Inactivation of SSIIIa enhances the RS content through altering starch structure and accumulating C18:2 in japonica rice.

SSIIIa was the key gene responsible for RS formation in rice endosperm. The higher RS content in ssIIIa mutant has been proposed to be majorly due to the increased amylose-lipid complexes (RS5). However, the formation of RS5 elicited by ssIIIa mutation and the importance of RS5 for total RS content in rice are still unclear. With japonica ssIIIa loss-of-function mutants created by CRISPR/Cas9 gene editing, the effects of SSIIIa mutation on RS5 were furtherly evaluated through investigating the transcriptome and metabolites. Inactivation of SSIIIa caused significant enhancement in amylose and RS content but without depletion in starch reserves. SSIIIa mutation modulated the genes involved in carbohydrate and lipid metabolisms and the redistribution of substances, led to accumulated protein, glucose, fructose, and C18:2. Besides the increased amylose content and altered amylopectin structure, the increased C18:2 contributed greatly to the enhancement in RS content in japonica ssIIIa mutants through complexing with amylose to form RS5, while the existence of lipid counted against the enhancement of RS content in indica rice. RS5 showed discrepant contributions for the total RS in rice with different genetic background. Inactivation of SSIIIa has great potential in improving RS5 content in japonica rice without great yield loss.