Functional characterization of TaWRKY254 in salt tolerance based on genome-wide analysis of the WRKY gene family in wheat core parent Zhou8425B.

The WRKY gene family plays a pivotal role in regulating plant growth, development, and stress responses. Zhou8425B, a core wheat parent in Chinese breeding programs known for its superior agronomic traits, remains underexplored in terms of its WRKY functional landscape. In this study, we identified 294 WRKY transcription factors in the Zhou8425B genome and conducted comprehensive bioinformatics analyses covering gene structure, protein properties, phylogenetic relationships, conserved motifs, and cis-regulatory elements. RNA-seq analysis across 12 tissues revealed that 274 WRKY genes are highly expressed and form distinct tissue-specific clusters. Notably, TaWRKY254 (TraesZ8425B6B01G167200) was significantly upregulated under various environmental stresses. RT-qPCR confirmed that TaWRKY254 expression under salt stress was substantially higher in Zhou8425B compared to Chinese Spring. Sequence diversity analysis revealed a 513 bp deletion in the promoter region and a T-to-C nonsynonymous mutation in the exon, resulting in an isoleucine-to-valine substitution in Zhou8425B. Based on this 513 bp difference, we developed a specific molecular marker and genotyped the recombinant inbred lines (RILs) from a Zhou8425B × Chinese Spring. Phenotypic analysis showed that RILs carrying the TaWRKY254 genotype exhibited enhanced salt tolerance, as evidenced by increased catalase, proline, and soluble protein levels, reduced lipid peroxidation, and significantly higher thousand kernel weight compared to those with the TaWRKY254 genotype. These findings suggest that TaWRKY254 may play an important role in salt stress adaptation and yield-related traits, highlighting its potential as a genetic resource for salt-tolerant wheat breeding.