Mechanism Research Of OsGPAT3 In Regulating Anther Programmed Cell Death And Male Sterility In Rice

The male reproductive development of rice is a highly complex and precisely regulated biological process,which is directly related to the reproduction and yield of rice.A large number of genes are involved in the process and any mutation or abnormal expression of these key factors may cause abnormal microspore development,resulting in pollen abortion and spikelet sterility.At present,a series of genes related to male sterility have been reported,but it is far from enough to further explore the molecular mechanism and complete regulatory network of rice male reproductive development.In this study,phenotype analysis and map-based cloning of a no pollen male sterile mutant,gpat3-2,which was screened from Zhonghui 8015,an indica rice restorer line induced by ethyl methanesulfonate(EMS),were carried.On this basis,the function mechanism of the glycerol-3-phosphate acyltransferase gene OsGPAT3 in regulating rice anther programmed cell death and male sterility was preliminarily elucidated.The main results are as follows:1.The vegetative growth and spikelet structure of the gpat3-2 mutant were basically same as that of Zhonghui 8015,but the anthers were thin and milky white without visible pollen.The semi thin sections analysis of anther showed that the degradation of tapetum cells was delayed,the endothecium and middle layer of anther did not degrade,and the microspore cells degraded into lines resulting in the failure of microspore cell maturation process at the late stage of anther development.Electron microscope observation and TUNEL test further confirmed that the degradation process of cells in each layer of anther wall in the gpat3-2 mutant were totally abnormal.The synthesis of nutritive substances such as Ubisch bodies and sporopollenins were blocked,and the microspore cells degraded during the pollen filling stage and unable to develop into mature pollen due to the insufficient nutrients supply.These cytological observation suggested that the male sterile phenotype of the gpat3-2 mutant was probably caused by the abnormal development of tapetum and anther wall,which leads to the lack of nutrition supply in the later stage.2.Genetic analysis showed that the gpat3-2 phenotype was controlled by a pair of recessive nuclear genes.The GPAT3-2 male sterile gene was mapped into a 26 KB region between the markers ZH-3 and ZH-6 on the long arm of rice chromosome 11,which contained three open reading frames.Sequencing results revealed that the mutant carried a single nucleotide transformation(G to A)occurred in the first exon of a glycerol-3-phosphate acyltransferase gene(LOC_Os11g45400),which resulted in the mutation of tryptophan into termination codon and early termination of final protein translation.Functional complementation test,CRRSPR/Cas9 gene knockout and further phenotype analysis of allelic mutants obtained from our mutant library further confirmed that OsGPAT3 was responsible for the male sterility phenotype of the mutant.3.The qPCR analysis results showed that the expression of OsGPATi was increased significantly at pollen mother cell stage and slightly decreased during meiosis stage,but increased significantly in the later anther development stage in the mutant.In addition,regulators involved in tapetum development and fatty acid metabolism of the anther sac wall also showed significant changes in the mutant.Based on these genetic and cytological analyses,OsGPAT3 is proposed to coordinate the differentiation and degradation of the anther wall and pollen grains in addition to regulating lipid biosynthesis.This study provides insights for further understanding the function of GPATs in regulating rice male reproductive development and also lays a material resourse and theoretical basis for hybrid rice breeding.