Natural haplotype variation in OsGSK3 fine-tunes grain length via phosphorylating and stabilizing OsRanBP1 in rice

Rice (Oryza sativa L.) is a staple cereal crop whose grain size substantially influences yield. The kinase OsGSK3, a known negative regulator of brassinosteroid (BR) signaling, was previously shown to modulate grain length. Here, we characterize the functional divergence between its two major haplotypes, OsGSK3^Hap3 and OsGSK3^Hap5. A single-nucleotide polymorphism in OsGSK3^Hap5 extends the OsGSK3 open reading frame, adding 13 C-terminal amino acids and correlating with longer grains, whereas OsGSK3^Hap3 is associated with shorter grains. Consistently, overexpression of OsGSK3^Hap3 reduced grain size, while overexpression of OsGSK3^Hap5 or knockout of OsGSK3^Hap3 increased it. Both haplotypes physically interact with OsRanBP1, a positive regulator of grain length. Biochemical analyses demonstrated that OsGSK3^Hap3 possesses stronger kinase activity than OsGSK3^Hap5 and more efficiently phosphorylates OsRanBP1, triggering its degradation and thereby suppressing grain length. Conversely, the weaker kinase activity of OsGSK3^Hap5 results in attenuated OsRanBP1 phosphorylation and accumulation, promoting longer grains. Genetic and physiological evidence further demonstrates that OsGSK3 regulates rice grain development within the BR signaling pathway in an OsRanBP1-dependent manner. Our findings reveal that natural variation in OsGSK3 differentially fine-tunes grain size by modulating OsRanBP1 protein stability via phosphorylation, thereby providing a molecular mechanism underlying grain size diversity in rice.