Figure legends
Figure 1. Tissue-specific expression of SmGRAS5 and determination of tanshinones. (A) Tissue-specific expression of SmGRAS5 in xylem, phloem and periderm tissues of S. miltiorrhiza roots. The expression levels were normalized to values in the xylem. (B) Contents of tanshinones in different tissues of the roots. (C) A Sectional view of the SmGRAS5 and tanshinones distribution in the root tissues. Standard errors were calculated from three sets of biological replicates. Significant differences using one-way ANOVA and S-N-K comparison tests, P < 0.05.
Figure 2. Subcellular localization of SmGRAS5 in protoplasts of tobacco leaves (A) and onion epidermal cells (B). Upper images represent the GFP control, while lower images represent the SmGRAS5-GFP fusion proteins. GFP: green fluorescence; DAPI: fluorescence of DAPI nuclear dye; Cholorophyll: chloroplast autofluorescence; Bright field: field observations; Merged: merge of bright field and relevant fluorescence.
Figure 3. SmGRAS5 regulates the biosynthesis of tanshinones in transgenic hairy roots. (A) Relative quantitative analysis ofSmGRAS5 expression in the transgenic lines and controls. (B) Analysis of tanshinones production from SmGRAS5 OE hairy root lines. (C) Analysis of tanshinones production from SmGRAS5 AE hairy root lines. (D) Relative expression levels of the genes involved in tanshinones biosyntheses in the SmGRAS5 transgenic lines. Standard errors were calculated from three sets of biological replicates. Significant differences using Student’s t-test, * 0.01< P < 0.05, ** P < 0.01.
Figure 4. GA affects the tanshinones and GA contents in SmGRAS5OE hairy roots. (A) Analysis of GA3 production fromSmGRAS5 OE lines and controls with or without 100 μM GA treatment for 6 d. (B) Analysis of tanshinones production from SmGRAS5 OE lines and controls with or without 100 μM GA treatment for 6 d. (C) Relative expression levels of genes involved in tanshinones and GA biosyntheses in the SmGRAS5 OE lines with 100 μM GA treatment for 2/24 h. Standard errors were calculated from three sets of biological replicates. Significant differences using one-way ANOVA and S-N-K comparison tests, P < 0.05.
Figure 5. SmGRAS5 binds to the GARE motif of the SmKSL1 promoter and activates its expression. (A) Y1H assay shows the interaction between SmGRAS5 and the SmKSL1 promoter. SmKSL1promoter+pGADT7 as the negative control and SmKSL1promoter+pGADT7-SmERF6 as positive control. (B) Dual-LUC assay shows the effects of SmGRAS5 on SmKSL1 promoter activation. (C) EMSA analysis of SmGRAS5 binding to the GARE-motif of the SmKSL1promoter. Schematic diagram showing the GARE motif in the SmKSL1promoter. Standard errors were calculated from three sets of biological replicates. Significant differences using Student’s t-test, * 0.01< P < 0.05.
Figure 6. Differentially expressed genes (DEGs) regulated by SmGRAS5 from the RNA-seq data. (A) Volcano plot of the DEGs regulated by SmGRAS5 (G5O14 vs ATCC). (B) GO term classifications of the 3910 DEGs from G5O14 vs ATCC. (C) KEGG classification of the 3910 DEGs from G5O14 vs ATCC. (D) Expression levels of representative DEGs of secondary metabolism. The differentially expressed genes demonstrated significantly differential expression (FDR-adjusted P- value < 0.05, |fold-change| ≥ 2). The average FPKM (log10 scale) of each gene is shown in the heat map.
Figure 7. The differentially expressed genes (DEGs) involved in tanshinones, GA biosynthetic pathway and signaling pathway. DEGs demonstrated significantly differential expression (FDR-adjustedP- value < 0.05, |fold-change| ≥ 2). The average FPKM (log10 scale) of each gene is shown in the heat map.
Figure 8. Model for SmGRAS5 regulates the GA-mediated accumulation of tanshinones in S. miltiorrhiza . GA could induce SmGRAS5response and promote the accumulation of tanshinones. SmGRAS5 could inhibit GA biosynthetic, and catalyze the precursor GGPP to synthesize more tanshinones through directly binding to the promoter ofSmKSL1 .