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.