Genome-wide characterization and expression profiling of MAPK cascade genes in Salvia miltiorrhiza reveals the function of SmMAPK3 and SmMAPK1 in secondary metabolism

Abstract

Background: The contribution of mitogen-activated protein kinase (MAPK) cascades to plant growth and development has been widely studied, but this knowledge has not yet been extended to the medicinal plant Salvia miltiorrhiza, which produces a number of pharmacologically active secondary metabolites.

Results: In this study, we performed a genome-wide survey and identified six MAPKKK kinases (MAPKKKKs), 83 MAPKK kinases (MAPKKKs), nine MAPK kinases (MAPKKs) and 18 MAPKs in the S. miltiorrhiza genome. Within each class of genes, a small number of subfamilies were recognized. A transcriptional analysis revealed differences in the genes' behaviour with respect to both their site of transcription and their inducibility by elicitors and phytohormones. Two genes were identified as strong candidates for playing roles in phytohormone signalling. A gene-to-metabolite network was constructed based on correlation analysis, highlighting the likely involvement of two of the cascades in the synthesis of two key groups of pharmacologically active secondary metabolites: phenolic acids and tanshinones.

Conclusion: The data provide insight into the functional diversification and conservation of MAPK cascades in S. miltiorrhiza.

Keywords: Co-expression analysis; Gene family; MAPK cascades; Phenolic acid synthesis; Salvia miltiorrhiza; Tanshinone synthesis.

Fig. 1

The phylogeny of the SmMAPK gene family. The dendrograms were constructed using the neighbour-joining method applied to full-length A. thaliana and S. miltiorrhiza MAPK sequences. Bootstrap (500 replicates) values appear at each branch. a MAPK sequences, b MAPKK sequences, c MAPKKKK sequences, d MAPKKK sequences

Fig. 2

The intron/exon structure of the SmMAPK gene family members. a MAPK sequences, b MAPKK sequences, c MAPKKKK sequences, and d MAPKKK sequences. Exons are shown as yellow boxes and introns with a simple line. Untranslated regions are indicated by thick blue lines. 0, 1, and 2 represent the intron phase. Gene models are drawn to scale

Fig. 3

The domain content of the SmMAPK gene family products. a TxY, b CD domain, c D(L/I/V)K S/T-× 5-S/T, d G(T/S)P-x-(W/Y/F)MAPEV, e GT-× 2-(W/Y)MAPE, f GTPEFMAPE(L/V)Y, and g HR/HDL/I/VK-× 2-N/S GT/S-× 2-WMAPE

Fig. 4

The motif content of the SmMAPK gene family products. a-d Putative motifs in a) MAPK, b MAPKK, c MAPKKKK, and d MAPKKK sequences, as predicted by MEME software

Fig. 5

Heat maps illustrating patterns of gene coexpression. Genes encoding (a) key phenolic acid pathway enzymes, transcription factors and members of the SmMAPK family, b enzymes involved in the synthesis of tanshinones, transcription factors and members of the SmMAPK family. Transcript abundance was estimated in the roots, leaves and flowers of S. miltiorrhiza plants, some of which were exposed to salicylic acid, methyl jasmonate or yeast extract. Pearson correlation coefficient (PCC) values were calculated for these genes. Blue: low abundance, red: high abundance

Fig. 6

Probable MAPK cascades in S. miltiorrhiza. a Heat maps illustrating patterns of gene coexpression generated by Multi Experiment Viewer. Blue: low abundance, red: high abundance. b Network built on the basis of the correlation among SmMAPKKKKs, SmMAPKKKs, SmMAPKKs and SmMAPKs. Pearson correlation coefficient (PCC) values were calculated for each pair of genes. MAPK cascade reaction map constructed using Cytoscape 3.6.1.0 software. c MAPK cascade pattern diagram

Fig. 7

qRT-PCR analyses of coexpressed genes. a Heat maps of coexpressed genes based on qRT-PCR analyses. b Heat maps of coexpressed genes based on RNA-Seq analyses

Fig. 8

SmMAPK3 physically interacts with SmMYBs and SmAREB1. Y2H assay to detect the interactions of SmMAPK3 with SmAREB1, SmbHLH10, SmbHLH37, SmbHLH51, SmbHLH148, SmERF1L1, SmERF6, SmMYB9b, SmMYB36, SmMYB39, SmMYB111, SmMYC2a, SmMYC2b, SmPAP1, SmTTG1 and SmWRKY1. Transformed yeast was grown on selective medium lacking adenine, histidine, leucine, and tryptophan (SD-LWHA) with AbA and x-a-gal to test protein interactions