Moderate Regulation of <i>SBEIIb</i> Gene Expression by Editing Cis‐Regulatory Elements Enhances the Resistant Starch Content in Rice

As the staple food of over half the world's population, rice with a high resistant starch (RS) content may be beneficial in the dietary prevention of diabetes and hyperlipidemia. Mutating key enzymes in starch metabolism to increase the RS content has been successful in other crops. However, there are always adverse effects, such as low grain weight, wrinkled seeds, and powdery endosperm that accompany the high RS content in rice. Here, we identified two cis-regulatory elements (CREs) related to SBEIIb by combining assay-for-transposase-accessible-chromatin sequencing, DNAase I hypersensitive site sequencing and bioinformatics analysis. The RS content of cre mutants increased by ~5.31-fold compared to the wild type, but the starch morphology, grain shape, and major agronomic traits of the cre mutants were not affected. Analysis of the starch structure revealed that the increase in medium- and long-chain amylopectin contributed to the high RS phenotype of the cre mutants. Broad transcriptomics and metabolomics showed some changes in sugars, lipids, and amino acids in the mutant endosperm, suggesting that regulation of the starch metabolism pathway triggers the distribution of other metabolic pathways, but with a relatively small difference. The genetic study revealed that transcription factors NAC20, NAC23 and NAC26 bind to the CRE1 and CRE2 sequences, co-regulating SBEIIb gene expression. These results provide support for mildly developing high-RS varieties of rice, and possibly of other cereals.