SBEIIb is responsible for the chalk2 phenotype by regulating the formation of resistant starch in indica rice

Rice high in resistant starch is a valuable food for human health, especially for individuals with type 2 diabetes, as it supports effective blood sugar control and provides cardiovascular and intestinal benefits. However, developing rice varieties with a high resistant starch content remains a major challenge. In this study, we identified a mutant with increased chalkiness, chalk2, from the mutant library of indica rice Zhongjian 100. The chalk2 mutants exhibited significantly higher amylose and protein contents, while the total starch and lipid contents were reduced. An analysis of resistant starch in chalk2 revealed substantial increases in two resistant starch (RS) types, RS2 and RS3. Electron microscopy revealed abnormal starch granule development in the endosperm. The chalk2 mutant also showed reduced grain length, width, and thickness, as well as a lower seed-setting rate, which ultimately led to a significant reduction in grain yield. Through physical localization, Mut-Map analysis, and transgene complementation, we found that SBEIIb was responsible for the chalk2 phenotypes, and it is a member of the starch branching enzyme (SBE) family specifically expressed in the endosperm. Furthermore, the expression levels, enzymatic activity, and protein abundance of SBEIIb were significantly reduced in chalk2 mutants. These findings suggest that SBEIIb plays a crucial role in regulating the composition of starch and resistant starch formation in indica rice.