GA signaling involved in the biosynthesis of tanshinones and SAs
GA is a diterpene phytohormone that modulates growth and development throughout the whole life cycle of the plant (Sun, 2011). A study has shown that GA could promote the accumulation of tanshinones in the wild-type hairy roots of S. miltiorrhiza (Bai et al. , 2017), but its regulatory mechanisms are poorly understood. We found the SmGRAS5 was the most sensitive genes to GA response of the five SmGRASs genes in our previous study (Bai et al. , 2017). We speculated that it might play a major role in SmGRASs in the GA signaling. As a key regulator of GA signaling, GRAS has been classified into 13 different subgroups in Arabidopsis (Zhang et al ., 2018). Our phylogenetic analysis showed SmGRAS5 belonged to SCL3 subfamily, which is involved in the regulation of Arabidopsis root cell elongation, GA homeostasis and signaling (Heo et al. , 2011). AtSCL3 inhibited GA biosynthesis and antagonized with DELLA in maintaining GA homeostasis by feedback regulating upstream GA biosynthesis genes, as well as in modulating downstream GA responses by direct protein-protein interaction (Zhang et al. , 2011). DELLA protein interacted with AtMYB12 activating expression of flavonol biosynthetic genes (Tan et al. , 2019). The tissue-specific expression of GRAS genes often indicates their functional roles in development. It has been reported that diterpenoid tanshinones not only accumulated but also biosynthesized in the roots periderm ofS. miltiorrhiza (Xu et al. , 2015). We found thatSmGRAS5 highly expressed in the periderm and co-localized with tanshinones, which indicated that SmGRAS5 might be involved in the regulation of tanshinones biosynthesis. Overexpression of SmGRAS5significantly improved tanshinones accumulation through upregulating the expressions of biosynthetic genes, while in the SmGRAS5 AE lines the tanshinones accumulation was decreased. Notably, the promotion of SmGRAS5 to the tanshinones biosynthesis is the most significant in all of the SmGRASs, which we are currently studying. So we speculated that it might play a vital role in the regulation of tanshinones biosynthesis. Considering the correlation between tanshinones and GA biosynthetic pathways, we found that the expressions of the most of GA biosynthetic pathway genes were downregulated, which resulted in the decrease of GA content in the SmGRAS5 OE lines. Similarly, overexpressingHaGRASL also reduced the metabolic flow of GAs inArabidopsis (Fambrini et al. , 2015). SilencingSlGRAS26 inhibited the GA biosynthetic pathway, promoted the GA inactivation pathway, and resulted in GA deficiency in tomato (Zhouet al. , 2018). SlGRAS24 could downregulate GA biosynthetic genes, disrupt GA homeostasis and participate in a series of developmental processes through modulating GA and auxin signaling (Huanget al. , 2017). We speculated that SmGRAS5 might regulate the metabolic flow of diterpenoid GA and tanshinones biosynthesis. In addition, the GA application could promote the tanshinones biosynthesis in SmGRAS5 OE lines as well as control lines. Therefore, these results implied that SmGRAS5 could be induced by GA and might catalyze the precursor GGPP to synthesize more tanshinones. Moreover, transcriptome data also revealed that SmGRAS5 could regulate many secondary metabolites biosynthesis. It could inhibit the GA biosynthetic and signaling pathway at the same time, and induce tanshinones biosynthetic pathway through regulating biosynthetic genes.