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/Thrprotein kinases, namely, MAP kinase kinase kinase, MAP kinase kinase and MAP kinase,is a universal module in signal transduction pathways in all eukaryotes. These proteinshave been implicated in diverse cellular processes including cell growth, proliferation,differentiation, survival, development and in responses to a diversity of environmentalstimuli including cold, heat, reactive oxygen species, UV, drought and pathogen attack.Although MAPKs and MAPKKs have been systematically investigated in Arabidopsisand rice, no systematic analysis has been conducted in the emerging monocot model plantBrachypodium distachyon. B. distachyon is an annual weed, which has many attributesincluding small genome (272Mbp) diploid accessions, simple growth requirements, smallphysical stature, short lifecycle, self-fertility, ease of cultivation and transformation.Recently, the B. distachyon genome (diploid inbred line Bd21) has been sequenced, whichprovides an unprecedented opportunity for genome-wide analysis of the MAPK cascadesin 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. Wecharacterized them based on nomenclature, classification, phylogeny, protein conservedmotif, chromosomal distribution, gene duplication, gene structure, promoter region,synteny map with their orthologs in Arabidopsis and rice, expression patterns, theirprotein interaction profiles between these two families. The main results are as follows:1） Through homology alignment and HMMER search,16MAPK and12MAPKKgenes were identified from B. distachyon genomes, respectively. Multiple sequencealignment, conserved domain and phylogenetic analyses showed that B. distachyonMAPKs could also be classified into four groups corresponding to the group A, B, C andD in Arabidopsis and rice. Of them, there were six members containing TEY motif, ninemembers containing TDY motif and one member containing MEY motif. Similarly, theMAPKKs 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 thetwo family genes were non-random and certain chromosomes and chromosomal regionshad a relatively high density of MAPK or MAPKK genes. The results of gene duplicationevent revealed that tandem duplication or segmental duplication played a major role in theexpansion of the two families in B. distachyon. Through large-scale synteny analysisbetween B. distachyon and the other two species of rice and Arabidopsis,50%of MAPKgenes and42%of MAPKK genes from B. distachyon were identified to be synteny withtheir orthologs of rice genome, while there was only one gene in these two families foundbeing synteny with its orthologs of Arabidopsis. The result was consistent with the currentunderstanding of plant evolutionary history.3） The exon/intron structure analysis showed that all the members of MAPK familyhad introns with numbers varying from three to eleven, while58.3%of the total MAPKKgenes had no intron and the intron number of the remaining MAPKK genes averaged8.8.The members with close relationships shared much more similar structure organizationsthan others. In addition, we also analyzed the four regulatory elements including theWBOX cassettes, CBF boxes, DRE boxes and GCC motifs implicated in either responseto pathogens or plant stress in the1kb region upstream of the two family genes. Theresults showed that WBOX element existed in all the members of the two families; eachmember had the most WBOX and the fewest DRE elements. However, we see no clearevidence 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 fromalternative splicing) were cloned into the corresponding yeast hybrid vectors and werethen used to study their interactions. The results showed that a MAPK may haveinteraction 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 comparativelyanalyzed the expression profiles of the two family genes of B. distachyon, rice andArabidopsis in different tissues and in response to different abiotic stresses and conducted the pairwise comparisons of the expression profiles of putative orthologous or paralogspairs existing in the two family genes of B. distachyon and rice under different light andtemperature conditions. The expression pattern of each gene was associated with itsunique function but was not associated with their EST counts or sequence similarity.These results would provide a foundation for further evolutionary and functionalcharacterization of MAPK and MAPKK gene families in B. distachyon and other plantspecies to unravel their biological roles．...