Abstract
Perennial ryegrass, the most widely cultivated forage and turfgrass, exhibits a relatively poor temperature tolerance, which restricts its distribution and utilization in warmer regions. WRKY transcription factors regulate diverse biological processes in plants, including development and stress responses. Here, we report the comprehensive identification of 97 WRKY genes (LpWRKYs) in perennial ryegrass. The LpWRKYs were unevenly distributed across seven chromosomes, with one gene anchored to a scaffold. These genes were classified into three major groups and five subgroups based on phylogenetic relationships and structural domain diversity. All LpWRKYs contained either one or two WRKY domains and the LpWRKYs within the same group had similar motif compositions. A total of 12 tandem duplicates and 27 segmental duplicates were identified, suggesting that gene duplication might have contributed to the expansion of the LpWRKY gene family. Ka/Ks analysis revealed that all duplicated LpWRKY gene pairs were subjected to purifying selection during the evolution. Co-linearity analysis showed strong co-linearity between LpWRKYs and OsWRKYs from rice. Cis-acting elements in the LpWRKY promoters were related to growth and development, phytohormone responses, and stress responses. RNA-seq and qRT-PCR analysis revealed that the expression levels of LpWRKY5/16/33/35 were significantly up-regulated, while LpWRKY21/92 were significantly down-regulated in roots under heat stress. Additionally, LpWRKY12/21/33/35/43/89/92/96 displayed significant down-regulation in leaves under heat stress. It was suggested that these LpWRKYs were potentially associated with the heat-stress response in perennial ryegrass. Notably, LpWRKY33 and LpWRKY35 exhibited distinct tissue-specific transcriptional patterns in response to heat stress, implying their participation in complex tissue-dependent regulatory networks. Protein interaction prediction further suggested that LpWRKY96 may function as a potential central regulator in the heat response signaling network. Taken together, this study provides clues into the regulatory roles of the WRKY gene family in perennial ryegrass.