Promoter Editing of Starch Branching Enzyme IIb and Granule-Bound Starch Synthase I Balances Resistant Starch Content and Amylose Content in Rice

The resistant starch content (RSC) of a starch-rich food product is an important indicator of its glycemic index (GI). Foods with high RSC have low GI and are beneficial to human health. In recent years, some studies revealed that the loss function of single or multiple key enzymes in ‌the primary pathway of starch synthesis substantially increases RSC in rice, a staple food for about half of the world population. However, a noteworthy negative characteristic of these high RSC mutants is the substantially increased apparent amylose content (AAC). AAC as a major determinator of rice eating quality, must not be higher than an acceptable limit for most consumers. To date, the research on optimizing AAC or taste quality of high RSC rice was scarcely reported. To address this issue‌, two promoter editing (PE) strategies were adopted in this study to develop rice germplasms with a better balance between RSC and AAC: one is to edit the promoter of BEIIb in a low AAC rice variety, another is to edit the promoter of Waxy ( Wx ) gene in a BEIIb function-defective mutant. Using AAC ≤ 20%, which is the range of premium quality rice in China as the criterion, two homozygous lines with significantly increased RSC (≥ 5%) in the NG46 background by promoter editing of BEIIb and one homozygous line in the YT2 background by promoter editing of Wx gene were finally obtained. Further analysis revealed that the proportion of long-chain branches of amylopectin exhibited a stronger positive correlation with RSC than AAC in the population of BEIIb PE lines, even when wild-type data are taken into consideration. Furthermore, unexpectedly, a Wx PE-line with substantially decreased AAC (17.7%) but increased long‑chain amylopectin proportion also showed significantly increased RSC (16.9%). These data suggest that, rather than AAC, long-chain amylopectin is more critical for RSC in rice. Our study not only produces useful germplasms for high RSC rice breeding in the future but also provides an insight into understanding the relationship between starch molecular structure and RSC in defective BEIIb rice.