The OsMADS18-OsbZIP60 module plays a critical role in influencing grain chalkiness in rice.

Grain chalkiness is a detrimental trait that significantly reduces the quality and economic value of rice, yet its regulatory mechanisms remain poorly understood. To identify genetic loci regulating grain chalkiness, we performed high-throughput activation tagging-based T-DNA insertion mutagenesis in the model rice variety (Oryza sativa L.) Kitaake. We found that a loss-of-function mutation in OsMADS18 resulted in enhanced grain chalkiness, reduced plant height, and delayed flowering time. Through yeast two-hybrid (Y2H) screening, we identified OsbZIP60 as an interacting partner of OsMADS18. Further analysis revealed that OsMADS18 directly bound to and positively regulated the expression of chalkiness-related genes, including Chalk5, FLO7, OsPK2, and OsNF-YB1, which are also transcriptional targets of OsbZIP60. Genetic analysis revealed that the osbzip60-1 osmads18-1 double mutant phenocopied the chalkiness of osbzip60-1, with more severe defects than osmads18-1, placing OsbZIP60 downstream of OsMADS18 in grain chalkiness control. Additionally, OsMADS18 positively regulates grain weight, starch content, amylose content, and protein levels under high-temperature conditions by modulating the expression of unfolded protein response (UPR) genes, including OsbZIP50, OsBiP1, OsBiP2, OsBiP3, OsBiP4, and OsBiP5. The double mutant showed increased chalkiness under heat stress, underscoring the role of the OsMADS18-OsbZIP60 interaction in thermotolerance. These findings indicate that OsMADS18-mediated UPR, enhanced by its interaction with OsbZIP60, is critical for mitigating grain chalkiness under stress. We propose a novel model wherein the OsMADS18-OsbZIP60 transcriptional module centrally governs this trait in rice.