Genome-Wide Identification and Analysis of MAPK and MAPKK Gene Families in <em>Brachypodium distachyon</em>

<div><p>MAPK cascades are universal signal transduction modules and play important roles in plant growth, development and in response to a variety of biotic and abiotic stresses. Although MAPKs and MAPKKs have been systematically investigated in several plant species including <em>Arabidopsis</em>, rice and poplar, no systematic analysis has been conducted in the emerging monocot model plant <em>Brachypodium distachyon</em>. In the present study, a total of 16 MAPK genes and 12 MAPKK genes were identified from <em>B. distachyon</em>. An analysis of the genomic evolution showed that both tandem and segment duplications contributed significantly to the expansion of MAPK and MAPKK families. Evolutionary relationships within subfamilies were supported by exon-intron organizations and the architectures of conserved protein motifs. Synteny analysis between <em>B. distachyon</em> and the other two plant species of rice and <em>Arabidopsis</em> showed that only one homolog of <em>B. distachyon</em> MAPKs was found in the corresponding syntenic blocks of <em>Arabidopsis</em>, while 13 homologs of <em>B. distachyon</em> MAPKs and MAPKKs were found in that of rice, which was consistent with the speciation process of the three species. In addition, several interactive protein pairs between the two families in <em>B. distachyon</em> were found through yeast two hybrid assay, whereas their orthologs of a pair in <em>Arabidopsis</em> and other plant species were not found to interact with each other. Finally, expression studies of closely related family members among <em>B. distachyon</em>, <em>Arabidopsis</em> and rice showed that even recently duplicated representatives may fulfill different functions and be involved in different signal pathways. Taken together, our data would provide a foundation for evolutionary and functional characterization of MAPK and MAPKK gene families in <em>B. distachyon</em> and other plant species to unravel their biological roles.</p> </div

Phylogenetic and domain analyses of MAPKKs.

<p>(a) Phylogenetic relationships of <i>B. distachyon</i>, <i>Arabidopsis</i> and rice MAPKK genes. The phylogenetic tree was constructed by NJ (Neighbor–joining) method using the MEGA 4 program. Different color patternings indicate different gene clusters or superclusters. (b) Schematic diagram of amino acid motifs of <i>B. distachyon</i> MAPKKs. The black solid line represents the corresponding BdMAPKK and its length. The different-colored boxes represent different motifs and their position in each BdMAPKK sequence.</p

Phylogenetic and domain analyses of MAPKs.

<p>(a) Phylogenetic relationships of <i>B. distachyon</i>, <i>Arabidopsis</i> and rice MAPK genes. The phylogenetic tree was constructed by NJ (Neighbor-joining) method using the MEGA 4 program. Different color patternings indicate different gene clusters or superclusters. (b) Comparison of CD domain among four BdMAPKs (c) Schematic diagram of amino acid motifs of <i>B. distachyon</i> MAPKs from different groups. Motif analysis was performed using MEME 4.0 software as described in the methods. The black solid line represents the corresponding BdMAPK and its length. The different-colored boxes represent different motifs and their position in each BdMAPK sequence. A detailed motif introduction for all the <i>B. distachyon</i> MAPKs is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046744#pone.0046744.s001" target="_blank">Figure S1</a>.</p

Expression profiles of 20 MAPK genes (a) and 10 MAPKK (b) genes in different <i>Arabidopsis</i>

<p><b>tissues.</b> Details of the experimental conditions are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046744#pone.0046744.s007" target="_blank">Table S3</a>. Log2 based value was used to create the heatmap. Difference in gene expression changes is shown in color as the scale.</p

Comparative yeast two-hybrid interaction analyses of six BdMPKs with five BdMKKs.

<p>The yeast strain YH2Gold containing the indicated plasmid combinations was grown on nutritional selective medium minus His, Leu, Trp and plus X-α-Gal (SD/-His-Leu-Trp/X-α-Gal, TDO/X). Positive or negative control on the left corner showed the interaction between SV40 large T-antigen and murine p53 or SV40 large T-antigen and human lamin C (Lam) examined on TDO/X, respectively.</p

Phylogenetic (A), EST (B) and RT-PCR (C) analyses of <i>Brachypodium</i> MAPK genes.

<p>The gene names of each gene cluster originated from tandem or segmental duplication event were indicated with the same color.</p

Pairwise comparisons of the expression profiles of putative orthologs between rice and <i>B. distachyon</i>

<p><b>MAPKs and MAPKKs.</b> The mRNA relative amount represents the log 2 based value of expression intensity and the expression profiles of the two family genes in rice and <i>B. distachyon</i> were provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046744#pone.0046744.s010" target="_blank">Table S6</a>. The treatment time (h) under the particular light and temperature condition is presented on the x-axis. R indicates the correlation coefficient of the expression profiles between orthologs or paralogs pairs under the corresponding light and temperature treatments. The positive (A), negative (B) and no obvious correlation (C) were detected from our identified 17 pairs of putative orthologs or paralogs in rice and <i>B. distachyon</i> MAPKs and MAPKKs.</p

Phylogenetic (A), EST (B) and RT-PCR (C) analyses of MAPKK genes.

<p>The gene names of each gene cluster originated from tandem or segmental duplication event were indicated with the same color (except the black color).</p

Pearson correlation coefficients for the expression profiles of orthologs or paralogs pairs.

a<p>represents the similarity of amino acid.</p

Nomenclature for MAPKs and MAPKKs in <i>Arabidopsis</i>, <i>Brachypodium</i> and <i>Oryza.</i>

*<p>From Hamel et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046744#pone.0046744-Hamel1" target="_blank">[19]</a>. At: <i>Arabidopsis thaliana</i>, Os: <i>Oryza sativa</i>, Bd: <i>B.distachyon</i>.</p