Genome-wide identification of MAPK, MAPKK, and MAPKKK gene families and transcriptional profiling analysis during development and stress response in cucumber

Abstract

Background: The mitogen-activated protein kinase (MAPK) cascade consists of three types of reversibly phosphorylated kinases, namely, MAPK, MAPK kinase (MAPKK/MEK), and MAPK kinase kinase (MAPKKK/MEKK), playing important roles in plant growth, development, and defense response. The MAPK cascade genes have been investigated in detail in model plants, including Arabidopsis, rice, and tomato, but poorly characterized in cucumber (Cucumis sativus L.), a major popular vegetable in Cucurbitaceae crops, which is highly susceptible to environmental stress and pathogen attack.

Results: A genome-wide analysis revealed the presence of at least 14 MAPKs, 6 MAPKKs, and 59 MAPKKKs in the cucumber genome. Phylogenetic analyses classified all the CsMAPK and CsMAPKK genes into four groups, whereas the CsMAPKKK genes were grouped into the MEKK, RAF, and ZIK subfamilies. The expansion of these three gene families was mainly contributed by segmental duplication events. Furthermore, the ratios of non-synonymous substitution rates (Ka) and synonymous substitution rates (Ks) implied that the duplicated gene pairs had experienced strong purifying selection. Real-time PCR analysis demonstrated that some MAPK, MAPKK and MAPKKK genes are preferentially expressed in specific organs or tissues. Moreover, the expression levels of most of these genes significantly changed under heat, cold, drought, and Pseudoperonospora cubensis treatments. Exposure to abscisic acid and jasmonic acid markedly affected the expression levels of these genes, thereby implying that they may play important roles in the plant hormone network.

Conclusion: A comprehensive genome-wide analysis of gene structure, chromosomal distribution, and evolutionary relationship of MAPK cascade genes in cucumber are present here. Further expression analysis revealed that these genes were involved in important signaling pathways for biotic and abiotic stress responses in cucumber, as well as the response to plant hormones. Our first systematic description of the MAPK, MAPKK, and MAPKKK families in cucumber will help to elucidate their biological roles in plant.

Fig. 1

Phylogenetic analysis and domain organization of cucumber MAPKs. a The unrooted phylogenetic tree was generated based on the amino acid sequences by the NJ method using MEGA 5. Bootstrap supports from 1000 replicates are indicated at each branch. The members of each subfamily are indicated with the same color. b Domain organization was analyzed by scanning the protein sequences for the presence of known motifs and domains with PlantsP. Different subgroups of CsMAPKs are represented by the capital letter A-D

Fig. 2

Phylogenetic analysis (a) and domain organization (b) of cucumber MAPKKs. Different subgroups of CsMAPKKs are represented by the capital letter A-D. For other details, see Fig. 1

Fig. 3

Phylogenetic analysis (a) and domain organization (b) of cucumber MAPKKKs. For other details, see Fig. 1

Fig. 4

Sequence alignment and motif analysis of CsMAPKs. a Multiple sequence alignment analysis of the peptides of MAPK proteins in cucumber. The highlighted part shows the conserved signature motif obtained with the ClustalX program. b Schematic diagram of amino acid motifs of CsMAPKs. Motif analysis was analyzed by MEME program online. Different colors of the boxes represent different motifs in the corresponding position of each CsMAPK proteins. Different subgroups of CsMAPKs are represented by the capital letter A-D. The detailed information of 10 motifs was illustrated in Additional file 6

Fig. 5

Sequence alignment and motif analysis of CsMAPKKs. Different subgroups of CsMAPKKs are represented by the capital letter A-D. The detailed information of 10 motifs was illustrated in Additional file 7. For other details, see Fig. 4

Fig. 6

Sequence alignment and motif analysis of CsMAPKKKs. The detailed information of 10 motifs was illustrated in Additional file 8. For other details, see Fig. 4

Fig. 7

Phylogenetic analysis and gene structure of CsMAPKs in cucumber. Right part illustrates the intron/exon configurations of the corresponding CsMAPK genes. The green boxes indicate the exons, and lines indicate the introns. Gene structures of CsMAPKs in different subgroups are shaded by different colors. Different subgroups of CsMAPKs are represented by the capital letter A-D

Fig. 8

Phylogenetic analysis and gene structure of CsMAPKKs in cucumber. Different subgroups of CsMAPKKs are represented by the capital letter A-D

Fig. 9

Phylogenetic analysis and gene structure of CsMAPKKKs in cucumber

Fig. 10

Chromosomal distributions and gene duplications of CsMAPKs, CsMAPKKs and CsMAPKKKs in cucumber genome. Dotted lines connect different MAPK, MAPKK, or MAPKKK genes that are present as duplicated gene pairs. Triangles indicate the upward or downward direction of transcription

Fig. 11

Expression profiles of CsMAPKs in different organs/tissues using qRT-PCR analysis. R: roots, S: stems, L: leaves, M-FL: male flowers, F-FL: female flowers, FR: fruits. All samples were run in triplicate and the data were normalized relative to the EF1a (accession number EF446145) related protein transcript levels. The expression levels of genes are presented in heatmap using fold-change values transformed to Log2 format by MeV4.8. The color scale and Log2 values (fold-change values) are shown at the bottom of heatmap. Genes were clustered according to their expression profiles

Fig. 12

Expression profiles of CsMAPKKs in different organs using qRT-PCR analysis

Fig. 13

Expression profiles of CsMAPKKKs in different organs using qRT-PCR analysis

Fig. 14

Expression patterns of CsMAPKs under abiotic and biotic stress treatment in cucumber by qRT-PCR analysis in heatmap. Details of the treatments are reported in Materials and Methods

Fig. 15

Expression patterns of CsMAPKKs under abiotic and biotic stress treatment in cucumber by qRT-PCR analysis in heatmap

Fig. 16

Expression patterns of CsMAPKKKs under abiotic and biotic stress treatment in cucumber by qRT-PCR analysis in heatmap