Cloning Of The Thermo-Sensitive Male Sterility Gene OsTMS19 In Rice And Its Photothermo-Sensitive Mechanis

Rice is the staple food for over 60%of the world’s population.Increasing rice yield and improving rice quality are effective ways to address global food security issues.Hybrid breeding is the most important and popular technology in crop breeding,which significantly improves crop yield and quality by utilizing hybrid vigor.In China,three-line and two-line hybrid rice have made great contributions to increasing yield and ensuring food security.However,due to limitations such as limited restorer lines and cumbersome seed production processes have hindered the further development of three-line hybrid rice.The discovery of photo/thermo-sensitive genic male sterile（P/TGMS）lines has pioneered two-line rice breeding,as most conventional varieties carry dominant restorer genes,the male sterile line could receive restorers with a wider range of germplasm resources to obtain superior hybrid combinations.P/TGMS lines lay the foundation for the development of two-line hybrid rice.Investigation of new P/TGMS loci helps promote further development of two-line hybrid breeding.Research on the function of P/TGMS genes will provide further insights into the molecular basis of plant male sterility and P/TGMS mechanism.In this study,we identified a novel rice TGMS line,ostms19,through mutants screening.Agronomic trait analysis indicated its potential for application in two-line hybrid rice.Gene mapping and genetic complementation experiments revealed that the TGMS phenotype of ostms19 was caused by a single nucleotide mutation（T-C）in a PPR gene.Cytological analysis showed that under high temperature,ostms19 exhibited excessive expansion of the tapetum and collapse of microspores during the stage 10-11of anther development.Further pollen wall staining revealed that defective intine layer of the mutant pollen may be the reason of its sterility phenotype.RT-PCR and q RT-PCR indicated that OsTMS19 is a ubiquitously expressed gene,with highest expression in anthers.In situ hybridization and immunohistochemistry experiments showed that the transcripts and protein expression of OsTMS19 were presented in the tapetum and microspores at stage 10-11 during anther development.Subcellular localization experiments demonstrated that OsTMS19 protein is localized in mitochondria,suggesting that it might be involved in energy metabolism during anther development.Through phenotype observation during the nutritional growth stage and the construction of its strong mutants,we proposed that OsTMS19plays a role in early embryo development and nutritional growth.We further analysed the activities of electron transport chain（ETC）complexes of ostms19.The results showed that the activities of mitochondrial complexes I,III,IV and V were obviously reduced in the ostms19 mutant compared with ZH11 under high temperatures.Mitochondrial dysfunction is frequently associated with oxidative bursts.The NBT and H₂DCF-DA staining showed excessive ROS accumulation was presented in ostms19during stages 10-11 of anther development under high temperature conditions.Excessive ROS leads to the premature phenotype of PCD in the tapetal layer of ostms19anthers,with strong PCD signals appearing as early as stage 7.Comet assay showed that a large number of pollen nuclei in the mutant underwent degradation during high temperatures,which significantly restored under low temperature.These results indicate that the mutation of OsTMS19,as a PPR protein localized in the tapetum mitochondria,lead to abnormal mitochondrial function,causing excessive accumulation of ROS under high temperatures and resulting in pollen abortion.Subsequently,we specifically overexpressed the ROS scavenging genes Os SODA and Os CATB in ostms19 pollen,which restored pollen vitality and fertility under high temperature.This result indicated that the ROS clearance in gametophyte of ostms19was able to rescue its sterile phenotype.Therefore,we proposed that the tolerance of haploid microspores to ROS in the locule is crucial for the fertility conversion of ostms19.During the growth process of rice,the ROS accumulation in the anthers under high temperature and long-day photoperiod conditions are significantly higher than those in low temperature and short-day photoperiod.We further analyzed the accumulation of ROS in the anthers of other three TGMS lines（tms5,ostms15,and ostms18）under different temperature conditions through NBT staining.The results showed that the accumulation of ROS in these mutants was consistent with ostms19.This study demonstrates that the ROS accumulation combined by effects of gene mutations and inducing by high temperatures damage the tapetum and pollen,leading to male sterility.Temperature and light may regulate pollen development in crops by affecting ROS homeostasis,which could be a common mechanism for the sterility–fertility conversion of rice P/TGMS lines.