OsSPL14 and OsNF-YB9/YC8-12 subunits cooperate to enhance grain appearance quality by promoting Waxy and PDIL1-1 expression in rice

The identification of seed development-related regulators is critical for the genetic improvement of yield and grain quality in cereal crops. SQUAMOSA PROMOTER BINDING PROTEIN-LIKE14 (OsSPL14) is a well-studied, plant-specific transcription factor; however, its roles in controlling rice grain appearance quality and the underlying molecular mechanisms have not been fully elucidated. In this study, we demonstrate that OsSPL14 positively regulates appearance quality by controlling grain chalkiness in rice. Genetic analysis revealed that knockdown or knockout of OsSPL14 leads to a chalky grain phenotype, which is associated with significant defects in compound starch granules and notable changes in both starch and protein contents in the endosperm. Transcript analysis identified multiple genes regulated by OsSPL14, including the key granule-bound starch synthase gene Waxy (Wx) and the protein disulfide isomerase-like enzyme-encoding gene PDIL1-1. Both in vitro and in vivo assays demonstrated that OsSPL14 directly binds to the GTAC-box motif in the Wx and PDIL1-1 promoters to enhance their expression. Protein-protein interaction experiments further revealed that OsSPL14 interacts with the nuclear transcription factor Y (NF-Y) heterodimer OsNF-YB9/YC8-12 to promote the transcription of Wx and PDIL1-1, thereby enhancing rice grain appearance quality. Our findings uncover a novel regulatory pathway controlled by OsSPL14 and provide new insights into the molecular mechanisms underlying rice grain appearance quality, with promising implications for genetic improvement in rice.