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

1. A novel OsSBEIIb allele was identified in an indica rice mutant. 2. The OsSBEIIb mutation increases resistant starch (RS) content in raw rice flour, as well as in retrograded and heated rice. 3. The chalk2 mutant serves as a valuable indica rice germplasm for breeding high-RS rice varieties. High-resistant starch rice 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 high resistant starch content remains a major challenge. In this study, we identified a mutant, chalk2 , with increased chalkiness from the mutant library of indica rice ZJ100. The chalk2 mutants exhibited significantly higher amylose and protein contents, while total starch and lipid contents were reduced. 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 decreased 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 phynotypes, a member of the starch branching enzyme (SBE) family, specifically expressed in the endosperm. Furthermore, the expression levels, enzyme 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.