LpDREB2A-LpbZIP41/LpbZIP66 module orchestrates heat stress tolerance by modulating ABA biosynthesis and carbonic anhydrase activity in perennial ryegrass.

Heat stress is a major environmental constraint limiting the productivity of perennial ryegrass (Lolium perenne), a widely cultivated forage and turfgrass. Here, through a genome-wide association study (GWAS) analysis of a diverse perennial ryegrass population, we identified LpbZIP41 as a major heat tolerance quantitative trait locus (QTL) for heat tolerance. Natural variation at this locus comprises three haplotypes showing differential thermotolerance, with evidence of positive selection during breeding. LpbZIP41 localizes to the nucleus and exhibits rapid heat-inducible expression. Functional validation via CRISPR-Cas9-mediated knockout confirmed that LpbZIP41 is essential for heat stress. Overexpression of LpbZIP41 in perennial ryegrass enhanced thermotolerance, as evidenced by improved survival, reduced lipid peroxidation, maintained membrane integrity, and global transcriptional reprogramming under heat stress. Mechanistically, we uncovered a transcriptional cascade that LpDREB2A directly activates LpbZIP41 transcription by binding to dehydration-responsive element (DRE) motifs in the promoter. LpbZIP41, in turn, regulates LpNCED4, a key abscisic acid (ABA) biosynthesis gene, and LpβCA2, encoding a stress-responsive carbonic anhydrase. Furthermore, LpbZIP66 functions as a negative regulator that antagonizes LpbZIP41 activity via heterodimerization. Collectively, our findings established an LpDREB2A-LpbZIP41/LpbZIP66 regulatory module as a central hub that integrates ABA biosynthesis and metabolic signals to confer heat stress tolerance in a cool-season grass.