The type-2C protein phosphatases (PP2Cs), negatively regulating ABA responses and MAPK cascade pathways, play important roles in stress signal transduction in plants. Brachypodium distachyon is a new model plant for exploring the functional genomics of temperate grasses, cereals and biofuel crops. To date, genome-wide identification and analysis of the PP2C gene family in B. distachyon have not been investigated. In this study, 86 PP2C genes in B. distachyon were identified. Domain-based analyses of PP2C proteins showed that they all contained the phosphatase domains featured as 11 conserved signature motifs. Although not all phosphatase domains of BdPP2C members included all 11 motifs, tertiary structure analysis showed that four residues contributing to magnesium/manganese ions (Mg2+/Mn2+) coordination were conserved, except for two noncanonical members. The analysis of their chromosomal localizations showed that most of the BdPP2C genes were located within the low CpG density region. Phylogenetic tree and synteny blocks analyses among B. distachyon, Arabidopsis thaliana and Oryza sativa revealed that all PP2C members from the three species can be phylogenetically categorized into 13 subgroups (A–M) and BdPP2Cs were evolutionarily more closely related to OsPP2Cs than to AtPP2Cs. Segmental duplications contributed particularly to the expansion of the BdPP2C gene family and all duplicated BdPP2Cs evolved mainly from purifying selection. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis showed that BdPP2Cs were broadly expressed in disparate tissues. We also found that almost all members displayed up-regulation in response to abiotic stresses such as cold, heat, PEG and NaCl treatments, but down-regulation to biotic stresses such as Ph14, Guy11 and F0968 infection. In the present study, a comprehensive analysis of genome-wide identification and characterization of protein domains, phylogenetic relationship, gene and protein structure, chromosome location and expression pattern of the PP2C gene family was carried out for the first time in a new model monocot, i.e., B. distachyon. Our results provide a reference for genome-wide identification of the PP2C gene family of other species and also provide a foundation for future functional research on PP2C genes in B. distachyon.

Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense response. However, the molecular mechanisms underlying MAPK cascades are still very elusive, largely because of our poor understanding of how they relay the signals. The MAPK cascade is composed of MAPK, MAPKK, and MAPKKK. They transfer signals through the phosphorylation of MAPKKK, MAPKK, and MAPK in turn. MAPKs are organized into a complex network for efficient transmission of specific stimuli. This review summarizes the research progress in recent years on the classification and functions of MAPK cascades under various conditions in plants, especially the research status and general methods available for identifying MAPK substrates, and provides suggestions for future research directions.

Group Ⅲ WRKY transcription factors (TFs) play pivotal roles in responding to the diverse abiotic stress and secondary metabolism of plants. However, the evolution and function of WRKY66 remains unclear. Here, WRKY66 homologs were traced back to the origin of terrestrial plants and found to have been subjected to both motifs’ gain and loss, and purifying selection. A phylogenetic analysis showed that 145 WRKY66 genes could be divided into three main clades (Clade A–C). The substitution rate tests indicated that the WRKY66 lineage was significantly different from others. A sequence analysis displayed that the WRKY66 homologs had conserved WRKY and C2HC motifs with higher proportions of crucial amino acid residues in the average abundance. The AtWRKY66 is a nuclear protein, salt- and ABA- inducible transcription activator. Simultaneously, under salt stress and ABA treatments, the superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities, as well as the seed germination rates of Atwrky66-knockdown plants generated by the clustered, regularly interspaced, short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system, were all lower than those of wild type (WT) plants, but the relative electrolyte leakage (REL) was higher, indicating the increased sensitivities of the knockdown plants to the salt stress and ABA treatments. Moreover, RNA-seq and qRT-PCR analyses revealed that several regulatory genes in the ABA-mediated signaling pathway involved in stress response of the knockdown plants were significantly regulated, being evidenced by the more moderate expressions of the genes. Therefore, the AtWRKY66 likely acts as a positive regulator in the salt stress response, which may be involved in an ABA-mediated signaling pathway.