Genome editing of OsSBEIIb rewires starch architecture to lower predicted glycemic index and enhance protein quality in rice endosperm.

Starch branching enzyme IIb (OsSBEIIb) plays a key role in determining amylopectin structure and starch digestibility. In this study, CRISPR/Cas9 was used for splice site disruption of OsSBEIIb in the elite indica cultivar IR64, generating a truncated protein with impaired function and reduced relative expression levels. The result showed extensive remodeling of starch fine structure, including reduced short-chain amylopectin chains, increased long-chain glucan fractions, higher apparent amylose content up to 39% and elevated resistant starch (RS) levels up to 10%. These modifications were accompanied with substantial 23-unit decreased predicted glycemic index (pGI), from 80 ± 1 in SBE2b_WT to 57 ± 1 in sbe2b mutant lines. Biophysical analyses showed reduced relative crystallinity but higher gelatinization temperatures, indicating more thermally stable starch structures. Beyond starch, sbe2b mutants displayed improved nutritional profiles, with crude protein content increasing by up to 10% and elevated levels of several essential amino acids, including lysine, methionine, threonine and histidine. Metabolite and lipid profiling revealed broader endosperm metabolic shifts affecting carbohydrates, amino acids, phenylpropanoids, fatty acids and triacylglycerols. However, these nutritional benefits were offset by grain quality and agronomic trade-offs, notably increased chalkiness and reduced seed weight. Overall, OsSBEIIb disruption effectively lowers glycemic properties while enhancing protein quality through coordinated metabolic changes in rice endosperm.