Transcriptional Profiles of SmWRKY Family Genes and Their Putative Roles in the Biosynthesis of Tanshinone and Phenolic Acids in Salvia miltiorrhiza

Abstract

Salvia miltiorrhiza Bunge is a Chinese traditional herb for treating cardiovascular and cerebrovascular diseases, and tanshinones and phenolic acids are the dominated medicinal and secondary metabolism constituents of this plant. WRKY transcription factors (TFs) can function as regulators of secondary metabolites biosynthesis in many plants. However, studies on the WRKY that regulate tanshinones and phenolics biosynthesis are limited. In this study, 69 SmWRKYs were identified in the transcriptome database of S. miltiorrhiza, and phylogenetic analysis indicated that some SmWRKYs had closer genetic relationships with other plant WRKYs, which were involved in secondary metabolism. Hairy roots of S. miltiorrhiza were treated by methyl jasmonate (MeJA) to detect the dynamic change trend of SmWRKY, biosynthetic genes, and medicinal ingredients accumulation. Base on those date, a correlation analysis using Pearson's correlation coefficient was performed to construct gene-to-metabolite network and identify 9 SmWRKYs (SmWRKY1, 7, 19, 29, 45, 52, 56, 58, and 68), which were most likely to be involved in tanshinones and phenolic acids biosynthesis. Taken together, this study has provided a significant resource that could be used for further research on SmWRKY in S. miltiorrhiza and especially could be used as a cue for further investigating SmWRKY functions in secondary metabolite accumulation.

Keywords: Salvia miltiorrhiza; SmWRKY; phenolic acid; secondary metabolites; tanshinones.

Figure 1

Phylogenetic tree of the WRKY from S. miltiorrhiza and other plants. The tree was constructed from amino sequences using MEGA 6.0 by the neighbor-joining (NJ) program with 1000 bootstrap replicates. Clades with different colors represent diverse group/subgroup.

Figure 2

Schematic diagram of conserved motifs in WRKY proteins of S. miltiorrhiza. (A) The number symbols in colored boxes (1–20) represent different motifs. Box size indicates the length of motifs; (B) sequence logo of twenty conserved motifs.

Figure 3

Expression of SmWRKY genes and biosynthetic genes in different tissues and flowering phase. Hierarchical clustering and expression profiles of the SmWRKYs and biosynthetic genes in different organs/tissues in S. miltiorrhiza. Blocks with colors indicate low/down expression (blue), high/up expression (red), or no expression/no change (white).

Figure 4

Expression of SmWRKY genes and biosynthetic genes in response the MeJA. Hairy roots of S. miltiorrhiza were treated by MeJA with final concentration of 100 μM for different times, and gene expression was measured via RT-qPCR analysis. The 0 h time point was used as control. Fold change in transcript abundance is illustrated as heat map on a natural log scale (treatment/control). Blocks with colors indicate low/down expression (blue), high/up expression (red), and non-expression/ no change (white).

Figure 5

Effects of MeJA on accumulations of tanshinones and phenolic acids in S. miltiorrhiza hairy roots. The vertical bars show the SD values (n = 3). The asterisks indicate statistically significant differences at p < 0.05 (** p < 0.01) between the content in the MeJA treated cultures and that in the corresponding controls.