The bHLH Transcription Factors MYC2, MYC3, and MYC4 Are Required for Jasmonate-Mediated Inhibition of Flowering in Arabidopsis

In plants, the floral transition is flexibly controlled by various environmental conditions and endogenous developmental cues. In Arabidopsis, six major flowering pathways respond to changes in these factors (Fornara et al., 2010Fornara F. Montaigu A. Coupland G. SnapShot: control of flowering in Arabidopsis.Cell. 2010; 141: 550Abstract Full Text PDF PubMed Scopus (467) Google Scholar). The photoperiod, vernalization, and ambient pathways monitor exogenous signals from the environment such as day length, minimum winter temperature, and ambient temperature (Fornara et al., 2010Fornara F. Montaigu A. Coupland G. SnapShot: control of flowering in Arabidopsis.Cell. 2010; 141: 550Abstract Full Text PDF PubMed Scopus (467) Google Scholar). By contrast, the autonomous, gibberellin, and age pathways respond to endogenous cues linked to developmental status (Fornara et al., 2010Fornara F. Montaigu A. Coupland G. SnapShot: control of flowering in Arabidopsis.Cell. 2010; 141: 550Abstract Full Text PDF PubMed Scopus (467) Google Scholar). Accumulating evidence indicates that the six flowering pathways converge in a network to regulate floral integrator genes FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (Fornara et al., 2010Fornara F. Montaigu A. Coupland G. SnapShot: control of flowering in Arabidopsis.Cell. 2010; 141: 550Abstract Full Text PDF PubMed Scopus (467) Google Scholar). Jasmonates (JAs) are a class of important phytohormones that control multiple physiological aspects of plant development, growth, and defense. Although a previous study has shown that JA receptor COI1 and JA-ZIM domain (JAZ) proteins are involved in regulation of floral transition (Zhai et al., 2015Zhai Q.Z. Zhang X. Wu F.M. Feng H.L. Deng L. Xu L. Zhang M. Wang Q.M. Li C.Y. Transcriptional mechanism of jasmonate receptor COI1-mediated delay of flowering time in Arabidopsis.Plant Cell. 2015; 27: 2814-2828PubMed Google Scholar), whether JA-activated transcription factors MYC2, MYC3, and MYC4 (MYC2/3/4), which are the most extensively studied components in the JA signaling pathway, play a regulatory role floral transition remains unclear. In this study, we therefore examined the role of MYC2/3/4 in floral transition. We first analyzed the flowering phenotypes of myc2 mutants, myc2/3, myc2/4, and myc3/4 double mutants, and a myc2/3/4 triple mutant. Under long day (LD) or short day (SD) conditions, myc2/3 and myc2/4 double mutants exhibited earlier-flowering phenotypes compared with the wild-type. In contrast, the flowering times of the myc2, myc3, myc4 single mutant and myc3/4 double mutant were comparable with that of wild-type plants. Moreover, the myc2/3/4 triple mutant exhibited an earlier-flowering phenotype compared with myc2/3 and myc2/4 double mutants under both LD and SD conditions (Figure 1A; Supplemental Figures 1 and 2). These results suggest that the three MYC proteins redundantly regulate flowering under both LD and SD conditions. Considering that the photoperiod pathway is a central signaling in flowering time regulation, we then tested whether flowering control in the myc2/3/4 mutant involves regulation of expression levels of the photoperiodic flowering pathway genes GI, CO, FT, and TSF. As shown in Supplemental Figure 3A and 3B, GI and CO transcription was unaltered in myc2/3/4 mutant plants compared with the wild-type. By contrast, FT transcript levels in the myc2/3/4 mutant were significantly increased at most time points compared with those in the wild-type (Figure 1B). Similarly, mRNA levels of TSF, the closest paralog of FT, were significantly higher in myc2/3/4 mutant plants at two time points compared with their wild-type counterparts (Supplemental Figure 3C). Further qRT–PCR analysis and GUS staining revealed that MYC2/3/4 and FT displayed overlapping expression patterns (Supplemental Figure 4). Under LD or SD conditions, the myc2/4 plants exhibited earlier-flowering phenotypes than those of myc2/3 plants (Figure 1), suggesting that MYC4 is more important in mediating flowering compared with MYC3. Consistent with this assumption, the spatial expression pattern of FT was more similar to that of MYC4 than MYC3. Moreover, the MYC2 protein level was increased at middle day (ZT3 and ZT15), while FT expression level was low under LD conditions (Supplemental Figure 4H). These results suggest that MYC2/3/4 regulate floral induction by modulating the transcription of FT. Having ascertained that MYC2/3/4 regulate FT expression and that MYC2/3/4 have expression patterns similar to that of FT, we further investigated whether MYC2/3/4 interact with chromatin regions of the FT gene. It has been demonstrated that MYC2/3/4 bind to G-box or G-box-related motifs of their target genes (Fernández-Calvo et al., 2011Fernández-Calvo P. Chini A. Fernández-Barbero G. Chico J.M. Gimenez-Ibanez S. Geerinck J. Eeckhout D. Schweizer F. Godoy M. Franco-Zorrilla J.M. et al.The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses.Plant Cell. 2011; 23: 701-715Crossref PubMed Scopus (737) Google Scholar). By examinig the genomic sequence of FT gene, we found that several putative G-box-related motifs are in the genomic region of FT gene, raising a possibility that MYC2/3/4 may interact with these sequences to regulate flowering (Figure 1C). To test this, we generated transgenic plants overexpressing MYC2 fused with the DNA sequence encoding a c-MYC tag. Under LDs, overexpression of MYC2 resulted in slightly delayed flowering and repressed FT expression (Supplemental Figure 5). Subsequently, we examined the interaction between MYC2 and the FT gene through chromatin immunoprecipitation (ChIP). As shown in Figure 1D and 1E, MYC2 was found to bind the two genomic regions (d and g) of the FT gene. In addition, our yeast-one-hybrid results showed that MYC2 is indeed able to bind the g region of the FT gene but not that with mutation in the G-box-related motifs (Supplemental Figure 6). These results demonstrate that MYC2 specifically associates with chromatin regions of the FT gene. Having determined that MYC2 binds the chromatin regions of the FT gene, we next examined whether MYC2/3/4 genetically interact with FT to regulate flowering. To test this, the myc2/3/4 mutant was crossed with the ft-10 to obtain the ft-10 myc2/3/4 quadruple mutant. As shown in Figure 1F and Supplemental Figure 7, the ft-10 mutant exhibited late flowering, while the myc2/3/4 mutant exhibited early flowering compared with the wild-type (Yoo et al., 2005Yoo S.K. Chung K.S. Kim J. Lee J.H. Hong S.M. Yoo S.J. Yoo S.Y. Lee J.S. Ahn J.H. CONSTANS activates SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 through FLOWERING LOCUS T to promote flowering in Arabidopsis.Plant Physiol. 2005; 139: 770-778Crossref PubMed Scopus (390) Google Scholar). Under the same conditions, the early flowering phenotype of myc2/3/4 was completely suppressed by ft-10 (Figure 1F; Supplemental Figure 7), suggesting that the early flowering of myc2/3/4 required functional FT. Previous studies and our results indicate that some of JA signaling pathway components (COI1, JAZ, MED25, and MYC2/3/4) are involved in the regulation of flowering time (Zhai et al., 2015Zhai Q.Z. Zhang X. Wu F.M. Feng H.L. Deng L. Xu L. Zhang M. Wang Q.M. Li C.Y. Transcriptional mechanism of jasmonate receptor COI1-mediated delay of flowering time in Arabidopsis.Plant Cell. 2015; 27: 2814-2828PubMed Google Scholar, Iñigo et al., 2012Iñigo S. Alvarez M.J. Strasser B. Califano A. Cerdán P.D. PFT1, the MED25 subunit of the plant Mediator complex, promotes flowering through CONSTANS dependent and independent mechanisms in Arabidopsis.Plant J. 2012; 69: 601-612Crossref PubMed Scopus (85) Google Scholar; Figure 1A; Supplemental Figure 1). We therefore hypothesized that exogenous application of JA affects flowering. To test this possibility, we recorded the flowering times of wild-type and mutant plants treated with 50 μM methyl jasmonate (MeJA). In the presence of MeJA, wild-type plants flowered after developing 11.7 rosette leaves, which was significantly later than the mock-treated plants that flowered with 9.8 rosette leaves (Figure 1G; Supplemental Figure 8). Interestingly, MeJA-treated myc2/3/4 triple mutant plants flowered after developing 7.8 rosette leaves, which was slightly later than mock-treated myc2/3/4 plants flowering with 7.2 rosette leaves, but significantly earlier than mock-treated wild-type plants (Figure 1G; Supplemental Figure 8). Thus, exogenous JA substantially represses flowering induction. We then investigated whether exogenous JA affects flowering by regulating FT expression. To this end, FT-promoter-controlled GUS transgenic plants were treated with MeJA or a mock solution for 10 days. As shown in Figure 1H, the MeJA-treated pFT:GUS plants displayed a GUS signal compared with the mock-treated plants. As a negative control, the pbHLH60:GUS transgenic plants were not responsive to MeJA. Consistent with their flowering phenotypes (Figure 1G; Supplemental Figure 7), the transcript level of FT in MeJA-treated myc2/3/4 plants was reduced compared with that in mock-treated plants but was still higher than that of mock-treated wild-type plants (Figure 1I). These observations indicate that exogenous JA represses flowering time and FT expression at least partially through the MYC2/3/4. As the transcriptional factors, MYC2/3/4 have been shown to bind the chromatin regions of their target genes and to repress or activate their transcription. To further dissect the regulatory function of MYC2/3/4 on FT transcription, we performed transient expression assays in wild-type plant mesophyll protoplasts. As MYC2 associates with the genomic regions of the FT gene (Figure 1C–1E), the FT genomic sequence was fused to the Luciferase (LUC) gene as a reporter, and the FT genomic sequence with G-box-related motifs mutated in the d and g regions was used as a control. In addition, the MYC2 gene was driven by the Cauliflower mosaic virus (CaMV) 35S promoter as the effector (Figure 1J). As shown in Figure 1K, co-expression of MYC2 effector with the reporter repressed LUC expression. However, co-expression of MYC2 effector with the control reporter has no influence on LUC expression (Figure 1K). These results suggest that MYC2 negatively regulate FT transcription. Previous studies have shown that treatment with exogenous gibberellin (GA) or abscisic acid (ABA) affects plant flowering (Griffiths et al., 2006Griffiths J. Murase K. Rieu I. Zentella R. Zhang Z.L. Powers S.J. Gong F. Phillips A.L. Hedden P. Sun T.P. et al.Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis.Plant Cell. 2006; 18: 3399-3414Crossref PubMed Scopus (579) Google Scholar, Wang et al., 2013Wang Y.P. Li L. Ye T.T. Lu Y.M. Chen X. Wu Y. The inhibitory effect of ABA on floral transition is mediated by ABI5 in Arabidopsis.J. Exp. Bot. 2013; 64: 675-684Crossref PubMed Scopus (156) Google Scholar). In this study, our results reveal that exogenous JA also represses flowering in Arabidopsis (Figure 1G; Supplemental Figure 8) partially in a MYC2/3/4-dependent manner (Figure 1). However, MeJA-treated myc2/3/4 plants flowered even slightly later than mock-treated plants, suggesting that other JA-mediated pathways are also involved in the control of flowering time in myc2/3/4 (Figure 1G; Supplemental Figure 8). Consistent with this possibility, Zhai et al., 2015Zhai Q.Z. Zhang X. Wu F.M. Feng H.L. Deng L. Xu L. Zhang M. Wang Q.M. Li C.Y. Transcriptional mechanism of jasmonate receptor COI1-mediated delay of flowering time in Arabidopsis.Plant Cell. 2015; 27: 2814-2828PubMed Google Scholar found that the APETALA2 transcription factors TARGET OF EAT1 (TOE1)/TOE2 interact with JAZs and repress the transcription of FT. Moreover, previous studies showed that JAZ proteins interact with MYC2/3/4 and inhibit their transcriptional function (Cheng et al., 2011Cheng Z. Sun L. Qi T. Zhang B. Peng W. Liu Y. Xie D. The bHLH transcription factor MYC3 interacts with the Jasmonate ZIM-domain proteins to mediate jasmonate response in Arabidopsis.Mol. Plant. 2011; 4: 279-288Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, Fernández-Calvo et al., 2011Fernández-Calvo P. Chini A. Fernández-Barbero G. Chico J.M. Gimenez-Ibanez S. Geerinck J. Eeckhout D. Schweizer F. Godoy M. Franco-Zorrilla J.M. et al.The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses.Plant Cell. 2011; 23: 701-715Crossref PubMed Scopus (737) Google Scholar). All these previous findings and the results in this study together suggest that JA-dependent degradation of JAZs could lead to the release of MYCs and TOEs, and subsequently modulate FT transcription and flowering (Figure 1; Cheng et al., 2011Cheng Z. Sun L. Qi T. Zhang B. Peng W. Liu Y. Xie D. The bHLH transcription factor MYC3 interacts with the Jasmonate ZIM-domain proteins to mediate jasmonate response in Arabidopsis.Mol. Plant. 2011; 4: 279-288Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, Zhai et al., 2015Zhai Q.Z. Zhang X. Wu F.M. Feng H.L. Deng L. Xu L. Zhang M. Wang Q.M. Li C.Y. Transcriptional mechanism of jasmonate receptor COI1-mediated delay of flowering time in Arabidopsis.Plant Cell. 2015; 27: 2814-2828PubMed Google Scholar). Because treatment with exogenous JA delayed flowering time, we also tested whether endogenous JA is also involved in this process. However, the JA biosynthesis mutants lox2 and aos exhibited the delayed flowering (Zhai et al., 2015Zhai Q.Z. Zhang X. Wu F.M. Feng H.L. Deng L. Xu L. Zhang M. Wang Q.M. Li C.Y. Transcriptional mechanism of jasmonate receptor COI1-mediated delay of flowering time in Arabidopsis.Plant Cell. 2015; 27: 2814-2828PubMed Google Scholar; Supplemental Figure 9). Therefore, AOS- and LOX2-mediated JA biosynthesis may be not necessary for the inhibition of floral transition at least in our experimental conditions. JA acts as a crucial defense signal and mainly regulates plant resistance against pathogens and insects. Interestingly, defense responses are often accompanied by significant growth inhibition including flowering in JA signaling (Yang et al., 2012Yang D.L. Yao J. Mei C.S. Tong X.H. Zeng L.J. Li Q. Xiao L.T. Sun T.P. Li J. Deng X.W. et al.Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade.Proc. Natl. Acad. Sci. USA. 2012; 109: E1192-E1200Crossref PubMed Scopus (560) Google Scholar, Zhai et al., 2015Zhai Q.Z. Zhang X. Wu F.M. Feng H.L. Deng L. Xu L. Zhang M. Wang Q.M. Li C.Y. Transcriptional mechanism of jasmonate receptor COI1-mediated delay of flowering time in Arabidopsis.Plant Cell. 2015; 27: 2814-2828PubMed Google Scholar). It is thus possible that the delayed flowering after JA application is a treatment-related stress response. However, further investigations to elucidate the mechanisms underlying the involvement of endogenous JA in flowering are required. In this study, we provide compelling evidence showing that MYC2/3/4 redundantly modulate flowering under both LD and SD conditions. However, Major et al., 2017Major I.T. Yoshida Y. Campos M.L. Kapali G. Xin X.F. Sugimoto K. de Oliveira Ferreira D. He S.Y. Howe G.A. Regulation of growth-defense balance by the JASMONATE ZIM-DOMAIN (JAZ)-MYC transcriptional module.New Phytol. 2017; https://doi.org/10.1111/nph.14638Crossref PubMed Scopus (138) Google Scholar recently reported that the myc2/3/4 triple mutant exhibited no obvious effect on flowering time. One possibility leading to the contradictory observation may that different myc2 mutant lines were used to construct the myc2/3/4 triple mutant: the jin1-2/myc2 was used in our study, but jin1-7/myc2 was used by Major et al., 2017Major I.T. Yoshida Y. Campos M.L. Kapali G. Xin X.F. Sugimoto K. de Oliveira Ferreira D. He S.Y. Howe G.A. Regulation of growth-defense balance by the JASMONATE ZIM-DOMAIN (JAZ)-MYC transcriptional module.New Phytol. 2017; https://doi.org/10.1111/nph.14638Crossref PubMed Scopus (138) Google Scholar. In our observations, myc2/3 and myc2/4 double mutants consistently showed earlier-flowering phenotypes than myc3/4, indicating that MYC2 plays a more important role than MYC3/4 in regulating floral transition (Figure 1). It is also possible that the flowering phenotype of the myc2/3/4 triple mutant is subjected to growth conditions (including light, temperature, photoperiod, etc.) or other unknown factors. We showed that the myc2/3 and myc2/4 double mutants as well as the myc2/3/4 triple mutant all consistently exhibited early flowering phenotype under both LD and SD, indicating that MYC2/3/4 are required for JA-mediated inhibition of flowering at least in our growth conditions. This work was supported by the Natural Science Foundation of China (U1202264 to D.Y.; 31401040 to Y.H.), the Youth Innovation Promotion Association CAS (to Y.H.), the CAS “Light of West China” Program (to Y.H.), the Chinese Society of Cell Biology (Early Career Fellowship to Y.H.), and the Program for Innovative Research Team of Yunnan Province (2014HC017 to D.Y.).