Genome-Wide Identification, Interaction, And Expression Analysis Of MAPK And MAPKK Gene Families In Brachypodium Distachyon

Mitogen-activated protein kinase (MAPK) cascade, which consists of three Ser/Thr protein kinases, namely, MAP kinase kinase kinase, MAP kinase kinase and MAP kinase, is a universal module in signal transduction pathways in all eukaryotes. These proteins have been implicated in diverse cellular processes including cell growth, proliferation, differentiation, survival, development and in responses to a diversity of environmental stimuli including cold, heat, reactive oxygen species, UV, drought and pathogen attack. Although MAPKs and MAPKKs have been systematically investigated in Arabidopsis and rice, no systematic analysis has been conducted in the emerging monocot model plant Brachypodium distachyon. B. distachyon is an annual weed, which has many attributes including small genome (272Mbp) diploid accessions, simple growth requirements, small physical stature, short lifecycle, self-fertility, ease of cultivation and transformation. Recently, the B. distachyon genome (diploid inbred line Bd21) has been sequenced, which provides an unprecedented opportunity for genome-wide analysis of the MAPK cascades in the B. distachyon species.In this study, we identified16MAPK genes and12MAPKK genes in the B. distachyon genome based on the conservation of plant MAPKs and MAPKKs. We characterized them based on nomenclature, classification, phylogeny, protein conserved motif, chromosomal distribution, gene duplication, gene structure, promoter region, synteny map with their orthologs in Arabidopsis and rice, expression patterns, their protein interaction profiles between these two families. The main results are as follows:1) Through homology alignment and HMMER search,16MAPK and12MAPKK genes were identified from B. distachyon genomes, respectively. Multiple sequence alignment, conserved domain and phylogenetic analyses showed that B. distachyon MAPKs could also be classified into four groups corresponding to the group A, B, C and D in Arabidopsis and rice. Of them, there were six members containing TEY motif, nine members containing TDY motif and one member containing MEY motif. Similarly, the MAPKKs of B. distachyon could also be classified into four groups. Of the four groups, one group had the most members (five), while the number of other groups was relatively uniform.2) Chromosomal distribution analysis results showed that the distributions of the two family genes were non-random and certain chromosomes and chromosomal regions had a relatively high density of MAPK or MAPKK genes. The results of gene duplication event revealed that tandem duplication or segmental duplication played a major role in the expansion of the two families in B. distachyon. Through large-scale synteny analysis between B. distachyon and the other two species of rice and Arabidopsis,50%of MAPK genes and42%of MAPKK genes from B. distachyon were identified to be synteny with their orthologs of rice genome, while there was only one gene in these two families found being synteny with its orthologs of Arabidopsis. The result was consistent with the current understanding of plant evolutionary history.3) The exon/intron structure analysis showed that all the members of MAPK family had introns with numbers varying from three to eleven, while58.3%of the total MAPKK genes had no intron and the intron number of the remaining MAPKK genes averaged8.8. The members with close relationships shared much more similar structure organizations than others. In addition, we also analyzed the four regulatory elements including the WBOX cassettes, CBF boxes, DRE boxes and GCC motifs implicated in either response to pathogens or plant stress in the1kb region upstream of the two family genes. The results showed that WBOX element existed in all the members of the two families; each member had the most WBOX and the fewest DRE elements. However, we see no clear evidence of a correlation between the arrangement of these promoter cassettes (WBOX, CBF, DRE and GCC) and in silico expression via EST counts.4) Six MAPK genes and four MAPKK genes (five proteins generated from alternative splicing) were cloned into the corresponding yeast hybrid vectors and were then used to study their interactions. The results showed that a MAPK may have interaction with multiple MAPKKs and vice versa. The cross interaction between MAPK-MAPKK reflected the diversity of MAPK signal transduction pathways.5) By using RT-PCR and microarray data in the database, we comparatively analyzed the expression profiles of the two family genes of B. distachyon, rice and Arabidopsis in different tissues and in response to different abiotic stresses and conducted the pairwise comparisons of the expression profiles of putative orthologous or paralogs pairs existing in the two family genes of B. distachyon and rice under different light and temperature conditions. The expression pattern of each gene was associated with its unique function but was not associated with their EST counts or sequence similarity.These results would provide a foundation for further evolutionary and functional characterization of MAPK and MAPKK gene families in B. distachyon and other plant species to unravel their biological roles.