Regulatory Function Of Heat Stress Transcription Factor HsfA2c Conferring Heat Tolerance In Tall Fescue

Turfgrass has extensive values of application in landscape,football field,water and soil conservation,and ecological protection.Turfgrass can be classified into two classes,cool-season turfgrass and warm-season turfgrass.In the regions along the middle and lower reaches of the Yangtze,cool-season turfgrass and warm-season turfgrass both can be used,but cool-season turfgrass are used more widely due to their longer green period.However,the poor performance in hot summer is the limitation of cool-season turfgrass.High temperature is the critical environmental factor affecting the growth and development of cool-season turfgrass,which can lead to withering even death of turfgrass.This severely affects the landscape capacity of cool-season turfgrass.Therefore,improving heat tolerance and summer performance of cool-season turfgrass is an issue that needs to be addressed urgently.Heat stress transcription factor(HSF)is the central controller on transcriptional regulation of heat-responsive genes in higher plant,and it is irreplaceable in heat signal transduction and establishment of heat tolerance.HSF can regulate the transcriptional activity of heat-responsive genes by specifically binding the heat shock element(HSE)on the promoter of heat-responsive genes.HSF has a large family,but HsfA2s are the most responsive members in heat regulation.In Arabidopsis or tomato,there is only one HsfA2,while there are up to five members of HsfA2s in Poaceae species,such as rice and turfgrass.And it needs more research for the specific function of each member of the HsfA2s.In this study,we isolated HsfA2c gene from the cool-season turfgrass tall fescue and transformed it into Arabidopsis to investigate its regulative function in heat tolerance before overexpressed it in itself to improve heat tolerance.In addition,we cloned the promoter of FaHsfA2c and found many ABA-responsive cis-elements,and further studied the function of ABA in heat response of tall fescue.The main research contents and results briefing as follows:1.To obtain the FaHsfA2c gene sequence and investigate its heat-responsive characteristic,we isolated the FaHsfA2c cDNA sequence from tall fescue,which contains 1102-bp length and 1068-bp open reading frame with encoding 356 amino acids.The cluster analysis revealed that FaHsfA2c has the closest evolutionary relationship with HvHsfA2c of barley and followed by BdHsfA2c,OsHsfA2c and SiHsfA2c.FaHsfA2c has the specific structures of DNA binding domain,oligomerization domain,nuclear localization signal,nuclear export signal,and activator motif.FaHsfA2c was found to localize in nucleus by cellular localization in Arabidopsis protoplast with GFP-fused plasmid.And FaHsfA2c could be induced by heat in leave and root of tall fescue by qRT-PCR analysis,indicating its possible involvement in heat regulation of tall fescue.2.The FaHsfA2c was introduced into the pEarleyGate 103 and the integrated plasmid was transformed into Arabidopsis.The heat-tolerant lines were selected by using young seedlings growing on plate.The Adult seedlings of transgenic lines performed higher survival rate,net photosynthesis rate,chlorophyll content and photochemical efficiency than wild type under middle and severe heat stress,which demonstrating reduced heat damage of photosynthetic and membrane systems.To identify the regulated genes by FaHsfA2c in Arabidopsis,related genes were analyzed by qRT-PCR.The results revealed that AtApx2?AtHsp18.1-CI?AtHsp22.0-ER?AtHsp25.3-P?AtHsp26.5-P(r)?AtHsp70b and AtHsp101-3 had higher transcription levels in transgenic lines than wild type plants,indicating their possible roles in antioxidant system and protein protection regulated by FaHsfA2c in transgenic lines.Moreover,FaHsfA2c could rescue the heat-sensitive defects of hsfa2 mutant with improving survival rate and chlorophyll content of transgenic lines under heat stress,which further proved the regulative function of FaHsfA2c in heat response.3.To examine the heat regulation of FaHsfA2c in tall fescue,the plant expression vector pEarleyGate 103-FaHsfA2c was transformed into tall fescue calli through agrobacterium infection.Sixteen resistant lines were obtained after resistance screening,among which 11 transgenic lines were confirmed by PCR detection of the reporter gene insertion.Two heat-tolerant lines OE#2 and OE#7 were selected after heat screening and used for exploring the heat-tolerant mechanisms.The net photosynthesis rate,chlorophyll content,photochemical efficiency,growth rate,tiller numbers and growing activity of non-transgenic plants were lower than that of transgenic lines after 42 d of heat stress,while the yellowing rate was on the contrary.The gene regulation analysis revealed that FaHsp18.1?FaHsp22.0?FaHsp26.5?Fasp70 and FaHsp101all up regulated after 1 h of 37? heat treatment in all plants,but the transcription levels were higher in transgenic lines than non-transgenic plants.These results demonstrated that overexpression of FaHsfA2c could improve heat tolerance of tall fescue by enhancing response of downstream HSPs,triggering fast protection mechanism,and reducing damages of photosystem caused by heat.4.In order to investigate the upstream regulators of FaHsfA2c,the 2333 bp sequence upstream of the ATG in FaHsfA2c gene was isolated by genome walking method.The cis-elements on the promoter were predicted by using online analysis software of PlantCARE and PLACE,and the specific conserved elements for higher plants(e.g.,CAAT-box and TATA-box)were discovered.Moreover,there were large amounts of inducible elements related to stress,including ABA,anaerobic,JA,GA,Cytokinin,SA,and calcium responsive elements.This indicates that FaHsfA2c expression may be induced by multiple factors and it may be involved in various stress responses.To identify the ABA function in FaHsfA2c-mediated heat response,the transcription level of FaHsfA2c was detected after treatment of 5-?M ABA.The results revealed that ABA could induce up-regulation of FaHsfA2c and its downstream gene of HSPs.In addition,exogenous application of ABA improved heat tolerance of tall fescue and reduced damages of photosynthetic and membrane system caused by heat stress.During the heat stress progress,ABA treatment enhanced the expression of ABA-responsive genes and heat-responsive genes.Drought preconditioning also could improve heat tolerance of tall fescue with increased ABA content after drought and enhanced gene expressions in the early phase of heat stress.The yeast one hybrid assay gave the evidence that AREB and DREB could bind to the cis-elements on FaHsfA2c promoter,indicating the possibility of ABA regulating FaHsfA2c-mediated heat response indirectly through ABA-responsive factors.