Mechanism Of Rice (Oryza Sativa L.) HSTL Gene In High Temperature Response

The high temperature stress has seriously affected the yield of rice.Therefore,the selection and cultivation of high-temperature-tolerant rice varieties has become an effective method to cope with high-temperature stress.C2H2 zinc finger protein transcription factors are a relatively large family of transcription factors in plants,which are involved in plant growth and development and responses to a variety of abiotic stresses such as salt,and drought.Our previous studies obtained a heat stress tolerance（hst）mutant of rice Zhonghua 11,and found that the C2H2 zinc finger protein transcription factor HST had a frameshift mutation.HSTL（heat stress tolerance like）is the homologue of HST,both of which have the same zinc finger domain.We used CRISPR/Cas9 technology to obtain hstl knockout mutants and hstl/hst double mutants.Therefore,this paper took the HSTL transcription factor as the research object,and used wild-type ZH11,mutants hst,hstl 3-1,hstl 12-1,and double mutants hstl/hst 3-2,hstl/hst 15-2 as experimental materials,in order to explore the regulatory mechanism of HSTL in the response of rice to high temperature stress,to provide a theoretical basis for revealing the molecular mechanism of high temperature tolerance in rice,and to provide a reference for breeding new rice varieties with high yield,high quality and stress resistance.The research results of this paper were as follows:1.HSTL expression characteristics and subcellular localization analysisThe expression of HSTL in roots of WT at seedling stage basically showed an upward trend after 0.5 h of high temperature treatment.And the expression of HSTL decreased after 1 h of high temperature treatment in leaves,then the expression of HSTL basically showed an upward trend.The expressions of HSTL in WT and mutant hst anthers were down-regulated after high temperature treatment at heading and flowering stage.The subcellular localization was observed by laser confocal microscopy after transient expression in tobacco.The results showed that the HSTL protein was localized in the nucleus and cell membranes.2.Identification and phenotypic analysis of hstl mutants and hstl/hst double mutantsWe botained hstl and hstl/hst homozygous mutants,and through the phenotype observation at different periods.It was found that the double mutants hstl/hst 3-2 and hstl/hst 15-2 had the highest plant height,followed by the mutants hstl 3-1,hstl 12-1,and then the mutant hst,WT the shortes.It was indicated that HSTL gene mutation could promote the growth of plant height.Double mutants hstl/hst 3-2 and hstl/hst15-2 had the highest number of grains per panicle,followed by mutants hstl 3-1 and hstl 12-1 and hst,WT the least.The double mutants hstl/hst 3-2 and hstl/hst 15-2 had the earliest heading time,followed by the mutants hstl 3-1,hstl 12-1 and hst,and WT the last.And the expression levels of Cytokinin oxidases（Os CKXs）were down-regulated in rice anthers of mutants hst,hstl 3-1,hstl 12-1 and double mutants hstl/hst 3-2 and hstl/hst 15-2.3.Phenotypic and physiological analysis of hstl mutants and hstl/hst double mutants under high temperature stressAfter high temperature stress at 42℃,the survival rate of double mutants hstl/hst3-2 and hstl/hst 15-2 were the highest,followed by mutant hst,then mutant hstl 3-1,hstl 12-1,and finally WT.Compared with wild-type ZH11,the expression of the peroxidase gene Prx24（encoding an H₂O₂scavenger）decreased in mutant hstl and double mutant hstl/hst rice plants,leading to the increase of H₂O₂.Physiological analysis of rice showed that under high temperature stress,HSTL gene mutation reduced the stomatal conductance,maintained higher relative water content,reduced the loss of water in rice,and increased the high temperature tolerance of hstl mutant rice.4.Transcriptome sequencing analysis of hstl mutants and hstl/hst double mutants under high temperature stressWe used transcriptome sequencing technology to analyze the differential experssion genes and GO enrichment analysis of hstl mutants and hstl/hst double mutants under high temperature stress.The following genes were screened for quantitative PCR validation,including 5 genes related to plant hormone metabolism（Os GA3ox2,Os GA20ox1,Os GA20ox4,Os EATB and Os ERF68）;3 genes related to photosynthesis（Os TLP27,Os PLGG1b and Ospsb S2）;3 genes related to heat shock response（Os Hsf A2a,Os Hsp140 and Os Hsf A3）;1 gene related to amino acid transport（Os ATL7）.We speculated that HSTL may regulate gibberellin and ethylene metabolism,photosynthesis-related pathways and heat shock response to regulate high temperature tolerance of rice.In summary,HSTL gene mutation could reduce the expression of Os CKXs,thereby increasing rice plant branches and grains per spikes.In addition,HSTL gene mutation could reduce the expression of Prx24 and increase the content of H₂O₂and stomatal closure,thereby increasing the high temperature tolerance of hstl mutant rice.By transcriptome sequencing analysis,we speculated that HSTL may also regulate gibberellin and ethylene metabolism,photosynthesis and heat shock response to regulate high temperature tolerance of rice.