Abstract Excessive temperatures during grain filling can compromise endosperm starch biosynthesis and decrease grain quality and yield in rice. However, the molecular mechanisms underlying these remain unclear. Here, we show that heat shock protein OsHsp40-1 interacts with and elevates the ATPase activity of OsHsp70-2 in rice. OsHsp40-1 also interacts with the key starch biosynthetic enzymes OsGBSSI and OsPPDKB and thereby enhances their stability and activity, which is essential for maintaining rice quality and grain yield under moderate high-temperature (HT) conditions. Overexpression of OsHsp70-2 and OsHsp40-1 in rice significantly improved grain quality and yield at HT. Furthermore, a haplotype analysis identified favorable alleles of OsHsp70-2 and OsHsp40-1, which could be used for improving thermotolerance in rice. Collectively, our findings reveal a novel mechanism by which OsHsp70-2–OsHsp40-1 module ameliorates the effects of HT on starch biosynthesis, providing a new strategy for genetic improvement of rice quality and yield under HT conditions.
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