Peng Li

Key message Plant-specific HD2s have been characterized; HDT1 orthologs were dicot-specific. AtHDT4 interacts with AtILR3 to increase Cd tolerance. Abstract The plant-specific histone deacetylase 2 (HD2) family is crucial for growth and stress responses, yet its evolutionary origins and functional diversification remain largely unknown. Here, we systematically elucidated the evolutionary trajectories of HDT homologs. We found the emergence of HDT1 orthologs as a dicot-specific innovation, characterized by unique motif acquisition and accompanied by relaxed purifying selection. Synteny analysis indicated that the duplication events establishing the HDT1 and HDT3 lineages were associated with whole-genome duplications (WGDs) specific to the dicot lineage. In contrast, the expansion of the HDT gene family in monocots appears to rely primarily on local duplication mechanisms, such as tandem duplications. Codon usage analysis revealed distinct species-specific preferences: lycophytes, bryophytes, and algae exhibited higher frequencies of G3s, C3s, GC3, CBI, Nc, and overall GC content, suggesting potential adaptive evolution or optimization for translational efficiency. Functional validation demonstrated that AtHDT4 contributes to the plant response to cadmium (Cd) stress. Specifically, AtHDT4 expression was significantly upregulated under CdCl₂ treatment. Compared with wild-type (WT) plants, the hdt4 mutant exhibited markedly reduced activities of key antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Inductively coupled plasma mass spectrometry (ICP-MS) analyses confirmed that AtHDT4 regulates Cd accumulation. Furthermore, AtILR3 expression was significantly downregulated in the hdt4 mutant, implicating it in the Cd stress response. As anticipated, direct protein–protein interaction between AtHDT4 and AtILR3 was verified. This study not only uncovers the critical role of AtHDT4 in mediating plant responses to Cd stress but also provides a broader evolutionary perspective on the functional diversification and specialization of HDT homologs across plant lineages.

Main conclusion TL63 orthologs were angiosperm specific and had undergone motifs loss and gain, and increased purifying selection. AtTL63 was involved in the response of yeast and Arabidopsis plants to oxidative stress. Abstract The Tóxicos en Levadura (TL) family, a class of E3 ubiquitin ligases with typical RING-H2 type zinc finger structure, plays a pivotal role in mediating physiological processes and responding to stress in plants. However, the evolution and function of TL63 remain unclear. In this study, TL63 homologs were dated roughly back to the origin of land plants and confirmed to have subjected to the gain and loss of motifs and increased purifying selection. Phylogenetic analysis displayed that 279 TL63s could be divided into four main clades (Clade A-D). Notably, the ancestral tandem TL40/41 cluster contributed to the expansion of modern Brassicaceae TL40/41. The substitution rate tests revealed that the TL63 lineage was evidently different from other lineages. The codon usage index exhibited that monocotyledons preferred to use not A3s and T3s, but C3s, G3s, CAI, CBI and Fop. Sequence analysis showed that the TL63 homologs had conserved TM and GLD motifs and RING-H2 domain whose key amino acid residues accounted for the high average abundance. Particularly, Arabidopsis thaliana TL63 (AtTL63) was located in the nuclei, cell membranes and peroxisomes and expressed universally and significantly throughout A. thaliana development. Under H₂O₂ treatment, low or moderate expression of the AtTL63 held beneficial effects on the growth and viability of yeast cells and the mutation or overexpression of the AtTL63 positively affected the growth of A. thaliana plants. In brief, this study could supply useful insight into the evolution of the plant TL63s and the AtTL63 functions under oxidative stress.

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.

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.

Background WRKY transcription factor is involved in regulation of plant growth and development, response to biotic and abiotic stresses, including homologous WRKY3 and WRKY4 genes which play a vital role in regulating plants defense response to pathogen and drought stress. Methods and results To investigate the function of AtWRKY3 and AtWRKY4 genes in regulating salt and Me-JA stresses, the loss-of-function mutations were generated by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system in Arabidopsis thaliana. Several independent transgenic lines with single or double mutations were obtained via Agrobacterium-mediated transformation. The knockout lines of AtWRKY3 and AtWRKY4 genes were successfully achieved and confirmed by qRT-PCR technology. Expression analysis showed that AtWRKY3 and AtWRKY4 genes had significantly up-regulated under salt and Me-JA stresses. The growth of double mutant plants under salt or Me-JA stresses were significantly inhibited compared with corresponding wild type (WT) plants, especially their root lengths. Moreover, the double mutant plants displayed salt and Me-JA sensitivity phenotypic characteristics, such as the increased relative electrolyte leakage (REL) and a substantial reduce in the activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities. Conclusion Taken together, these data suggested that the simultaneous modification of homologous gene copies of WRKY are established using CRISPR/Cas9 system in A. thaliana and the loss of AtWRKY3 and AtWRKY4 has an effect on ROS scavenging pathways to reduce stress tolerance.

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.