Gain-of-function OsGLW3 : A dominant positive regulator of grain length improving hybrid rice quality

Size is a critical determinant of rice grain appearance quality and yield. In this study, we isolated a dominant mutant of rice grain size, RE10498, from an EMS-induced mutant library of ZH8015 characterized by long grains. Using the MutMap method, we cloned the causal gene, OsGLW3, which encodes an expressed protein of unknown function. Targeted editing of position 3119 in the OsGLW3 coding region to 'A' in the wild-type ZH8015 produced positive transgenic lines whose grain size matched that of RE10498. Knocking out OsGLW3 in the wild-type ZH8015 did not alter the grain size of positive plants (KO-OsGLW3ZH), whereas knocking out OsGLW3 in the mutant RE10498 resulted in decreased grain length and increased grain width in its positive plants (KO-OsGLW3RE), reverting to a phenotype consistent with the wild-type. Overexpression of the OsGLW3RE allele in ZH8015 resulted in a highly significant increase in grain length. These findings indicate that RE10498 is a gain-of-function mutant. OsGLW3RE positively regulated the level of brassinolide, which affected grain size by regulating the proliferation and extension of glume cells. Crossing ZH8015 and RE10498 with the three-line sterile lines Fu387A (F387A), Nei5A (N5A), and Zhongxiang131A (ZX131A) showed that the hybrids F387A/RE10498, N5A/RE10498, and ZX131A/RE10498, respectively, exhibited improved rice appearance quality: longer grains with reduced chalkiness. The OsGLW3RE allele identified in this study, along with its functional marker dCAPs-G, holds significant reference value for breeding high-quality hybrid rice varieties.