Regulation Mechanism And Cold Tolerance Of LGS1,A Grain Shape Gene In Rice

Rice(Oryza sativa L.)can grow in a wide diversity of environments and provides the main calorific intake for more than half of the world's population.As the world population is predicted to reach 9 billion by the middle of this century,requiring a 70 to 100%increase in food production relative to the current levels.It is a great challenge to ensure sustainable food production and supplies in the world.Among the yield-associated traits,the thousand grain weight is restricted by the grain shape that can be further characterized by grain length,grain width and grain thickness.In addition,rice grain shape is known to greatly affect the milling quality and taste of the cooked food product.Thus,grain shape is an important trait that affects yield component and commercial value of rice.The rice genetics and breeding group of School of Life Sciences in Xiamen University have been working on developing rice cultivars with high yield,big grain shape and improved grain quality for the last three decades,such as Jiazhao No.l and Jiafuzhan.A combination of various approaches including discovery of new germplasms through gamma-ray irradiation mutagenesis,traditional hybridization and molecular assisted selection breeding techniques have been incorporated into the genetics and breeding program.A series of long grain mutants were isolated from the progeny population.However,the genetic basis underlying the long grain appearance is unclear.In the previous work of our team,we chose three of the long and large grain rice mutants,JF171,JF178 and JF3,to cross with Samba,a rice cultivar with very short grains.F2 and advanced backcross populations were generated for genetic analysis of grain traits.QTLs associated with grain shape traits were mapped to a 19.8-kb region between markers RM13838 and RM13840 on the long arm of rice chromosome 2,which contains only the LGS1 gene.In the current work,the LGS1 was cloned,characterized and analyzed.The main results of this study are summarized as the following:(1)LGS1 encoding growth regulating factor OsGRF4.Analysis the structure of LGS1,which possessed 5 exons,4 introns,5 'UTR and 3' UTR,with a total of 3825 bp of its genomic DNA.LGS1,encoding a protein with 394 amino acids,which has two conserved domains on the N-termini,namely QLQ(Gln,Leu,Gln)and WRC(Trp,Arg,Cys),and FFD(Phe,Phe,and Asp)and TQL(Thr,Gln,and Leu)domain on the C-termini.(2)Analysis of OsGRF4 alleles of 47 indica or japonica rice germplasms such as Ma85,JF178 and Samba revealed that 2-bp mutation(TC-AA)on its third exon exists lead to amino acid change(Serine-Lysine)on the WRC domain,which is assoiated with large grain shape in rice.Meanwhile,1-bp(G-A),2-bp(GC-TT),1-bp(G-T)replacement were found on the 1st,2nd,4th exon,respectively.These three polymorphisms may associate with the different evolutionary process of OsGRF4 between indica and japonica subspecies.(3)Analysis of grain traits based on reciprocal experiments between JF178 and Saiba,and NILs-LGS1JF178 and recurrent parent Samba showed that LGS1 is a typical nuclear inheriting locus on grain traits.It regulates grain length,grain width and grain weight,and showed pleiotropic effects on other traits(e.g.panicle length,tillers),exhibits as an incomplete dominant gene on grain traits.(4)Genetic complementation tests of LGS1 and LGS1 RNAi transgenic show that LGS1 involve in regulating rice spikelet development(pollen mother cell meiosis stage),influence rice grain shape development,and regulate rice grain length,grain width and grain weight.LGS1 is a gain-of-function gene.(5)Analysis of the temporal and spatial expression of lgsl showed that lgsl widely expressed through rice growth and development.It expressed in the process of meristem(young roots,stems,nodes,leaves,panicle,and rice filling process)and highly expressed in the spike differentiation period(pollen mother cell meiosis stage).LGS1 positive control of rice growth,accumulation of biomass,promoting the development of rice grain shape.(6)LGS1 regulates rice vegetative organs and developmental organs(spike),and glume cell division,cell elongation and cell expansion,and spikelet hull volume,improve the filling rate and increase the number of endosperm cells,thus enhance rice yield.(7)LGS1 locates to the cell nucleus and has the transcription activation activity on its carboxyl-terminus.(8)Transcript abundance of lgsl was severely altered by osa-miR396a-i,and further demonstrates that the expression of miR396 members impedes the transcript levels of lgs1.(9)The expression of Igsl was significantly downregulated under cold stress,which were correlated with the upregulation of osa-miR396,and affecting the growth retardation in rice seedlings.(10)The rice seedlings of mutant type LGS1 can enhance the cold tolerance and showed relatively high survival rate under cold stress,compared to wild type lgs1.