Three transcription factors, OsNF-YC8, OsNF-YC9, and OsNF-YC10, function redundantly to promote amylopectin synthesis by activating SBE3 expression in rice endosperm.

Starch branching enzyme (SBE) is a key enzyme in starch biosynthesis that introduces branch points into starch molecules. Among its three isoforms, SBE3 plays a pivotal role, as its loss of function significantly alters starch granule structure and accumulation in rice, leading to chalky grains and reduced grain weight. However, the molecular regulatory network governing SBE3 expression remains largely unknown. In this study, three NF-YC family transcription factors-OsNF-YC8, OsNF-YC9, and OsNF-YC10-were identified as transcriptional activators of SBE3, each directly binding to the AAGAGG motif in the SBE3 promoter. Functional analysis revealed functional redundancy among these NF-YC members, as disruption of one or two genes had little effect on SBE3 expression. In contrast, simultaneous disruption of all three genes in the osnf-yc8/9/10 triple mutant resulted in a pronounced reduction in SBE3 transcription, accompanied by a substantial decrease in short-chain amylopectin with degrees of polymerization ranging from 6 to 24. The endosperm of osnf-yc8/9/10 appeared milky white, and resistant starch content was 3.7-fold higher than that of the wild type, without a significant reduction in grain weight. These results elucidate the regulatory roles of OsNF-YC8, OsNF-YC9, and OsNF-YC10 in SBE3 expression and short-chain amylopectin biosynthesis, highlighting their potential for developing functional rice cultivars.