SmGRAS5 directly regulated tanshinones biosynthetic gene but not GA
TFs usually regulate the transcription of target genes by directly binding to the elements in the promoter. Studies have shown that GRAS could interact with the promoter of downstream genes and regulate their expression (Smit et al. , 2005; Hirsch et al. , 2009). For instance, OsGRAS23 could bind to the potential target genes’ promoters to further positively modulating rice drought tolerance (Xu et al. , 2015). AtSCL14 was shown to be essential for activating the stress-inducible promoters (Fode et al. , 2008). Moreover, SlGRAS2 also regulated the fruit development downstream genes (Li et al. , 2018). Therefore, we speculated that SmGRAS5 might directly regulate the expressions of the tanshinones and GA biosynthetic pathway genes. Previous work has indicated that tanshinones biosynthesis is initiated by cyclization of general diterpenoid precursor GGPP to copalyl diphosphate (CPP) by SmCPS1 and subsequent further cyclization to the abietane miltiradiene by SmKSL1 (Cui et al. , 2015).SmCPS1 and KSL1 , as the downstream key enzyme genes in tanshinones biosynthetic pathway, were the most highly expressed in the periderm and played a vital role in tanshinones biosynthesis (Xuet al. , 2015). Considering the significant response ofSmCPS1 and SmKSL1 to SmGRAS5 and GA, Y1H, Dual-LUC and EMSA assays were performed and demonstrated that SmGRAS5 recognized the GARE motif of the SmKSL1 promoter, leading to the activation of expression. However, we verified several GA biosynthetic genes and found that none of their promoters interacted with SmGRAS5. These results showed that SmGRAS5 could only directly regulate tanshinones biosynthesis by interacting with SmKSL1 . However, SmKSL1was likely not the only target gene for SmGRAS5 regulation. GRAS could also interact with other TFs to mediate the regulation of the transcription activity of other target genes. For instance, GA inhibited flavonol biosynthesis via DELLA protein, which could interact with SG7 MYBs, to regulate the transcriptional levels of the biosynthesis pathway key genes (Tan et al. , 2019). In addition, the specific regulatory mechanism of SmGRAS5 on GA biosynthesis genes is also unclear. SmGRAS was likely to co-regulate GA biosynthesis by interacting with other negative regulatory factors and forming complexes. Therefore, identifying new interactive TFs or downstream targets of SmGRAS5 may provide further insight into the molecular mechanism of SmGRAS5-mediated regulation of secondary metabolite biosynthesis.
Finally, a potential mechanism for the SmGRAS5-mediated GA-promoted tanshinones biosynthesis was proposed (Fig. 8). GA could induceSmGRAS5 response and promote the biosynthesis of tanshinones through directly activating the expression of SmKSL1 . SmGRAS5 could inhibit the GA biosynthetic and signaling pathway at the same time and might promote metabolic flow to catalyze the precursor GGPP to synthesize more tanshinones.