Mechanism And Regulation Of The Impairment In Spikelet-Opening And Fertilization Of Photo-thermo-sensitive Male Genetic Sterile Rice Lines Under High Temperature Stress

"Two-line-method" hybrid rice,namely a hybrid produced by a photo-thermo-sensitive genic male sterile(PTSGMS)rice(Oryza sativa L.)line crossed with a restorer line,can easily maintain sterile line production without using maintainer lines.Moreover,two-line hybrid rice also has many other advantages,such as a wider range of germplasm resources which can be used as breeding parents,greater heterosis,and simpler procedures for breeding and hybrid seed production,compared with three-line hybrid rice with nuclear-plasma interaction pollen sterility as the core technology.Today,PTSGMS rice lines have become a main way using heterosis in rice because of its broad application in China.However,the fertility of PTSGMS rice lines is easily affected by high temperature(HT)stress in the reproductive growth period.With the increase in the global greenhouse effect recently,HT stress happens frequently and results in spikelet-opening and pistil fertilization impairments of PTSGMS rice lines,leading to decreased seed-setting rate and seed yield of their hybrids.Therefore,it is of great significance for ensuring hybrid seed production security to understand the mechanism of spikelet-opening and fertilization impairments of PTSGMS rice lines under HT stress during the reproductive growth period,and to explore an effective regulation technique to reduce the high temperature hazards during the reproductive growth period of PTSGMS rice lines.In this study,typical PTSGMS rice lines and one restorer line were used and subjected to normal temperature(NT)and HT treatments at different reproductive stages to systematically investigate the mechanism underlying the spikelet-opening and fertilization barriers of PTSGMS rice lines under HT stress,and to explore regulation techniques,including chemical regulation and cultivation regulation,mitigating the HT hazards to the PTSGMS rice lines.The main results are as follows.1.The mechanism underlying the impairment in spikelet-opening of PTSGMS rice lines under HT stress during anthesisTwo PTSGMS rice lines,Peiai 64S and Shen 08S,and one restorer line,Yangdao 6,were used and subjected to NT and HT treatments during anthesis.The spikelet-opening characteristics and some physiological traits were observed.The results showed that compared with NT,HT significantly reduced the spikelet-opening angle,the length of spikelet-opening time,the percentage of opened spikelets of PTSGMS rice lines,and the fertilization rate and the seed-setting rate of the hybrid of PTSGMS rice lines,with more effect on PTSGMS rice line than on the restorer line.HT significantly reduced the contents of soluble sugar,starch,jasmonic acid(JA),methyl jasmonate(MeJA)and the water content of the lodicules.The contents of soluble sugar,JA,MeJA in the lodicules before and during flowering were significantly and positively correlated with the number of opened spikelets and spikelet-opening rate.Under HT stress during anthesis,application of JA or MeJA significantly increased the contents of JA,MeJA,ascorbic acid(AsA),soluble sugars in the lodicule,and the activities of catalase(CAT)and ?-amylase in lodicules,the rate of opened spikelets,the length and the angle of spikelet-opening,whereas significantly decreased the content of H2O2 in lodicules in comparison with water application.The results indicate that a lower rate of opened-spikelets is closely associated with a lower content of Jasmonates(JAs)in the lodicules of PTSGMS rice lines.JAs can alleviate the spikelet-opening impairment caused by HT stress during anthesis by enhancing the ability of antioxidation and osmotic regulation in lodicules of the PTSGMS rice lines,thereby improving the rates of fertilization and seed-setting,and increasing the yield of the hybrids of PTSGMS rice lines.2.The role of jasmonates in regulating stigma vitality of PTSGMS rice lines under HT stress during anthesisPeiai 64S,Shen 08S and Yangdao 6 were pot grown and subjected to NT and HT treatments during anthesis.The stigma fresh weight,stigma area,rate of stigma exertion,the number of pollens germinated on single stigma,the contents of JA and MeJA in the stigmas of the PTSGMS lines,and the rate of fertilization and the seed production of the hybrids of PTSGMS lines were observed.The results showed that under HT stress during anthesis,a PTSGMS rice line with higher contents of JA and MeJA in stigmas had better stigma characteristics reflecting the stigma vigor,including higher stigma fresh weight,larger stigma area,higher rate of stigma exertion and more number of pollens germinated on single stigma,higher fertilization rate,seed setting rate and seed yield when the line was crossed with the restorer line.Under the HT stress during anthesis,MeJA application significantly decreased the temperature of opened spikelets with exerted stigmas,the transpiration rate of the flag leaf,H2O2 content in stigmas,and significantly increased the stigma fresh weight,stigma area,and the contents of JA,MeJA,and AsA,the activity of CAT in stigmas,the rate of stigma exertion,stigma receptivity,the number of pollens germinated on single stigma,the rate of fertilization,and increased the seed production of PTSGMS rice lines when they were crossed with the restorer rice line.Under the HT stress during anthesis,applying MeJA significantly decreased the temperature of opened spikelets with exerted stigmas,the transpiration rate of the flag leaf,H2O2 content in stigmas,and significantly increased the stigma fresh weight,stigma area,and the contents of JA,MeJA,AsA,the activity of CAT in the stigmas,the rate of stigma exertion,stigma receptivity,the number of pollens germinated on single stigma,and the rate of fertilization,and increased the seed production of PTSGMS rice lines when crossed with the restorer rice line.The results imply that JAs(JA,MeJA)in stigmas can enhance the antioxidant ability and inhibit the production of reactive oxygen species(ROS)in the stigma of the PTSGMS rice lines,and thereby alleviating the damage of HT during anthesis to the stigma vitality.3.The role of brassinosteroids in regulating pistil activity of PTSGMS rice lines under HT stress during anthesisThree PTSGMS rice lines,Peiai 64S,Guangzhan 63S,and Shen 08S,and one restorer line,Yangdao 6,were used and subjected to NT and HT treatments during anthesis,and the changes in pistil activity were investigated.The results showed that HT stress during anthesis significantly reduced the contents of 24-epibrassinolide(24-EBL)and 28-homobrassinolide(28-HBL)in the pistil of PTSGMS rice lines.However,the response of pistil activity to HT stress during anthesis varied with rice lines.A PTSGMS rice line with higher BRs(24-EBL,28-HBL)contents in the pistil showed lower contents of 1-aminocyclopropane-1-carboxylic acid(ACC,the precursor of ethylene synthesis)and H2O2,higher CAT activity,higher AsA content in the pistil of PTSGMS lines,and higher fertilization rate,seed-setting rate and seed yield after the PTSGMS rice line crossing the restore rice line.Application of 24-EBL or 28-HBL under HT stress during anthesis significantly decreased contents of ACC and H2O2 in the pistils,whereas significantly increased the contents of 24-EBL,28-HBL and AsA,and the activity CAT in the pistils of PTSGMS lines,and significantly increased the rates of fertilization and seed-setting when the PTSGMS lines were crossed with the restorer line.The results demonstrate that BRs can enhance antioxidant system(AOS),and inhibit the excess production of ROS,and consequently,alleviate the harm caused by HT stress during anthesis to the pistils of PTSGMS rice lines.4.The mechanism underlying HT stress during meiosis impairing fertilization of PTSGMS rice linesTwo PTSGMS rice lines,Peiai 64S,Shen 08S,and one restorer rice lines,Yangdao 6,were pot grown and subjected to NT and HT treatments during meiosis.Agronomic and physiological traits were observed and compared between NT and HT.The results showed that HT stress during meiosis significantly increased the panicle temperature,the degree of wrapped panicles,the contents of H2O2,MDA,and abscisic acid(ABA)in the young panicles of PTSGMS rice lines,and significantly decreased the SPAD value,photosynthetic rate of the top first expanded leaf on the stem,and the dry matter weight,CAT activity,the contents of non-structural carbohydrates(NSC),nitrogen(N),AsA,indole-3-acetic acid(IAA),gibberellin-3(GA3),zeatin+zeatin riboside(Z+ZR),24-EBL,28-HBL,JA,and MeJA in young panicles,with more effects on PTSGMS lines than on a restorer line.The HT stress significantly decreased the fertilization rate and seed-setting rate of the hybrids produced form the PTSGMS lines.The results suggest that the decreases in fertilization ability of PTSGMS rice lines and the reduction in seed-setting rate and seed yield of the hybrids from PTSGMS lines are mainly attributed to the decreases in growth-promoting hormonal levels and increases in ROS production in the young panicles of rice,especially for PTSGMS lines,under the HT stress during meiosis.5.The role of applying N-fertilizer in regulating spikelet-opening and fertilization of PTSGMS rice lines under HT stressPeiai 64S,Shen 08S and Yangdao 6 were pot-grown and subjected to HT stress either during meiosis or during anthesis.Either spikelet-protecting N-fertilizer that was applied at the stamen and pistil differentiation stage or grain-enhancing N-fertilizer that was applied at seven days before heading was conducted,and no application of N-fertilizer was taken as control.The effects of the N-fertilizer applications on the spikelet-opening and fertilization of PTSGMS rice lines under HT stress were investigated.The results showed that under HT stress during meiosis,application of spikelet-protecting N-fertilizer significantly decreased the surface temperature of rice canopy,especially surface temperature of young panicles,the contents of H2O2,MDA,ABA in young panicles,and significantly increased the SPAD value and photosynthetic rate of leaves,the dry weight of young panicles,the contents of N,NSC,and growth-promoting hormones,such as IAA,GA3,Z+ZR,24-EBL,28-HBL,JA,MeJA,and the activities of key enzymes involved in N metabolism and carbohydrates synthesis in young panicles,and the number of spikelets per pot,and the rates of fertilization and seed-setting and seed yield of the hybrids of PTSGMS rice lines.The results demonstrate that increasing application of the spikelet-protecting N-fertilizer can effectively alleviate the damage to the young panicles caused by HT stress during meiosis.Under HT stress during anthesis,application of grain-enhancing N-fertilizer significantly decreased the temperature of opened spikelets with exerted stigmas,the contents of H2O2 and MDA in pistils,and significantly increased the contents of AsA,the activities CAT,nitrate reductase,glutamine synthetase,glutamine oxoglutrate aminostransferase,ADP glucose pyrophosphorylase,soluble starch synthase,the contents N,soluble sugars and starch in pistils,and increased the rates of fertilization and seed-setting and seed yeid of the hybrids of PTSGMS rice lines.The results suggest that the application of grain-enhancing N-fertilizer can improve the tolerance of pistils to HT stress during anthesis and reduce the harm of the HT stress to fertilization of PTSGMS rice lines.