Natural Alleles Of A UDP-Glucosyltransferase Gene Responsible For Differential Endosperm Development And Abiotic Stress Tolerance Between Upland Rice And Paddy Rice

Traditional upland rice generally exhibits insufficient grains resulting from abnormal endosperm development compared to paddy rice.The endosperm is an important part of rice seeds and the main part of nutrient storage.The main process of endosperm development is transporting the monosaccharide or oligosaccharide synthesized by photosynthesis to endosperm and synthesizing starch.Many genes and QTLs(Quantitative Trait Locus)regulating endosperm development have been reported,including sugars-transport pathway genes,starch-synthesis pathway genes and other genes,but it is little known about the mechanism of abnormal endosperm development in upland rice.To isolate the key QTL responsible for abnormal endosperm development,a traditional upland rice variety,YZN(Yanzhinuo,Oryza sativa,japonica),and a japonica paddy rice variety,YD1(Yundao1,Oryza sativa,japonica)with normal endosperm,was used as the donor parent and the recurrent parent,respectively.We obtained a set of introgression lines(ILs)by backcrossing YZN with YD1,and one of the ILs,J77 exhibited a significant decrease in 1000-grain weight,grain width,grain thickness,and grain length compared with YD1 and showed similar YZN phenotypes.By backcrossing J77 and YD1,we obtained an BC3F2 mapping population of 472 individuals.Using over 500 published SSR markers covering the whole genome,a major QTL on chromosome 5 was identified,named qEDR5.1(Endosperm Development in Rice),in the interval between two molecular markers,RM18829 and RM19121.To fine map the candidate gene for qEDR5.1,we constructed the near isogenic lines for qEDR5.1 by SSR molecular marker-assisted method,and named NIL-qEDR5.1YZN,which harbored a YZN-derived qEDR5.1 allele in the YD 1 genetic background.By phenotype and genotype linkage analysis of BC4F2 and BC4F3 populations from backcrossing NIL-qEDRS.1YZN with YD1,qEDR5.1 was finally mapped to a 48 kb region between markers C5029 and C5030.In this region,six ORFs were annotated.Through the sequence comparison of these six ORFs,we identified several sites that varied in the LOC_Os05g47950 coding region between YD1 and NIL-qEDRS.1YZN and resulted in amino acid sequence changes.The functional verification of genetic transformation materials such as mutants,over expression and complementarity,indicated that LOC_Os05g47950 is responsible for endosperm development in rice.LOC_Os05g47950 encodes a UDPglucosyltransferase,and we named it EDR1.Defects in endosperm development always cause grain quality changes.By analyzing grain quality we found that NIL-qEDR5.1YZN showed a significant decrease in protein content and transparency but an increase in grain chalkiness compared with YD1.On the other hand,SEM images of grains with high chalkiness from NILqEDR5.1YZN showed that the endosperm was filled with loosely packed spherical starch granules with large air spaces,whereas the grains with low chalkiness from YD1 consisted of densely and regularly packed polyhedral starch granules.By enzyme activity assay,we found the glucosyltransferasu activity of EDR1YD1 was more than 12 times higher than EDR1YZN,indicated that natural variation in the amino acid sequence of EDR1 greatly reduced the UDP-glucosyltransferase activity of EDRIYZN,thereby causing the abnormal endosperm development in NIL-qEDR5.1YZN.Analyses of the two alleles EDR1YD1 and EDR1YZN between paddy rice and upland rice ’showed that EDR1 was conservative in upland rice,but segregated in paddy rice.Further analyses of grain chalkiness in alleles of EDR1YD1 and EDR1YZN varieties indicated that rice varieties harbored EDR1YZN and EDR1YD1 preferentially showed high chalkiness,and low chalkiness,respectively.These results suggested that EDR1 responsible for diferential endosperm development between upland rice and paddy rice.The expression pattern showed that the EDR1 was highly expressed not only in developing seeds but also in radicle and coleoptile during germination,indicating that EDR1 might play an important role in seed germination.We performed seeds germination assay under different abiotic stress,and the seeds of NIL-qEDR5.1YZN and crisp1,crisp2 exhibited higher germination rate under salt stress and drought stress,indicating that EDR1YD1 might negatively regulate rice seed germination in response to salt stress and drought stress.Taken together,we mapped and cloned a UDP-glucosyltransferase gene EDR1.Natural variation of EDR1 greatly reduced the UDP-glucosyltransferase activity of EDRⅠYZN,thereby causing the abnormal endosperm development in NIL-qEDR5.1YZN,accompanied with insufficient grains and changes in grain quality.On the other hand,NIL-qEDR5.1YZN exhibited higher germination rate under abiotic stress.Our founding suggested that the UDP-glucosyltransferase gene EDR1 is an important regulator responsible for endosperm development and abiotic stress tolerance in rice.