Fine-tuning Of Wx And SSII Expression To Improve Rice Eating And Cooking Quality

Eating and cooking quality(ECQ),the most concerned aspect of consumers,breeders,researchers and farmers,is the most important part of rice grain quality.Physicochemical properties of starch are directly correlated to the formation of rice ECQ.Rice with lower amylose content(AC),lower gelatinization temperature(GT)and higher gel consistency(GC)tend to have higher ECQ.Among the starch synthesis-related genes(SSRGs),Wx gene encoding GBSSI(granule-bound starch synthase ?)is considered as the major determinant of rice ECQ,with major roles in controlling AC and GC,and minor role in controlling GT.The natural allelic variations of Wx in different rice varieties have led to the wide variations in rice grain AC and ECQ.The application of elite Wx natural alleles has contributed a lot for rice ECQ improvement.Recently,the mining of natural allelic variation of Wx has entered a bottleneck stage,while the defects of existing Wx alleles have gradually caused the attention of breeders and researchers.To prevent the high homogeneity of high-ECQ rice breeding and meet the various requirements of consumers from different areas,novel Wx alleles are urgently required by breeders to further fine-tune and improve rice ECQ.The development of CRISPR/Cas technology makes it possible to achieve this aim.SS? genes,including SS?-1,SSII-2 and SSII-3,are the coding genes of soluble starch synthase ?(SS?),which can regulate rice ECQ by modulating the ratio of amylopectin A-chain and B1-chain.Previous research on SSII genes mainly focused on SS?-3(also known as ALK)gene,which has been proved to be the major gene controlling GT.However,our previous study showed that down-regulation of SSII-2 expression by RNA interference(RNAi)could result in improved rice ECQ,indicating that SSII-2 also plays an important role in the formation of rice ECQIn this study,we used CRISPR/Cas9 system to edit the key sites in the promoter and the non-catalytic coding region of Wx gene,hoping to fine-tune rice grain AC by regulating Wx gene expression or GBSSI activity.We also used hybridization and CRISPR/Cas9 technology to reveal the function of SS?-2 and its role in the formation of rice ECQ,so as to harvest a novel target gene for rice ECQ improvement.The main findings are as follows:1.Based on the analysis of the cis-acting elements in Wx promoter and the domains in GBSSI protein,we selected nine target sites(S1-S9)in both of Wx promoter and 3'-end of Wx coding sequence for genome editing in japonica Nipponbare(Wxb).The results of apparent amylose content(AAC)showed that the AAC of S7-edited lines were significantly decreased,indicating S7-editing may has the potential to improve rice ECQ.We finally obtained six homozygous S7-edited lines with fine-tuned AC but without T-DNA insertion,designated as Wxb-C/A,Wxb-A/C,Wxb-i1,Wxb-d2,Wxb-d8 and Wxb-d15.Under the winter planting conditions in Hainan,the novel Wx alleles showed decreased AC distributed gradiently between the conventional japonica rice(Wxb)and soft rice(Wxmp),but their grains remained transparent instead of the opaque endosperm as shown in those of soft rice cultivars.2.Data from Wx gene expression and in vitro promoter activity analysis showed that the AC declines of the novel Wx alleles are mainly caused by the reduction of Wx transcription,suggesting that editing the S7 site in Wx gene core promoter can effectively manipulate Wx gene expression.We speculate that editing the S7 site in Wx core promoter may destroy one or some recognition or binding sites of general transcription factors(GTFs)in RNA polymerase ? core transcription mechanism,thus resulted the lower basic transcription level of Wx.Sequence alignment of S7 and core promoter of Wxnatural alleles showed that the S7 site is highly conserved in the Wxlv,Wxa,Wxin,Wxmp and Wxop alleles,indicating that a similar strategy could be adopted to create more novel Wx alleles with a wider range of grain AC.These results provide a new method for further enriching variations of rice grain quality.3.The S7-edited lines exhibited different AC in different growth seasons.In the winter in Hainan,the AC of six S7-edited lines declined markedly compared with the wild type.However,no significant or slight changes were observed in AC in S7-edited lines grown in the summer at Yangzhou.The temperature treatment experiment proved that the temperature difference is the main reason for the AC difference,indicating that manipulation of Wx gene expression by S7-editing was responded to temperature.We speculate that this was caused by the reduced thermal stability of the recognition and/or binding of GTFs to Wx core promoter.In-depth exploration of the mechanism provides possibilities to breed high-ECQ and extreme temperature-insensitive rice varieties in the future.4.In this study,we designed a gradient drying experiment to monitor and compare the grain appearance of SS?-2 RNAi transgenic lines and soft rice controls with similar AC.After drying,the grains of both SS?-2 RNAi transgenic rice and the wild-type Nip(Wxb)remained transparent,and the starch granule morphology was similar to that observed before drying;both exhibited intact and polyhedral shapes,packed tightly together.However,the endosperms of both Nip(Wxmp)and Nip(wx)grains became opaque,and we also observed some holes in the core of the starch granules,with a larger number of holes in Nip(wx).We believe that the formation of holes in starch granules after drying might lead to light refraction and thus cause the opaque endosperm of soft or waxy rice.The relative crystallinity showed significant increments in the SS?-2 RNAi lines when compared with Nip(Wxb),which could be the reason for no holes in their starch granules.5.The SS?-2 RNAi was introduced into two Nipponbare-derived near-isogenic lines Nip(Wxa)and Nip(wx)by crossong.The physicochemical properties analysis results showed that inhibiting the expression of SS?-2 can synergistically regulate the synthesis of both amylose and amylopectin to improve ECQ in non-waxy rice and modulate the synthesis of amylopectin with decreased GT in waxy rice,indicating that Wx gene was involved in the grain quality formation of SS?-2 RNAi transgenic rice.The ssii-2,ssii-3 and ssii-2ssii-3 mutants were further created through CRISPR/Cas9.Although the AC of the ssii-2ssii-3 mutant was not reduced,its amylopectin structure,GT and RVA profile showed high similarity with those of SS?-2 RNAi transgenic rice,suggesting that SS?-3 gene was also involved in the grain quality formation of SSII-2 RNAi transgenic rice.Thus,the improved grain quality of SS?-2 RNAi transgenic lines was resulted from the coordinately down-regulated expression of SS?-2,SS?-3 and Wx genes,hence enhanced rice ECQ.6.The absence of SS?-2 caused mild decrease in AC,GT and RVA profile,as well as slight increase in the ratio of amylopectin chains with DP 10-12 and DP>34,and slight decrease in the ratio of amylopectin chains with DP 6-9 and DP 13-33.Besides,SS?-2 mutation also caused a significant increase in starch crystallinity and significant expression reductions of Wx and SS?-3.These results suggested that SS?-2 plays an important role in the synthesis of both arnylose and amylopectin in rice endosperm.Thus,SS?-2 could be a novel biotechnological target for improving both ECQ and appearance quality of rice.