Interaction of SmGRAS5 with tanshinones and GA biosynthetic gene
To further investigate how SmGRAS5 regulates tanshinones biosynthesis, we performed Y1H, Dual-LUC and EMS assays. According to our qRT-PCR results, the expressions of most genes in the tanshinones biosynthetic pathway were upregulated in the SmGRAS5 OE lines. Among them,SmCPS1 and SmKSL1 , which are the key downstream genes in tanshinones biosynthesis, their expressions were remarkably upregulated. The Y1H Gold reporter strains that have the SmKSL1 promoter transformed with SmGRAS5 prey plasmid and positive control could grow normally on SD/-Leu (700 ng/ml AbA) (Fig. 5A). However, the Y1H Gold reporter strains that have the SmCPS1 promoter transformed with SmGRAS5 prey plasmid and negative control could not grow on SD/-Leu (700 ng/ml AbA) (Fig. S6). These results provided in vivo evidence that SmGRAS5 participates in tanshinones biosynthesis by directly binding to the SmKSL1 promoter. We further confirmed this by dual-luciferase report system. The Dual-LUC assay showed that SmGRAS5 could directly activate the SmKSL1 promoter (Fig. 5B).
Our promoter analysis indicated that the promoter sequence ofSmKSL1 contains a GARE motif at 774 bp upstream of the ATG codon (Fig. 5C). To further confirm binding between SmGRAS5 and the fragment containing the SmGRAS5 recognition element in vitro, purified SmGRAS5-MBP fusion proteins were combined with the fragment containing the GARE motif, and they were analyzed by EMSA. Subsequently, specific DNA–SmGRAS5 protein complexes were strongly detected (Fig. 5C). These results confirmed that SmGRAS5 regulated tanshinones biosynthesis by directly binding to the GARE motif of the SmKSL1 promoter. All these data together indicated that SmGRAS5 could directly bind to the GRAE in the promoter of SmKSL1 to induce its expression.
Furthermore, in order to further study whether SmGRAS5 directly regulates the expressions of GA biosynthesis genes. We analyzed some promoters of GA biosynthesis genes that were significantly downregulated by SmGRAS5. We designed GA response element probes for some GA biosynthesis genes and conducted EMSA experiments (Fig. S8). Unfortunately, none of the promoters of these GA biosynthesis genes interacted with SmGRAS5. We speculated that SmGRAS5 might inhibit the expressions of GA biosynthesis genes by interacting with other negative regulatory factors or inhibit GA biosynthesis by other pathways.