Loss-of-function of SSIIa and SSIIIa confers high resistant starch content in rice endosperm

Rice (Oryza sativa L.) endosperm accumulates huge amounts of starch. Rice starch is highly digestible, potentially enhancing the occurrence of blood sugar- and intestine-related diseases such as type 2 diabetes. Resistant starch (RS) is hardly digestible in small intestine but can be converted into beneficial short-chain fatty acids in large intestine, potentially reducing the incidence of these diseases. However, it is still difficult to produce a high RS rice variety. Here, we report that simultaneous deficiency of soluble starch synthases IIa and IIIa confers high RS content in rice endosperm. The ssIIa ssIIIa exhibited higher RS content than did the ssIIIa ssIIIb, a mutant reported currently to have remarkably higher RS content than parental ssIIIa, under our experimental conditions. Loss-of-function of SSIIa and SSIIIa significantly elevated the activity of granule-bound starch synthase I and thus content of amylose. Furthermore, total lipid content increased in mutant seeds, implying that intermediate metabolites spilled out from starch biosynthesis into lipid biosynthesis. The increased amylose content and improved lipid synthesis coordinately contributed to high RS content in mutant seeds. These results further reveal the molecular mechanism of RS occurrence in rice endosperm and provide a critical genetic resource for breeding higher RS rice cultivars.