A MYB61-SWB9-KOs module regulates grain chalkiness via gibberellin biosynthesis in rice endosperm

Grain chalkiness leads to the deterioration of grain appearance quality, which affects grain processing quality and the market value of rice. Gibberellin plays a crucial role in seed germination and plant growth, but its mechanism on endosperm starch synthesis and rice grain chalkiness formation remains largely elusive. Here, we identified a grain white belly (chalkiness in the belly area of grain) gene, SWB9, which encodes a kinesin-4 protein with a conserved ATPase domain and a coiled-coil domain. The mutation of SWB9 affects the starch structure, resulting in a grain white belly. SWB9 regulates endogenous gibberellin synthesis and accumulation in endosperm by directly binding to the promoter of ent-kaurene oxidase genes (KO1, KO2 and KOL5) encoding gibberellin-biosynthetic enzymes, and negatively regulates their expression. The loss of SWB9 function resulted in higher gibberellin content in the endosperm of swb9 than that of the wild type. Besides, a MYB transcription factor, MYB61 binds to the promoter of SWB9 and activates its expression. The grain of myb61 showed the same white belly phenotype as swb9, while overexpression of SWB9 in myb61 inhibited the grain white belly phenotype. Furthermore, the exogenous GA₃ treatment showed increased grain chalkiness, and high gibberellin treatment can induce the reduced expression of MYB61, and then weaken the inhibitory effect of SWB9 on the expression of KO1, KO2 and KOL5, so as to break the homeostasis of endogenous gibberellin in the endosperm. Meanwhile, MYB61 directly binds to the promoter of amylopectin synthesis-related genes, SSIIa, BEIIb, ISA1 and PUL, at the GAMYB element and activates their expression, further affecting the distribution of amylopectin chain length. Our findings uncover a new insight into the gibberellin dose-dependent feedback regulation loop in rice endosperm that determines grain chalkiness formation.