Chloroplast CASEIN KINASE 2 enhances maize thermotolerance via sHSP26 phosphorylation and retrograde HSFA3 regulation

Chloroplasts are highly sensitive to heat stress. While we have previously demonstrated that chloroplast-localized small heat shock protein 26 (sHSP26) protects maize (Zea mays) photosynthetic systems from thermal damage and undergoes heat stress-induced phosphorylation, the responsible kinases and functional significance of this phosphorylation remain unknown in maize thermotolerance. In this study, we identify a chloroplast-localized casein kinase 2 (cpCK2) in maize. ZmcpCK2 expression is up-regulated by ABA and heat stress. Genetic evidence reveals that Zmcpck2-KO lines exhibit more severe chloroplast structural damage, reduced ABA content, and increased heat sensitivity compared to wild-type plants under thermal stress. Notably, ABA pretreatment restores thermotolerance in knockout lines to wild-type levels. Furthermore, biochemical characterization demonstrates that ZmcpCK2 specifically phosphorylates sHSP26 at Ser32. Structure-function analysis shows that while the phospho-dead mutant sHSP26S32A maintains normal subcellular localization and protein interactions, its overexpression confers significantly less protection of photosystem II (PSII) activity and chloroplast protein stability during heat stress compared to the wild-type sHSP26. Integrated phosphoproteomic and transcriptomic analyses reveal that ZmcpCK2 dysfunction triggers retrograde signaling that regulates the expression of nuclear-encoded genes, including those involved in ABA biosynthesis, sHSP26, and heat shock factor HSFA3. Importantly, we establish a regulatory cascade where HSFA3 activates sHSP26 expression by directly binding its promoter, while ABA positively regulates HSFA3 and sHSP26 expression. Our findings elucidate a crucial ZmcpCK2-ABA-HSFA3-sHSP26 module in maize chloroplast thermotolerance, providing valuable molecular targets for developing heat-resistant maize varieties.