The natural variation allele OsGSW3.2 in Oryza rufipogon is involved in brassinosteroid signaling and influences grain size and weight.

Oryza rufipogon is the ancestor of cultivated rice and harbors many elite genes; thus, this plant is an important germplasm for improving rice varieties. Grain size is an important factor in determining rice yield and quality. In this study, we identified a natural variation allele from the O. rufipogon inbred line Huaye3 (HY3), which is located on chromosome 3 and named it GRAIN SIZE and WEIGHT 3.2 (OsGSW3.2). The OsGSW3.2 knockout (KO) mutant presented increased grain size and weight, which was associated with decreased chlorophyll content and long awns. The overexpression of OsGSW3.2(HY3) caused a significant decrease in grain size and weight. OsGSW3.2 negatively regulates grain size through cell proliferation. Transcriptomic analysis of spikelet hulls from the KO lines and wild-type HY3 revealed that the differentially expressed genes (DEGs) were enriched mainly in plant-pathogen interactions, plant hormone signal transduction, and the plant MAPK signaling pathway, and so on. A laminar inclination experiment verified that OsGSW3.2 was involved in the BR signaling pathway. Yeast two-hybrid, BiFC, LAC, and pull-down experiments verified that OsGSW3.2 interacted with OsGSK4, which was related to BR signaling, in yeast and plant cells. OsGSW3.2 influenced rice grain size and weight via interaction with OsGSK4. Haplotype analysis of a core collection of cultivated rice revealed that transcriptional accumulation and differential SNPs in the coding region may influence grain size and weight. Our results provide new insight into the role of OsGSW3.2 in affecting grain size and weight, which will help elucidate the genetic basis of rice domestication.