| Rice(Oryza sativa L.)is the staple food for more than half of the world's population,and the continuous increase in rice production is required to meet the growing food demand due to population growth in the future.However,rice is increasingly cultivated in more marginal environmental stresses because of global warming,and heat stress is a major environmental limitation to plant growth and development.In these environments,temperatures frequently exceed the critical temperature of seed setting,resulting in spikelet sterility and drastic yield loss.Hence,with the rapid increase in atmospheric concentrations of CO2 resulting from the expansion of industrialization,rice will be grown under extreme high temperatures more frequently in many areas.High temperature is a major disastrous factor that leads to the serious rice yield decline and deterioration in quality.Therefore,understanding the mechanisms of heat injury will play an important role in crop improvement.Rice development during the reproductive stage is very sensitive to high temperature,but little is known about the transcriptional regulation of rice spikelet development in response to heat stress.Nitrogen,the most important inorganic nutrient for plant growth and development,iteractiong with temperature can affect the actual production of rice,and the molecular mechanisms of rice spikelet fertility in response to high temperature interacting with nitrogen are lacking.The effects of high temperature on rice at early milk ripe stage and late booting stage were mainly discussed in this study,and the main contents include:(1)The experiment was designed with in plant growth cabinet.One early rice variety(Ganxin 203)was exposed to six high temperature intensities(34~39 °C)and different duration(1~6 d),and the differences between different high temperature treatment intensities and duration on yield components and physiological items of at early milk ripe stage were analysed.Finally,the selection and quantitative evaluation for heat damage item of early rice were processed by subordination function method,stepwise regression analysis,and path analysis.(2)Four different rice varieties were exposed to different high temperature treatment intensities(35 °C and 38 °C)for 3 days at early milk ripe stage,and the differences between different high temperature treatment intensities on yield components,and physiological items of different early rice varieties were analyzed.(3)Six rice varieties were processed at 35 °C and 38 °C for 3 days at milk ripe stage,and the effects of high temperature on amylose content and starch RVA profile's characteristic parameters in different early rice varieties were discussed.Furthermore,the correlation between starch RVA profile's characteristic values and amylose content was analyzed.(4)In order to explore the compound effects of nitrogen level and high temperature at late booting stage on rice yield and physiological items,two nitrogen levels were set,and two rice cultivars were processed at 38 °C for 4 days at late booting stage.(5)The compound effects of nitrogen level and high temperature at late booting stage on yield and physiological characteristics of early rice were investigated at two nitrogen levels,with conventional rice cultivar(Zhong 531)as materials at 38 °C for 4 days at late booting stage.(6)This study received four treatments with zhong 531: normal nitrogen level with natural temperature(NN),high nitrogen level with high temperature(HH),and NH,HN,respectively.The transcriptome profiling analysis of rice spikelet at meiosis stage was processed using RNA sequencing as an attempt to gain insights into differentially expressed genes of rice spikelet fertility in response to temperature interaction with nitrogen.The main results in this study are as follows:1.The heat injury of rice was quantitatively evaluated using subordination function method,and seven high temperature treatments combinations(36 °C for 5 d and 6 d,37 °C for 4 d,38 °C for 3 d and 4 d,39 °C for 3 d and 4 d,respectively)were identified as severe disasters.The evaluated result of heat injury was demonstrated with the comprehensive evaluation of the physiological items by principal component analysis.2.The selection and quantitative evaluation for all items were processed by stepwise regression analysis and path analysis,and thirteen items(seed setting rate,unfilled grain rate,1000-grain weight,single plant yield,plumpness,ear weight per stem,harvest index,amylose content,SPAD value,proline content,soluble protein content,soluble sugar content,and superoxide dismutase(SOD)activity,respectively)were selected to establish the optimal regression equation.The direct path coefficient of character stress index of single plant yield,1000-grain weight,plumpness,chlorophyll content,proline content,and soluble protein content were highest,which can identify and predict the heat injury of rice at early milk ripe stage.3.Seed setting rate,1000-grain weight,plumpness,harvest index,and single plant yield of Ganxin 203 declined under high temperature,while the index of heat sensitivity and unfilled grain rate increased compared with the control.The range of its variation increased along with addition of high temperature intensity and prolongation of duration.Proline content,conductivity,and catalase(CAT)activity showed the uptrend,while SPAD value,soluble sugar content,soluble protein content,and SOD activity showed the downtrend under high temperature.4.Seed setting rate,1000-grain weight,plumpness,and single plant yield of all early rice varieties declined under high temperature treatment,while unfilled grain rate increased compared with the control.The range of its variation increased along with addition of high temperature treatment intensity.There were significant genotypic differences between the responses of high temperature to yield components among different early rice varieties.The influence of high temperature on hybrid rice Jinyou 402 was more than that of Ganxin 203,and conventional rice E134 was more than Zhong 531.The influences of high temperature on physiological items were consistent with the influence of yield components.5.There were significant difference in amylose content,breakdown,setback,and consistency between different rice varieties and temperatures.The rice starch viscosity curve under 38 °C high temperature was always lower than those of the controls,indicating that rice quality was decreased under higher temperature.While the RVA profile's characteristic values of Zhong 531(heat tolerant variety)differed slightly from those of the controls.Moreover,the amylose content of rice starch was significantly correlated with RVA profile's characteristic values.6.At the same nitrogen level,yield per plant,effective panicles per plant,spikelets per panicle,spikelets fertility,differentiated spikelets per panicle,and harvest index of two rice cultivars were decreased under high temperature at late booting stage,while the ratio of degenerated spikelets,leaf weight per stem,stem and sheath weight per stem were increased under high temperature.In addition,under the the same conditions of natural temperature and high temperature,yield per plant and effective panicles per plant of two cultivars with high nitrogen level were higher than those with normal nitrogen level.There were some difference among the contents of four endogenous hormones(ABA,IAA,GA3,ZR,repectively)under high nitrogen level and high temperature.The ratio of growth-inhibiting hormone to growth-promoting hormones i.e.ABA/(IAA+ GA3+ ZR)was greater than that of the control.It was worth noting that the decreased percentage of yield per plant of two cultivars treated by high nitrogen level was reduced compared with normal nitrogen level under high temperature.Therefore,appropriate high nitrogen level can lighten the effects of high temperature on the reduction of output.7.High temperature at late booting stage significantly decreased spikelet fertility,yield per stem,harvest index,SPAD value,and soluble sugar content,while proline content of Zhong 531 was significantly increased under high temperature.Spikelet fertility,yield per stem,harvest index,proline content,and soluble sugar content were decreased at high nitrogen level.It was worth noting that spikelet fertility,yield per stem,and yield per plant at high nitrogen level were lower than those at normal nitrogen level under high temperature,implying that appropriate high nitrogen levels contributed to increase the effects of high temperature on grain production.8.The de novo assembly generated 52,553,536 clean reads aligned with 72,667 unigenes.In these differentially expressed genes(DEGs),we found 149 and 323 temperature-responsive DEGs in NN-vs-NH and HN-vs-HH,and 114 DEGs were co-expressed.Meanwhile,203 and 185 nitrogen-responsive DEGs were focused in NN-vs-HN and NH-vs-HH,and 111 DEGs were co-expressed.In addition,spikelet fertility of rice was significantly decreased under high temperature,combined with high nitrogen,it was more decreased.Accordingly,numerous spikelet genes involved in pollen development,pollen tube growth,pollen germination,especially sporopollenin biosynthetic process,and pollen exine formation were mainly down-regulated under high temperature.Moreover,the expression levels of co-expressed DEGs including 5 sporopollenin biosynthetic process genes(Unigene43852,Unigene32623,Unigene23940,Unigene23939,Unigene23941),and 7 pollen exine formation genes(Unigene45103,Unigene32014,Unigene32018,Unigene32017,Unigene42236,Unigene42231,Unigene42234)of NN-vs-NH were lower than that of HN-vs-HH.Therefore,these spikelet genes may play important roles in response to high temperature with high nitrogen and may be good candidates for crop improvement.In this study,the combination of subordination function method,principal component analysis,stepwise regression analysis,and path analysis will be contribute to the selection and quantitative evaluation for heat damage item of early rice at early milk ripe stage.The compound effects of nitrogen level and high temperature at late booting stage on yield and physiological characteristics of early rice were investigated in our study.The present study is the first to attempt to perform a de novo assembly of transcriptome sequencing of the temperature and nitrogen's interactive effects in rice spikelet,which may extend our understanding of the complex molecular and cellular events in spikelet. |
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