CsJAZ6 represses catechin biosynthesis though physically interacting with CsEGL3 and CsTTG1
To investigate whether CsHSFs-regulated CsJAZ6 participates in catechin biosynthesis, we injected AsODN-CsJAZ6 solution into tea leaves. Compared with sODN treatment, the expression of CsJAZ6was significantly reduced (Figure 5a). Also, we found that inCsJAZ6 -silenced leaves, the expression of almost all flavonoid biosynthetic genes was up-regulated (Figure 5b). This finding was supported by HPLC analysis, which consistently showed the silenced leaves to accumulate more catechin than the control leaves (Figure 5c).
To decipher the molecular mechanisms underlying CsJAZ6-mediated regulation of catechin biosynthesis, we utilized CsJAZ6 protein as a bait to screen a yeast library constructed from tea leaves. Among the screened candidates, CsEGL3 and CsTTG1 stood out as known important components of a catechin biosynthesis regulator complex. After first verifying the CsJAZ6-CsEGL3 and CsJAZ6-CsTTG1 interactions by Y2H assay using full-length CsEGL3 and CsTTG1 (Figure 6a), we confirmed them in tobacco leaves by BiFC assay. Co-expression of CsJAZ6-YFPN and CsEGL3/CsTTG1-YFPCor CsJAZ6-YFPC and CsEGL3/CsTTG1-YFPN resulted in strong signals in the nucleus, while the negative control combinations, including CsJAZ6-YFPN/YFPC and CsJAZ6-YFPC/YFPN, did not produce any detectable fluorescence signal (Figure 6b). We then performed anin vivo co-immunoprecipitation assay by co-transforming CsJAZ6-Myc with CsEGL3-Flag and CsTTG1-Flag into tobacco leaves, with a GFP-Myc construct as control. This revealed CsJAZ6 to co-precipitate specifically with CsEGL3 and CsTTG1 (Figure 6c, d). Collectively, these results imply that CsJAZ6 physically interacts with both CsEGL3 and CsTTG1. It has been reported that CsEGL3 and CsTTG1 couple with CsAN1 and form the MBW complex to regulate the expression of genes involved in the catechin biosynthesis pathway (Sun et al., 2016; Wei et al., 2019). To answer whether the interactions of CsJAZ6 with CsEGL3 and CsTTG1 would interfere with MBW complex formation, we performed a yeast three-hybrid assay. CsEGL3 was pre-inserted in the pBridge vector as the bait, and CsJAZ6 was integrated into the CsEGL3/pBridge vector under control of the pMET25 promoter. This assay revealed that, with increasing methionine concentration, CsJAZ6 gradually reduced and thereby caused growth of the CsEGL3-CsAN1 yeast colonies (Figure 6e). These findings suggest that CsJAZ6 mediates catechin biosynthesis, at least in part, through affecting formation of the MBW complex.
To further elucidate the regulatory impact of the CsJAZ6-CsEGL3/CsTTG1 interaction on catechin biosynthesis, the CsANS promoter was fused to a luciferase gene to generate the CsANSpro-LUC reporter. In the subsequent Dual-Luc assay, CsAN1, CsEGL3, CsTTG1, and CsJAZ6 were used as effectors (Figure 6f). Compared to the control, the promoter activity was significantly elevated by 2.7-fold when co-expressed with MBW complex (CsAN1, CsEGL3 and CsTTG1). However, the addition of CsJAZ6 reduced the activation effect of the MBW complex on the CsANSpromoter by 48% (Figure 6g). These results hint that CsJAZ6 negatively regulates CsANS gene expression as well as anthocyanin biosynthesis by interfering with MBW formation.