The ss3a/be2b double mutant yields high resistant starch through cooperative regulation of starch fine structure in indica rice.

Rice starch biosynthesis is governed by multiple enzymes, among which soluble starch synthase IIIa (SSIIIa) and starch branching enzyme IIb (BEIIb) play critical but distinct roles in determining starch fine structure. However, their combined effects on starch fine structure and physicochemical properties are not fully understood. Herein, we generated a double mutant ss3a/be2b in the indica variety GLA4 by crossing a radiation-induced SSIIIa mutant with a CRISPR/Cas9-generated BEIIb knockout mutant. The double mutant displayed a floury endosperm with irregular starch granules, an amylose content of 50.8% (on starch basis) and a high resistant starch content of 5.92% (on flour basis), approximately 2.7-fold that of the wild type. Its amylopectin chain-length distribution resembling that of ss3a, characterized by increased A and B2 chains and decreased B1 and B3 chains, which resulted in a mixed B- and V-type crystalline pattern and markedly decreased relative crystallinity. The gelatinization temperatures of ss3a/be2b were comparable to those of the wild type, but it showed lower gelatinization enthalpy and extremely low pasting viscosities. These findings demonstrate that SSIIIa and BEIIb cooperatively but non-redundantly regulate starch fine structure and functionality. The double mutant provides a promising genetic resource for developing high resistant starch rice with potential health benefits.