The Roles Of Rice Sugar Transporter OsSTP26 In Regulating Rice Quality

Rice(Oryza sativa L.)is the main food source for half of the world’s population.With the improvement of living standards,people are no longer satisfied with food and clothing,but to improve the requirement of rice quality.Therefore,rice quality improvement has become the focus in agricultural production and food processing.Endosperm is the mainly edible part of rice grain.Endosperm filling process is mainly based on the monosaccharides or oligosaccharides transfer from photosynthetic tissue to the endosperm for starch synthesis,which is an important process to determine the rice quality.Sugar transporter protein(STP)is a key regulatory factor to maintain efficient sugar transport and utilization during plant growth and development.However,its roles in rice quality regulation are not clear.It has been reported that the expression level of OsSTP26 in rice seed is higher than that of other STP family members,which may play an important role in rice grain development and storage substances accumulation.Therefore,this study systematically analyzed the role of OsSTP26 in rice quality regulation,which can provide new clues for the improvement of rice quality and rice nutrition.The main findings are as follows:1.Bioinformatic analysis and expression pattern of OsSTP26.In this study,bioinformatic analysis of the OsSTP26 revealed that the promoter region of OsSTP26 contains a cis-element associated with seed development.The N-terminal of OsSTP26 protein has a conserved SUGAR TRANSPORT domain contains in the MFS superfamily.Subcellular localization experiments have shown that OsSTP26 is mainly localized to the cell membrane,with a small amount localized to the nucleus.RT-qPCR results revealed that OsSTP26 was mainly expressed in stems,panicles,leaf sheaths,leaves and seeds,but less in roots.GUS-reporter transgenic plants were further constructed using the OsSTP26 promoter,and their tissue staining also revealed that OsSTP26 was mainly expressed in root,stem,leaf,panicle and seed,especially in the vascular tissue of the seed at the filling stage.In addition,the expression of OsSTP26 was obviously increased when treated with exogenous sugar solution,indicating that OsSTP26 plays an important role in sugar transport.2.Phenotype analysis of osstp26 mutants and OsSTP26 overexpressing lines.In this study,two independent osstp26 homozygous mutant were successfully obtained via the CRISPR/Cas9 system.Compared with the wild-type,osstp26-1 and osstp26-2 mutants showed 11.8%and 10.7%reduction in plant height,37.5%and 25.0%reduction in tiller number,8.9%and 8.8%reduction in 1000-grain weight,respectively.The osstp26-1 and osstp26-2 mutants had shorter grain length and the rice chalkiness were increased from 16.7%to 51.7%and 55.0%,respectively.These results indicate that knockout OsSTP26 decreased rice yield and seed appearance quality.OsSTP26 overexpressing lines were constructed by using the maize Ubi promoter and two independent lines were successfully obtained.Compared with the wild type,OsSTP26-GFP-3 and OsSTP26-GFP-9 overexpressing lines showed 1.8%and 6.9%increase in plant height,16.7%and 65.6%increase in tiller number,1.8%and 2.5%increase in 1000-grain weight,respectively,but no significant chalkiness,indicating that the overexpression of OsSTP26 is beneficial to the improvement of rice yield and appearance quality.3.Cooking and eating quality analysis of osstp26 mutants and OsSTP26 overexpressing lines.The cooking and eating quality of osstp26 mutants and OsSTP26 overexpressing lines were further examined and analyzed in this study.Compared with the wild type,the total starch content of the two osstp26 mutants decreased by 2.3%and 1.9%,the soluble sugar content decreased by 78.3%and 75.7%,and the protein content decreased by 12.1%and 10.7%,respectively.In contrast,the two OsSTP26-GFP overexpressing lines increased total starch content by 4.4%and 15.7%,soluble sugar content by 69.0%and 41.4%,and protein content by 4.6%and 23.4%,respectively.In addition,the amylose content was significantly reduced in both the osstp26 mutants and OsSTP26-GFP overexpressing lines.Textural analysis showed that the osstp26 mutants had lower hardness and viscosity than the wildtype,while the OsSTP26-GFP overexpressing lines had significantly higher hardness than the wild-type.The gelatinization and pasting properties showed that the enthalpy of osstp26 mutants was significantly higher than wild-type,while the pasting temperature and enthalpy of OsSTP26-GFP overexpressing lines were significantly decreased.These results indicate that the overexpression of OsSTP26 is beneficial to the accumulation of endosperm storage substances and the improvement of cooking and eating quality.4.Molecular mechanism of OsSTP26 in regulating rice quality.In order to investigate the molecular mechanism of OsSTP26 in regulating rice quality,17 proteins that may interact with OsSTP26 were screened by using the yeast two-hybrid library system.The interaction between OsSTP26 and F-box protein OsFBX28,rice calcium-dependent protein kinase OsCPK21 and α-amylase KCG211D10 were further identified via one-by-one yeast two-hybrid analysis.It is speculated that OsSTP26 may interact with these three proteins to regulate rice seed development and rice quality.