Structural Basis And Genetic Regulation Of Rice Grain Transparency

The appearance quality of rice is the primary trait that consumers pay attention to,and it is also an important determinant of the value of rice products.Rice grain appearance mainly relates to rice grain shape,transparency,and chalkiness.The genetic regulation mechanism of rice transparency formation is complex,in which moisture content and amylose content(AC)are key influencing factors.Recently,studies found that the Wx gene which involved in the regulation of AC synthesis is directly related to rice grain transparency.Besides,the allelic variation of this gene is the direct cause of the rich variation of AC among rice varieties,and it is also the main factor determining the appearance of waxy,dark and transparent endosperm in rice varieties.In order to clarify the starch structure basis of rice transparency formation,a series of rice varieties with different ACs and transgenic plants with Wx site-directed mutation were generated and used for rice grain transparency,starch physicochemical and structural analysis.On the other hand,based on previous studies,that transgenic rice with low amylose content but improved grain transparency,we constructed a series of transgenic rice lines carrying different Wx alleles and SSⅡ-2 and SSⅡ-3 constructs.And rice grain from the above lines were used for grain transparency and starch structure analysis.The main results are summarized as follows:1.Rice varieties with different ACs and transgenic plants with Wx site-directed mutation were used for grain transparency analysis.By using moisture gradient drying treatment,we found that rice grain transparency is positively correlated with moisture content.Then the grain starch structure analysis showed that under similar moisture conditions,the higher the AC,the higher the grain transparency.Besides,scanning electron microscope(SEM)observation of starch granular showed that grain transparency was negatively correlated with numbers of cavities within a single starch granule,as well as the cavity area.Also the crystal structure of starches with high and low moisture were compared and we found that the starch crystallinity under high moisture conditions is significantly higher than that of dry starch,and the differences is more significant for rice starch with low AC.2.To further investigate factors affecting rice grain transparency,The japonica rice Nipponbare(Nip)and its Wx near-isogenic lines(NILs)Nip-Wxmw and Nip-Wxmp were used for comparison of grain appearance quality.The results show that there is a positive correlation between rice transparency and moisture content.Besides,starch grain structure analysis shows that under similar moisture conditions,the higher the AC,the better the grain transparency.Also,SEM observation of starch structure showed that the grain transparency was negatively correlated with of cavity number within a single starch granule,as well as the cavity area.Then the starch crystal structure was compared between different NILs.The results showed that the starch crystallinity under high moisture conditions is significantly higher than that of dry starch,and the differences is more significant for rice starch with low AC.The starch granular structure during endosperm development wee compared by using SEM,and we found that the cavity within a single starch granular increased gradually with the development process,and the cavity area showed a significant negative correlation with AC.3.To study the effects of Wx alleles combined with other starch synthesis related genes on rice grain transparency,the transgenic rice Nip-SSII-2 RNAi was used as the donor parent and crossed with the low AC recipient Nip-Wxmw Nip-Wxmp to generate rice lines Nip-Wxmw/SSⅡ-2 RNAi and Nip-Wxmp/SSⅡ-2 RNAi under Nip background.Meanwhile,the gene editing lines Nip-Wxmw/SSⅡ2(I1A)and Nip-Wxmp/SSⅡ-3(D2A)were generated by crossing NILs with the SSⅡ-2 and SSⅡ-3 mutants generated previous by Crispr/Cas9 system.Firstly,we analyzed the basic agronomic traits of the transgenic rice lines and found that there were no significant differences between the transgenic lines and the corresponding parents.Secondly,the grain quality traits were analyzed and the results showed that the introduction of SSⅡ-2 RNAi construct under the background of three Wx alleles Wxb(Nip),Wxmw and Wxmp could significantly reduce the rice AC of by 5-7%.Results from the gradient drying treatment showed that the rice grain transparency is positively correlated with the moisture content.Besides,we found that the introduction of SSⅡ-2 RNAi construct can significantly improve rice grain transparency under lower AC conditions(7-8%),but when rice grain with very low AC(～5%),the the improved effect is limited.Then the AC of rice grain from Nip-Wxmp,Nip-Nip-Wxnw/SSII-2(I1A)and Nip-Wxmp/SSⅡ-3(D2A)measured and we found no significant differences between the three lines.However,the transparency of rice grain from Nip-Nip-Wxmp/SSⅡ-3(D2A)lines increased than others,while the transparency of rice grain from Nip-Wxmw/SSⅡ-2(I1A)rice is not improved.The above rice grains during the development of endosperm from different lines were used for SEM analysis and the results showed that the cavity within starch granduncle gradually increased with the grain development,and the cavity area was significantly negatively correlated with AC.Besides,the starch crystal structure analysis showed that starch crystallinity is positively correlated with moisture content.