Reactive oxygen species induced by heat stress during grain filling of rice (Oryza sativa L.) are involved in occurrence of grain chalkiness

Heat stress during grain filling increases rice grain chalkiness due to increased activity of alpha-amylase, which hydrolyzes starch. In rice and barley seeds, reactive oxygen species (ROS) produced after imbibition induce alpha-amylase activity via regulation of gibberellin (GA) and abscisic acid (ABA) levels during seed germination. Here, we examined whether ROS is involved in induction of grain chalkiness by alpha-amylase in developing rice grains under heat stress. To elucidate the role of ROS in grain chalkiness, we grew post-anthesis rice plants (Oryza sativa L. cv. Koshihikari) under control (25 degrees C) or heat stress (30 degrees C) conditions with or without antioxidant (dithiothreitol) treatment. The developing grains were analyzed for expression of NADPH oxidases, GA biosynthesis genes (OsGA3ox1, OsGA20ox1), ABA catabolism genes (OsABA8'OH1, OsABA8'OH2) and an alpha-amylase gene (OsAmy3E), endogenous H2O2 content and the grain quality. In grains exposed to heat stress, the expression of NADPH oxidase genes (especially, OsRbohB, OsRbohD, OsRbohF and OsRbohI) and the ROS content increased. Heat stress also increased the expression of OsGA3ox1, OsGA20ox1, OsABA8'OH1, OsABA8'OH2 and OsAmy3E. On the other hand, dithiothreitol treatment reduced the effects of heat stress on the expression of these genes and significantly reduced grain chalkiness induced by heat stress. These results suggest that, similar to cereal seed germination mechanism, ROS produced under heat stress is involved in alpha-amylase induction in maturating rice grains through GA/ABA metabolism, and consequently caused grain chalkiness.