| High temperature damage occurs frequently in southern China,causing a decline in rice(Oiyza sativa L.)yield and quality.The purpose of this study was to explore the key candidate genes regulated by spermidine(Spd)involving in the modulation of rice seed development and maturation under high temperature stress,and to reveal the specific mechanism of polyamines involved in the modulation of heat tolerance and vigor formation during rice seed maturation through functional studies of candidate genes.In this study,hybrid rice cv."Y Liangyou 689"was used as the material,and the spikes were treated with exogenous Spd or its synthetic inhibitor cyclohexylamine(CHA)on the third day after pollination,and high temperature was applied in the early stage of seed maturation.The changes of seed quality,endogenous polyamines and starch metabolism of different treatments at different developmental stage were studied;Transcriptome sequencing analysis was used to explore the major metabolic processes and key candidate genes of Spd involved in modulating rice seed maturation under high temperature stress at grain filling stage;Genetic transformation technology was used to study the relationship between key candidate gene OsSAP5(Stress associated protein)and plant heat tolerance,and to explore the possible interaction between OsSAP5 and polyamine.Present study was the first time to explore the mechanism of Spd’s involvement in modulating rice seed vigor under high temperature stress during grain filling stage,and the possible interaction between polyamines and OsSAP5 in improving plant heat tolerance was investigated.The main findings are shown below:The effects of exogenous Spd on the quality of rice seeds under high temperature stress during grain filling were studied.Exogenous Spd could significantly reduce the chalky grain rate for 33.33%of mature seeds(35 days after pollination)under high temperature stress during filling stage.Compared with high temperature control(HT),after the application of Spd,the thickness,1000-grain weight,seedling dry weight and germination index of the mature seeds were increased by 11.46%;18.38%,21.15%and 36.96%,respectively.At the same time,exogenous Spd could significantly reduce the MDA content of rice seeds under high temperature stress and significantly increase its POD activity.In addition,exogenous Spd increased the expression levels of SPDS,SPMS1 and SPMS2 in rice seeds under high temperature stress during grain filling,which leads to an increase in endogenous Spd and Spm content,and significantly increased amylose and total starch content.The results showed that exogenous Spd could improve the quality of rice seeds by regulating endogenous polyamine metabolism and starch metabolism,and alleviate the damage caused by high temperature during seed filling stage.The key candidate genes of exogenous Spd involved in modulating rice seed maturation under high temperature stress at grain filling stage were studied.Through transcriptome sequencing analysis,Spd could significantly regulate the establishment of heat tolerance during rice seed maturation.Exogenous Spd improved the high temperature tolerance of rice seed during grain filling by:(1)activating photosynthesis-related genes(OsNPB,CHL9,etc.);(2)activating grain filling-related sugar metabolism genes(OsTPP9,OsSS Ⅱ-3,OsSS Ⅰ,etc.);(3)up-regulating the expression of genes transcripting stress-related proteins(OsSAP5,OsSAP17,etc.)and other plant hormone related genes(OsARFl,OsARF17,etc.)to reduce the damage caused by high temperature,enhance photosynthetic capacity and improve seed vigor and enhance the heat tolerance of seeds.OsSAP5 was involved in the regulation of heat tolerance in transgenic Arabidopsis thaliana.OsSAP5 protein is mainly located in the nucleus,and widely existed in various parts of the cell and participated in plant’s response to high temperature stress.Seeds of transgenic Arabidopsis lines had higher survival rates under high temperature stress.After treatment with high temperature,the activities of CAT,POD and APX in the transgenic lines were significantly increased,while the content of MDA was significantly decreased.Meanwhile,the transgenic lines significantly increased the expression levels of SPDS2 and SPMS in Arabidopsis thaliana leading to a decrease of Put content and an increase of Spd and Spm content.In addition,the seed weight,Spd and Spm content of the transgenic lines increased significantly under high temperature stress during grain filling stage.It indicated that OsSAP5 was involved in the regulation of heat tolerance of transgenic Arabidopsis thaliana by increasing antioxidant enzyme activity,reducing membrane lipid peroxidation and regulating polyamine metabolism.The possible interaction between polyamines and OsSAP5 in improving plant heat tolerance was studied.Under high temperature stress,exogenous Spd could significantly improve the survival rate and seedling quality of transgenic and mutant Arabidopsis thaliana,and significantly improve the seed vigor and seedling quality of Ossap5 mutant rice.It indicated that exogenous Spd could significantly improve the heat tolerance of Arabidopsis thaliana and rice.At the same time,the expression of SPDS and SPMS in mutant Arabidopsis thaliana and mutant rice was significantly increased by applying Spd under high temperature stress,which resulted in the increase of Spd and Spm content.It suggested that there might be a synergistic effect between polyamines and OsSAP5 in improving plant heat tolerance.In conclusion,exogenous Spd could alleviate the effect of high temperature stress during rice seed maturation and improve the quality of rice seeds by modulating endogenous polyamines and starch metabolism.OsSAP5,a stress-associated protein gene regulated by exogenous Spd under high temperature stress,was closely related to plant heat tolerance,and might play a synergistic role with polyamines in plant heat tolerance.The results of this study not only can be used for reference for other crops,but also can improve the quality of rice seeds genetically and provide theoretical basis for breeding high temperature resistant varieties. |
PDF Full Text Request