Cloning,Function,and Thermotolerant Mechanism Of Heat Stress Transcription Factor(Hsf)in Soybean,Wheat,and Arabidopsis

Due to their sessile lifestyle,plants have developed complicated response mechanisms to deal with the challenge of high temperature in the evolutionary process.With the perception of ambient temperature elevation,the heat stress signals are transmitted intracellularly and a large number of heat shock genes are induced in plants.Heat stress transcription factors(Hsf)and heat shock proteins(Hsp)are the terminal components of the heat stress signal transduction chain.Hsf regulates heat stress response through activating the expression of heat shock genes.Hsp genes are one family of the most important heat shock genes.Hsp,as molecular chaperone,plays significant roles not only in protecting targeted proteins from damage under heat stress condition,but also target proteins folding,intracellular distribution and degradation of denatured proteins.Most of previous studies about plant Hsf genes function and mechanism of stress resistance mainly were reported in Arabidopsis(Arabidopsis thaliana),tomato(Solanum lycopersicum),and rice(Oryza sativa).However,studies in wheat(Triticum aestivum L.)and soybean(Glycine max)are rare.In this study,we identified Hsf gene family in soybean,verified functions of Hsf genes in soybean,wheat,and Arabidopsis,and researched their thermotolerant mechanisms.The main contents are as follows:1.Genome-wide identification,structural analysis,expressions,cloning,subcellular localization,and function of GmHsf-34.(1)Identification and classification of soybean Hsfs: Through the scanning for soybean genomic data,soybean Hsf family members was collected.Several sequences were rejected due to the absence of typical Hsf DNA binding domain or oligomerization domain.Ultimately,we identified a family of nonredundant soybean Hsf including 38 members.Thirty-eight Hsf members were divided into A,B,and C classes and further divided into 12 subclasses(A1,A2,A3,A4,A5,A6,A8,B1,B2,B3,B4,and C1),after phylogenetic tree was produced using soybean,Arabidopsis,rice,and maize(Zea mays)Hsf protein sequences.(2)Analysis of soybean Hsf gene and protein structures: Most of the soybean Hsf genes contain only one intron.Stresses related cis-elements in promoters indicated that soybean Hsf genes might respond to abiotic stresses and hormones.Soybean Hsfs contained nuclear localization signal except subclass A8 and B4.Class A members contained activator motifs except subclass A8.(3)Expressions of soybean Hsf genes in different periods of development and tissues,under drought,and heat stress conditions: After analyzing of gene chip data,expression level in root was found highest.And among the 38 soybean Hsf genes,GmHsf-34 expression level was highest.Using qRT-PCR assay,expressions of 19 soybean Hsf genes under drought and heat stresses were analyzed,and 10 genes were identified as both heat and drought induced genes.(4)Cloning and subcellular localization of three Hsf genes and proteins: Three class A Hsf genes,GmHsf-04,GmHsf-33,and GmHsf-34 were cloned from soybean cDNA,and these 3 proteins were subcellular located into nucleus respectively.(5)Function of GmHsf-34: GmHsf-34(GmHsf-34 was a member of A2 subclass)was transformed into Arabidopsis,and it was found that in transgenic Arabidopsis plants expression of GmHsf-34 showed elevated survival rates under heat stress condition and germination rates under drought condition.These results confirmed that the expression of GmHsf-34 in transgenic Arabidopsis plants led to the improvement of thermotolerance and drought resistance.2.Function and thermotolerant mechanism of wheat TaHsf1 gene.(1)Function of TaHsf1 gene: After exposed to high temperature(37°C)for 48 hours,survival rates of Arabidopsis plants expressed wheat TaHsf1 gene were significantly higher than that of wild type(WT).After exposed to 37°C for 42 hours,and then transferred to normal conditions,it was found that at the harvest period the plant heights and yields of transgenic lines were significantly higher than WT.These results confirmed that the expression of TaHsf1 in transgenic Arabidopsis plants led to the improvement of thermotolerance.(2)Influence of thermotolerace related gene expressions in Arabidopsis plants by TaHsf1 gene: Using RNA-Seq analysis,many genes were up-regulated in the transgenic plants,including ERF(Ethylene responsive factor),NAC(NAM,ATAF,and CUC transcription factor),MAPKKK(Mitogen-activated protein kinase kinase kinase),LEA(Late embryogenesis abundant),cytochrome P450,aquaporin,peroxidase,and nitrate transporter family proteins coding genes.Using qRT-PCR analysis,we found that the expressions of a set of heat induced genes,including Hsf and Hsp genes,in transgenic lines were significantly higher than WT.(3)Sreening of Hsp and Hsf proteins interacting with TaHsf1: TaHsf1 was used as a bait to screening cDNA library for interacting proteins by the yeast two-hybrid system.And TaHsp40,TaHsp70,TaHsp90,and TaHsfA2 d were found as candidates and cloned.(4)Protein interactions between TaHsf1 and Ta Hsp40,TaHsp70,TaHsp90,and TaHsfA2d: Interactions between TaHsf1 and TaHsp40,TaHsp70,and TaHsp90 were confirmed by yeast two-hybrid,BiFC(bimolecular fluorescence complementation),and pull-down assay,which suggested that heat shock proteins might be feedback regulators of TaHsf1.Using yeast two hybrid,bimolecular fluorescence complementation,and pull-down assay,protein interaction between TaHsf1 and TaHsfA2d(a member of A2 subclasses)was also confirmed,which suggested that class C members might regulate the expression of downstream genes together with the class A members.3.Function and thermotolerant mechanism of Arabidopsis AtHsfC1 gene.(1)Expression analysis of AtHsfC1 gene under hormone treated and abiotic stress conditions: Using data analysis of Arabidopsis gene chip and qRT-PCR analysis of AtHsfC1,it was found that the expression of AtHsfC1 gene was influenced by heat,drought,NaCl,and abscisic acid(ABA).(2)Function of AtHsfC1 gene: After exposed to 37°C for 42 hours,survival rates of athsfc1 mutant Arabidopsis plants were significantly lower than that of WT.After exposed to 30°C for 6 hours every day until harvest period,yields of athsfc1 mutant plants were significantly lower than WT.In addition,AtHsfC1 gene was cloned and constitutively expressed in athsfc1 mutant plants.We found that constitutive expression of AtHsf C1 gene reestablished the lost thermotolerance of athsfc1 mutant.These results confirmed that AtHsfC1 gene was required for thermotolerance in Arabidopsis.(3)Influences of thermotolerance related gene expressions in Arabidopsis plants by AtHsfC1 gene: After exposed to 37°C for 42 hours,it was found that the expressions of a set of heat induced genes,including Hsf and Hsp genes,in athsfc1 mutant were significantly lower than WT using qRT-PCR analysis.(4)Interaction between AtHsfC1 and AtHsfA2: As AtHsfC1 in Arabidopsis was the only homolog of wheat TaHsf1,we were interested in whether AtHsfC1 interacted with AtHsfA2.Finally,using yeast two-hybrid,BiFC,and co-immunoprecipitation assay(Co-IP),protein interaction between AtHsfC1 and AtHsfA2 was confirmed.These results in our study may provide theoretical basis and some candidate genes to wheat genetic engineering breeding of thermotolerant varieties.