Multifaceted Effects of Starch Branching Enzyme and Soluble Starch Synthase Gene Editing in Rice with Different Wx Genotypes.

Rice endosperm, the major edible portion of the grain, plays an important role in regulating blood glucose and preventing intestinal diseases by increasing its resistant starch (RS) content. Previous studies have shown that suppressing amylopectin biosynthesis via genome editing can increase RS content. However, the influence of different Waxy (Wx) allelic backgrounds on RS accumulation in edited lines has not been systematically evaluated. In this study, we used glutinous rice Yunan Heixiangnuo (HXN) with a nonfunctional wx allele and indica rice Yixiang 1B (YX1B) with a weak Wxb allele as backgrounds. We simultaneously knocked out SSSIIIa, SBEI, SBEIIa, and SBEIIb using CRISPR/Cas9, and systematically analyzed changes in RS content, rice quality, and yield traits. The results showed that, in the HXN background, multigene knockout did not significantly alter amylose or RS content but largely maintained favorable eating quality. In contrast, in the YX1B background, quadruple-gene knockout lines exhibited an increase in amylose content from 17.7% to 53.7% and an increase in RS content to 2.48%, representing a 4.35-fold increase over the wild type, while gel consistency and seed-setting rate were significantly reduced. Scanning electron microscopy revealed that multigene knockout markedly remodeled starch granule structure, shifting from dense polygonal granules to loosely packed spherical particles and resulting in a floury endosperm. Collectively, enhancement of RS content through multigene editing was influenced by Wx gene function. Although the wx allele failed to increase RS content, it still participated in the regulation of grain quality and yield traits by modulating starch structure. This study provides a reference for breeding high-RS rice cultivars while balancing yield performance and eating quality.