Genome-wide identification and analysis of abiotic stress responsiveness of the mitogen-activated protein kinase gene family in Medicago sativa L

Abstract

Background: The mitogen-activated protein kinase (MAPK) cascade is crucial cell signal transduction mechanism that plays an important role in plant growth and development, metabolism, and stress responses. The MAPK cascade includes three protein kinases, MAPK, MAPKK, and MAPKKK. The three protein kinases mediate signaling to downstream response molecules by sequential phosphorylation. The MAPK gene family has been identified and analyzed in many plants, however it has not been investigated in alfalfa.

Results: In this study, Medicago sativa MAPK genes (referred to as MsMAPKs) were identified in the tetraploid alfalfa genome. Eighty MsMAPKs were divided into four groups, with eight in group A, 21 in group B, 21 in group C and 30 in group D. Analysis of the basic structures of the MsMAPKs revealed presence of a conserved TXY motif. Groups A, B and C contained a TEY motif, while group D contained a TDY motif. RNA-seq analysis revealed tissue-specificity of two MsMAPKs and tissue-wide expression of 35 MsMAPKs. Further analysis identified MsMAPK members responsive to drought, salt, and cold stress conditions. Two MsMAPKs (MsMAPK70 and MsMAPK75) responds to salt and cold stresses; two MsMAPKs (MsMAPK60 and MsMAPK73) responds to cold and drought stresses; four MsMAPKs (MsMAPK1, MsMAPK33, MsMAPK64 and MsMAPK71) responds to salt and drought stresses; and two MsMAPKs (MsMAPK5 and MsMAPK7) responded to all three stresses.

Conclusion: This study comprehensively identified and analysed the alfalfa MAPK gene family. Candidate genes related to abiotic stresses were screened by analysing the RNA-seq data. The results provide key information for further analysis of alfalfa MAPK gene functions and improvement of stress tolerance.

Keywords: Medicago sativa L.; MsMAPK gene family; Abiotic stress response; Genome-wide analysis; Tissue-specific expression.

Fig. 1

Chromosome distribution of the MAPK genes in M. sativa. Each color represents different groups of chromosomes. MsMAPK genes were renamed as MsMAPK1-MsMAPK80 according to the order of chromosomes and the position of MsMAPK gene on chromosomes. Rectangle represents a tandem duplication event

Fig. 2

Phylogenetic tree of MAPK genes in M. sativa, A. thaliana, G. max and O. sativa. Red, orange, green and blue represent the A, B, C and D subgroup, respectively. The red, blue, green and black circles represent A. thaliana, G. max, O. sativa, and M. sativa, respectively

Fig. 3

Basic structures and motifs of the MsMAPK genes. (A) The structure of MsMAPK protein motif. (B) Basic structures of MsMAPK genes

Fig. 4

Schematic diagram of the syntenic relationships of MsMAPK genes in M. sativa. The gray ribbons represent syntenic blocks in the alfalfa genome, and the segmental duplication events are marked in red

Fig. 5

Collinearity analysis of the MsMAPK genes with those of A. thaliana, Medicago truncatula and G. max. The gray ribbons represent syntenic blocks in the alfalfa genome, and the segmental duplication events are marked in red

Fig. 6

Cis-acting elements of the MAPK gene promoters in M. sativa.A-D: MsMAPK genes were divided into four groups

Fig. 7

Expression of 58 MsMAPK genes in different tissues. The expression levels were normalized by row using the Z − scores algorithm. The color scale on the right side of the heatmap indicates the relative expression levels, and the color gradient from blue to red indicates an increase in expression levels

Fig. 8

Expression of MAPK genes in M. sativa under stress. (A) Expression of MAPK genes in M. sativa under salt stress. (B) Expression of MAPK genes in M. sativa under drought stress. (C) Expression of MAPK genes in M. sativa under cold stress. (D) Venn diagrams of MsMAPK genes responding to three stresses. Under salt stress: 0 h as CK and 0.5, 1, 3, 6, 12 and 24 h as S1 to S6, respectively. Under drought stress: 0 h as CK and 1, 3, 6, 12 and 24 h as M1, M2, M3, M4 and M5, respectively. Under cold stress: 0 h as CK and 2, 6, 24 and 48 h as C1, C2, C3 and C4, respectively

Fig. 9

MsMAPK expression under drought, salt and cold stress conditions according to RT–qPCR. (A) Expression of MsMAPKs under drought stress. (B) Expression of MsMAPKs under cold stress. (C) Expression of MsMAPKs under salt stress