RESUMEN
KEY MESSAGE: Coordinated regulation of amylose and amylopectin synthesis via manipulation of SSII-2, SSII-3 and Wx expression in endosperm can improve rice eating and cooking quality. With increasing rice consumption worldwide, many researchers are working to increase the yield and improve grain quality, especially eating and cooking quality (ECQ). The rice ECQ is mainly controlled by the expression of starch synthesis-related genes (SSRGs) in endosperm. Although the Wx and SSII-3/SSIIa/ALK genes, two major SSRGs, have been manipulated to improve rice ECQ via various breeding approaches, new methods to further improve ECQ are desired. In our previous study, we enhanced rice ECQ by knocking down SSII-2 expression in the japonica Nipponbare cultivar (carrying the Wxb allele) via RNA interference. Herein, the SSII-2 RNAi was introduced into two Nipponbare-derived near-isogenic lines (NILs), Nip(Wxa) and Nip(wx), carrying Wxa and wx alleles respond for high and no amylose levels, respectively. Analysis of physicochemical properties revealed that the improved grain quality of SSII-2 RNAi transgenic lines was achieved by coordinated downregulating the expression of SSII-2, SSII-3 and Wx. To further confirm this conclusion, we generated ssii-2, ssii-3 and ssii-2ssii-3 mutants via CRISPR/Cas9 technique. The amylopectin structure of the resulting ssii-2sii-3 mutants was similar to that in SSII-2 RNAi transgenic lines, and the absence of SSII-2 decreased the amylose content, gelatinisation temperature and rapid visco-analyser profile, indicating essential roles for SSII-2 in the regulation of amylopectin biosynthesis and amylose content in rice endosperm. The effect of SSII-2 was seen only when the activity of SSII-3 was very low or lacking. Our study provides novel approaches and valuable germplasm resources for improving ECQ via plant breeding.