Evolution of the WRKY66 Gene Family and Its Mutations Generated by the CRISPR/Cas9 System Increase the Sensitivity to Salt Stress in Arabidopsis

Abstract

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.

Evolutionary studies of the basic helix–loop–helix (bHLH) IVc gene family in plants and the role of AtILR3 in Arabidopsis response to ABA stress

“…Particularly, the ILR3 orthologs were replicated only in flowering plants, predating radiation in extant angiosperms, dating back to the duplications and diversification of an ancestral gymnosperm gene (Figure 2B). Strictly speaking, the ILR3 orthologs were angiosperm‐specific, which were similar to the evolutionary patterns of the WRKY66 and Tóxicos en Levadura 63 ( TL63 ) gene families (Li et al, 2023; Zhang et al, 2023). As is well known, the WGDs, as well as other types of duplications such as tandem duplication, can provide rich genetic materials for the structural and functional evolutions of modern genes (Van de Peer et al, 2009; Jiao et al, 2011).…”

“…gene families (Li et al, 2023;Zhang et al, 2023). As is well known, the WGDs, as well as other types of duplications such as tandem duplication, can provide rich genetic materials for the structural and functional evolutions of modern genes (Van de Peer et al, 2009;Jiao et al, 2011).…”

A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts

“…For example, the genome editing of bread wheat led to obtaining heritable resistance to powdery mildew [9]. Its use in Arabidopsis facilitated a study of transcriptional control of salt tolerance [10]. CRISPR-Cas9 based approaches are already in use the therapy of β-thalassemia [11] and transthyretin amyloidosis [12,13].…”