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.