1-3 rgl2-1 rga-t2 (Yu et al., 2004Yu H. Ito T. Zhao Y.X. Peng J.R. Kumar P.P. Meyerowitz E.M. Floral homeotic genes are targets of gibberellin signaling in flower development.Proc. Natl. Acad. Sci. USA. 2004; 101: 7827-7832Crossref PubMed Scopus (203) Google Scholar). Dexamethasone enhanced the upregulation of TAT1 and PDF1.2 only under JA treatment (Figure 1B), which confirms that RGA promotes the expression of JA-responsive genes in the JA-signaling pathway. To understand how DELLA proteins affect JA signaling in plant development, we examined the inhibition of root elongation by JA in various DELLA mutants. Root elongation in ga1-3 was significantly reduced under JA treatment, whereas the extent of such reduction was attenuated in ga1-3 combined with various DELLA mutants (Figure 1C). Moreover, GA largely suppressed the inhibitory effect of JA on root growth in ga1-3, whereas such suppressive effect was less obvious in ga1-3 lacking various DELLA proteins (Figure S1C). These results, together with the expression patterns of JA-responsive genes, further corroborate that DELLA proteins in the GA pathway positively regulate JA response during Arabidopsis development. As putative transcriptional regulators, DELLA proteins have been shown to interact with bHLH-type transcription factors to coordinate the effect of light and GA on plant development (de Lucas et al., 2008de Lucas M. Daviere J.M. Rodriguez-Falcon M. Pontin M. Iglesias-Pedraz J.M. Lorrain S. Fankhauser C. Blazquez M.A. Titarenko E. Prat S. A molecular framework for light and gibberellin control of cell elongation.Nature. 2008; 451: 480-484Crossref PubMed Scopus (785) Google Scholar, Feng et al., 2008Feng S. Martinez C. Gusmaroli G. Wang Y. Zhou J. Wang F. Chen L. Yu L. Iglesias-Pedraz J.M. Kircher S. et al.Coordinated regulation of Arabidopsis thaliana development by light and gibberellins.Nature. 2008; 451: 475-479Crossref PubMed Scopus (689) Google Scholar). To investigate how DELLAs are involved in the interaction between GA and JA-signaling pathways, we performed a yeast two-hybrid screening to identify RGA-interacting partners. Because the full-length RGA protein exhibited strong self-activation activity, two truncated versions of RGA protein were used for bait optimization (Figure S2A). The N-terminal deletion version of RGA protein (RGAΔN), which contains the LZ1 and LZ2 protein-interacting domains (Itoh et al., 2002Itoh H. Ueguchi-Tanaka M. Sato Y. Ashikari M. Matsuoka M. The gibberellin signaling pathway is regulated by the appearance and disappearance of SLENDER RICE1 in nuclei.Plant Cell. 2002; 14: 57-70Crossref PubMed Scopus (364) Google Scholar, Thomas and Sun, 2004Thomas S.G. Sun T.P. Update on gibberellin signaling. A tale of the tall and the short.Plant Physiol. 2004; 135: 668-676Crossref PubMed Scopus (124) Google Scholar), abolished RGA self-activation completely and was thus chosen as a bait. After screening an Arabidopsis cDNA library (CD4-30 from ABRC), we found that JAZ1, a key repressor of JA signaling (Thines et al., 2007Thines B. Katsir L. Melotto M. Niu Y. Mandaokar A. Liu G. Nomura K. He S.Y. Howe G.A. Browse J. JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling.Nature. 2007; 448: 661-665Crossref PubMed Scopus (1494) Google Scholar), interacted with RGAΔN (Figure 2A ). Consistent with a previous report (Chini et al., 2007Chini A. Fonseca S. Fernandez G. Adie B. Chico J.M. Lorenzo O. Garcia-Casado G. Lopez-Vidriero I. Lozano F.M. Ponce M.R. et al.The JAZ family of repressors is the missing link in jasmonate signalling.Nature. 2007; 448: 666-671Crossref PubMed Scopus (1412) Google Scholar), yeast two-hybrid assays showed that JAZ1 interacted with MYC2, a key transcriptional activator of JA-regulated gene expression, whereas no interaction was detected between MYC2 and RGAΔN (Figure 2A). Because there are 12 JAZ family members in Arabidopsis, and they function redundantly in JA signaling (Chini et al., 2007Chini A. Fonseca S. Fernandez G. Adie B. Chico J.M. Lorenzo O. Garcia-Casado G. Lopez-Vidriero I. Lozano F.M. Ponce M.R. et al.The JAZ family of repressors is the missing link in jasmonate signalling.Nature. 2007; 448: 666-671Crossref PubMed Scopus (1412) Google Scholar, Thines et al., 2007Thines B. Katsir L. Melotto M. Niu Y. Mandaokar A. Liu G. Nomura K. He S.Y. Howe G.A. Browse J. JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling.Nature. 2007; 448: 661-665Crossref PubMed Scopus (1494) Google Scholar), we further chose JAZ3 and JAZ9 to test whether they could also interact with RGAΔN. Our results demonstrated an interaction between these two proteins and RGAΔN, though the interaction appeared weaker for the AD-RGA and BD-JAZ3 constructs (Figure S2B). In addition the other DELLA proteins, GAI, RGL1, and RGL2, were also found to interact with JAZ1 (Figure S2B). These data suggest that there may be widespread interaction between DELLAs and JAZ proteins. We next performed GST pull-down assays to verify the protein interaction between RGA and JAZ1 using purified GST- and His-tagged proteins (Figure S2C). Again, we found that RGA specifically interacted with JAZ1, but not MYC2, whereas JAZ1 also interacted with MYC2 (Figure 2B). In addition, bimolecular fluorescence complementation (BiFC) analysis revealed the direct interaction of RGA and JAZ1 in the nuclei of living plant cells (Figure 2C). Coimmunoprecipitation analysis on the protein extracts from ga1-3 rga-t2 35S:RGA-9Myc 35S:JAZ1-6HA further confirmed the in vivo interaction between RGA and JAZ1 (Figure 2D). Taken together, these results indicate that DELLAs might regulate JA-signaling pathway through their interaction with JAZs. Because JAZ proteins serve as the major molecular link between the SCFCOI1 E3 ubiquitin ligase and MYC2 in the JA pathway, the interaction between DELLAs and JAZs may affect either the degradation of JAZs or the interaction between JAZs and MYC2. To test the former possibility, we examined the effect of GA or PAC on the degradation of JAZ1-GUS (β-glucuronidase) protein induced by JA in 35S:JAZ1-GUS roots (Thines et al., 2007Thines B. Katsir L. Melotto M. Niu Y. Mandaokar A. Liu G. Nomura K. He S.Y. Howe G.A. Browse J. JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling.Nature. 2007; 448: 661-665Crossref PubMed Scopus (1494) Google Scholar). Evidently, JA-mediated degradation of JAZ1-GUS was not affected by GA or PAC (Figure S3A). Similarly, JA had no effect on RGA degradation in ga1-3 rga-t2 pRGA:GFP-RGA plants (Figure S3B). Furthermore, GA or PAC still affected the expression of JA-responsive genes in the JA-insensitive mutant coi1-1, wherein the degradation of JAZs was abolished (Figure S3C). These observations suggest that DELLAs affect JA signaling downstream of the degradation of JAZs. We then investigated whether DELLAs affect the interaction between JAZs and MYC2. In vitro pull-down assays demonstrated that the interaction between His-MYC2 and GST-JAZ1 was impaired by an increased amount of GST-RGA (Figure 3A ). Similarly, His-MYC2 also attenuated the interaction between GST-RGA and His-JAZ1 (Figure 3B). These results demonstrate that RGA and MYC2 compete in vitro for binding to JAZs, implying that DELLAs could facilitate releasing MYC2 from JAZ/MYC2 complex through competing with the binding of MYC2 to JAZs. To study how DELLAs affect MYC2 function in transcriptional regulation of JA-responsive genes, we created jin1-8 pMYC2:MYC2-FLAG plants (Figure S4A) that rescued the JA-insensitive phenotype of the MYC2 mutant, jin1-8 (Lorenzo et al., 2004Lorenzo O. Chico J.M. Sanchez-Serrano J.J. Solano R. JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis.Plant Cell. 2004; 16: 1938-1950Crossref PubMed Scopus (878) Google Scholar), for examining MYC2 binding to the promoters of its target genes by chromatin immunoprecipitation (ChIP) assays. MYC2 encodes a bHLH transcriptional activator that specifically binds to G-box (CACGTG) or G-box-like motifs (Brown et al., 2003Brown R.L. Kazan K. McGrath K.C. Maclean D.J. Manners J.M. A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis.Plant Physiol. 2003; 132: 1020-1032Crossref PubMed Scopus (305) Google Scholar, Dombrecht et al., 2007Dombrecht B. Xue G.P. Sprague S.J. Kirkegaard J.A. Ross J.J. Reid J.B. Fitt G.P. Sewelam N. Schenk P.M. Manners J.M. Kazan K. MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis.Plant Cell. 2007; 19: 2225-2245Crossref PubMed Scopus (669) Google Scholar, Lorenzo et al., 2004Lorenzo O. Chico J.M. Sanchez-Serrano J.J. Solano R. JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis.Plant Cell. 2004; 16: 1938-1950Crossref PubMed Scopus (878) Google Scholar). We found a G-box or G-box-like motif within the 1 kb 5′ promoter of LOX2, TAT1, and PDF1.2 and designed primer pairs near these motifs for measurement of DNA enrichment (Figure S4B). Because the MYC2-FLAG protein started to accumulate from 1 hr after JA treatment and peaked at 12 hr in jin1-8 pMYC2:MYC2-FLAG (Figure S4B), we performed ChIP assays after JA treatment for 4 hr and detected the association of MYC2-FLAG with the promoter of LOX2 and TAT1, but not with PDF1.2 (Figure S4B). This is consistent with a previous study (Dombrecht et al., 2007Dombrecht B. Xue G.P. Sprague S.J. Kirkegaard J.A. Ross J.J. Reid J.B. Fitt G.P. Sewelam N. Schenk P.M. Manners J.M. Kazan K. MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis.Plant Cell. 2007; 19: 2225-2245Crossref PubMed Scopus (669) Google Scholar), indicating that LOX2 and TAT1 are direct targets of MYC2. We then chose 4 and 12 hr after JA treatment for analyzing the effect of DELLAs on MYC2 binding. At both time points, GA or PAC treatment did not affect the abundance of the MYC2-FLAG protein in nuclear extracts or immunoprecipitated fractions used in ChIP assays (Figure 3C). ChIP assays demonstrated that MYC2-FLAG binding to LOX2 and TAT1 promoters was enhanced when DELLA protein levels were increased by PAC, whereas DELLA degradation by GA attenuated such binding (Figure 3D), suggesting that under JA treatment, DELLA proteins do not directly affect MYC2 protein levels but enhance MYC2 binding to the G-box or G-box-like motif. We further examined the effect of induced RGA activity on MYC2 binding to LOX2 promoter using ga1-3 rga-t2 35S:RGA-GR jin1-8 pMYC2:MYC2-FLAG plants. Similarly, we found that induced RGA activity by dexamethasone treatment did not immediately affect MYC2 protein levels (Figure 3E) but enhanced MYC2 binding to LOX2 promoter (Figure 3F). In addition, further ChIP analyses revealed that direct association of MYC2-FLAG to the promoters of other four primary JA-responsive genes, LOX3, LOX4, JAZ1, and JAZ3 (Chung et al., 2008Chung H.S. Koo A.J. Gao X. Jayanty S. Thines B. Jones A.D. Howe G.A. Regulation and function of Arabidopsis JASMONATE ZIM-domain genes in response to wounding and herbivory.Plant Physiol. 2008; 146: 952-964Crossref PubMed Scopus (300) Google Scholar), was also modulated by DELLA proteins (Figure S4B). These observations suggest that DELLA proteins enhance MYC2 binding to the G-box or G-box-like motifs in its target genes. To further test the effect of DELLAs and JAZ1 on MYC2 transcriptional activity, we performed transient transactivation assays using the TAT1 promoter fused to the GUS gene as a reporter. Effector constructs for MYC2, JAZ1-6HA, or RGA were expressed under the control of the 2 × 35S promoter and transfected together with the reporter construct into jin1-8 mesophyll protoplasts. Without MYC2, TAT1 expression was very low, whereas expression of MYC2 protein dramatically activated the expression of TAT1 (Figure 3G). Coexpression of JAZ1 and MYC2 proteins repressed TAT1 expression. An additional supply of RGA attenuated the repression of JAZ1 on TAT1 expression, whereas RGA alone did not affect MYC2's ability to regulate TAT1 expression. Moreover, GA treatment, which triggered degradation of RGA, abolished the inhibitory effect of RGA on JAZ1 (Figure 3G). Thus, MYC2 transcriptional activity is inhibited by JAZ1 but promoted by RGA. These results, together with the effect of DELLAs on the expression of JA-responsive genes (Figures 1A, and 1B; Figure S1A) and enhancing MYC2 binding to the promoters of its target genes (Figures 3D and 3F; Figure S4B), strongly suggest that DELLA proteins modulate JA signaling through affecting MYC2's ability to transcriptionally regulate its target genes. We further examined modulation of JA signaling by DELLAs in JA-overproducing and -insensitive mutants. Root elongation of ga1 was significantly reduced in the background of hy1-101, a JA-overproducing mutant (Zhai et al., 2007Zhai Q. Li C.B. Zheng W. Wu X. Zhao J. Zhou G. Jiang H. Sun J. Lou Y. Li C. Phytochrome chromophore deficiency leads to overproduction of jasmonic acid and elevated expression of jasmonate-responsive genes in Arabidopsis.Plant Cell Physiol. 2007; 48: 1061-1071Crossr