SmGRAS5 regulates GA-promoted biosynthesis of tanshinones
Since SmGRAS5 OE lines grow slower than the control, which was similar to the GA-deficient phenotypes (Fig. S4). Consistent with the phenotypes, overexpression of SmGRAS5 indeed decreased the GA content and downregulated the expressions of most GA biosynthetic genes (Fig. 4A,C). To further investigate whether the regulatory function of SmGRAS5 on tanshinones biosynthesis is mediated by GA, we then used exogenous GA to treat the SmGRAS5 OE and control lines. We found that there are some GA-responded motifs (GARE-motif and P-box) in the promoter of SmGRAS5. And we also found GA treatment could induce the expression of SmGRAS5 (Fig. S5). The results showed that the GA content of control and SmGRAS5 OE lines were all significantly increased after GA treated. However, the expressions of the downstream genes of GA biosynthesis were different in the SmGRAS5 OE lines. GA biosynthetic enzymes SmGA20ox2/6 were upregulated, whileSmGA3ox1 was downregulated in the SmGRAS5 OE lines. And the expressions of the GA deactivating enzymes SmGA2ox8/9 were different in the SmGRAS5 OE lines. The results after GA treatment indicated that the increased GA content in the SmGRAS5 OE lines maybe not have occurred by promoting GA biosynthesis but could have also been caused by exogenous GA entering the cell. Intriguingly, the four tanshinones total contents of SmGRAS5 OE and control lines were all significantly increased under GA treatments (Fig. 4B). As expected, the expression of most tanshinones biosynthesis genes was quickly induced by GA application in all the lines (Fig. 4C). Among the tanshinones biosynthesis genes, the downstream genes GGPPS1 and KSL1 had the most significantly increased in the SmGRAS5 OE and control lines. Collectively, these results indicated that GA could induceSmGRAS5 response to further promote tanshinone biosynthesis.