Transcriptome analysis of SmGRAS5-regulated genes
To gain a comprehensive overview of the changes regulated by SmGRAS5 at the transcript level, we performed a transcriptome analysis of G5O14 and ATCC hairy roots. A total of 3910 differentially expressed genes (DEGs) were designated as SmGRAS5-regulated genes (Fig. 6A). A functional gene ontology (GO) analysis indicated that the DEGs regulated by SmGRAS5 were mostly enriched in biological process, metabolic process and catalytic activity terms (Fig. 6B). The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that hundreds of genes for metabolic pathways and the biosynthesis of secondary metabolites were enriched. Moreover, there were many DEGs enriched in stilbenoid, diarylheptanoid and gingerol biosynthesis, phenylpropanoid biosynthesis, limonene and pinene degradation, flavonoid biosynthesis, diterpenoid biosynthesis and plant hormone signal transduction (Fig. 6C). Since many genes involved in secondary metabolites biosynthesis were identified as SmGRAS5 regulated genes in our transcriptomic results. We further analyzed the secondary metabolism pathway using Mapman software (Fig. 6D). We could see that most of the DEGs in MVA and non MVA pathways were upregulated. And there were more upregulated DEGs in the terpenoids, alkaloids-like, carotenoids, flavonoids, anthocyanins and glucosinolates pathways. However, most DEGs in wax and chalcones pathways were downregulated.
Furthermore, we further analyzed transcriptome data to determine the regulatory roles of SmGRAS5 in tanshinones, GA biosynthetic and GA signaling pathways. As shown in Figure 7, the expressions of most tanshinones biosynthetic pathway genes were upregulated while the expressions of most GA biosynthetic and signaling pathway genes were downregulated in the SmGRAS5 OE lines. The qRT-PCR analysis verified the transcriptomic data (Fig. S9). These results were consistent with our above data and supported the function of SmGRAS5 in promoting tanshinones biosynthesis but inhibiting the GA biosynthesis and GA signaling pathway. Taken together, our results suggested that SmGRAS5 might reduce GA biosynthesis by promoting the universal precursor GGPP to synthesize more tanshinones and further to inhibit downstream GA signaling pathway.