Function Characterization Of Heat Shock Transcription Factor AtHsfA1d In Arabidopsis Thaliana

Heat stress is one of the main abiotic stresses faced by plants.Heat stress often changes the morphological,physiological and biochemical state of plants,which in turn has adverse effects on plant growth and development,metabolism,reproduction and production.Therefore,in order to cope with heat stress,plants have evolved complex mechanisms to resist the damage.Heat shock transcription factor(HSF)can regulate the expression of stress response genes such as heat shock protein(HSP),which plays a vital role in the response to heat stress.There are 21 HSF genes in Arabidopsis thaliana,which can be divided into A,B and C groups.The AtHsfA1 subfamily,including AtHsfA1 a,AtHsfA1 b,AtHsfA1 d and AtHsfA1 e,are the main regulatory factors of heat shock response induced by Arabidopsis thaliana.In this study,the complementary experiment of AtHsfA1 family members in yeast hsf1 mutants preliminarily proved that the function of AtHsfA1 d gene was similar to that of yeast Sc Hsf1 gene.Then we identified the heat tolerance of Col-0,Hsf A1 s QK quadruple mutant and transgenic AtHsfA1 d gene quadruple mutant.The function of AtHsfA1 d gene was preliminarily analyzed by using the techniques of overexpression of AtHsfA1 d in wild-type Col-0 and mutant cry1-304,nuclear protein extraction and western blot,identification of stress resistance of transgenic Arabidopsis,chromatin immunoprecipitation sequencing and transcriptome sequencing.The main results are as follows:(1)When AtHsfA1 family genes were transferred into yeast hsf1 deletion mutants,the yeast mutants transferred into AtHsfA1 b,AtHsfA1 d and AtHsfA1 e could grow normally,while the yeast mutants transferred into AtHsfA1 a could not grow.AtHsfA1 b,AtHsfA1 d and AtHsfA1 e could restore the growth of yeast hsf1 deletion mutants.(2)We identified the heat tolerance of wild-type Col-0,Hsf A1 s QK quadruple mutant and quadruple mutant transformed with AtHsfA1 d gene.The results showed that both wild-type and quadruple mutant transformed with AtHsfA1 d gene could survive under 45? heat shock for 2hours,but quadruple mutant could not tolerate high temperature.Therefore,At Hsf1A1 d restored the heat tolerance of Hsf A1 s QK mutant.(3)We cultured the transgenic Arabidopsis with AtHsfA1 d gene under the background of wild type Col-0 and mutant cry1-304 in dark for 4 days,and then transferred into white light for1 hour.Western blot detection showed that the content of AtHsfA1 d protein in the nucleus of Col-0/AtHsfA1d-FLAG was significantly higher than that in dark,while the content of AtHsfA1 d protein in the nucleus of cry1-304/AtHsfA1d-FLAG was little different from that in dark.It suggested that At Cry1 is needed for the nuclear localization of light-induced AtHsfA1.Through the western blot detection of the nuclear protein of transgenic Arabidopsis under normal growth conditions,it was found that AtHsfA1 d was enriched in the nucleus in an At Cry1-dependent way,and the nuclear localization of AtHsfA1 d depended on At Cry1.(4)Through the identification of stress resistance of wild type,a1 d mutant and transgenic Arabidopsis under salt stress and drought stress,we found that compared with wild type and transgenic plants,the growth of a1 d mutant was smaller,and the leaves were withered and yellow,which indicated that the inhibition of a1 d mutant under Na Cl and mannitol stress was more obvious,and the stress resistance decreased after AtHsfA1 d gene was deleted.(5)54 genes regulated by heat shock transcription factor AtHsfA1 d were screened by transcriptome sequencing and chromatin co-immunoprecipitation sequencing analysis.GO and KEGG enrichment analysis showed that these genes were mainly involved in abiotic stress response(temperature,light,reactive oxygen species and osmotic stress,etc.),endoplasmic reticulum protein processing and some metabolic pathways(inositol phosphate metabolism of glyoxylic acid and dicarboxylic acid metabolism,etc.).To sum up,AtHsfA1 d heat shock transcription factors play an important role in Arabidopsis heat shock response,have diversified functions in plants,and may be involved in photoperiod,ABA and other signal pathways to regulate response to abiotic stress.