Summary The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature‐sensitive floury endosperm11‐2 (flo11‐2) mutant was isolated from ion beam‐irradiated rice of 1116 lines. The flo11‐2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole‐exome sequencing of the flo11‐2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid‐localized 70‐kDa heat shock protein 2 (cpHSP70‐2). The cpHSP70‐2 of the flo11‐2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11‐2 mutants that express the wild‐type cpHSP70‐2 showed significantly lower chalkiness than the flo11‐2 mutant. Moreover, the accumulation level of cpHSP70‐2 was negatively correlated with the chalky ratio, indicating that cpHSP70‐2 is a causal gene for the chalkiness of the flo11‐2 mutant. The intrinsic ATPase activity of recombinant cpHSP70‐2 was lower by 23% at Vmax for the flo11‐2 mutant than for the wild type. The growth of DnaK‐defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild‐type DnaK. The results indicate that the lowered cpHSP70‐2 function is involved with the chalkiness of the flo11‐2 mutant.

Chalky grains of rice are increased due to high temperature (HT) during a ripening period. However, the underlying mechanisms of the chalkiness are not well known, seemingly due to the obtuse response of wild type and lack of effective mutants. In this study, we isolated and characterized the flo11-2 mutant, which showed higher number of chalky grains than wild type under HT but quite small number of chalkiness under cool temperature as well as similar growth, development, and yield to that of the wild type. Using this high sensitivity of the flo11-2 mutant, we identified the most critical meteorological factor and developmental stage affecting chalkiness with 5 days HT treatments over 4 consecutive years. The results demonstrated that daily maximum temperature was more causative than daily mean or minimum temperatures which have been regarded as important factors before this. Besides, the developmental stage around 20 days after flowering (DAF) was most sensitive to HT rather than the early stage up to 15 DAF. In addition, we found that the flo11-2 mutant with a high chalky ratio was vulnerable to preharvest sprouting, which has never been reported before for chalky grains, but could cause significant yield and quality loss after extremely hot and dry summers followed by rainy cool autumns. The flo11-2 mutant is, therefore, a useful material for chalky grain research.