Physiological Responses Analysis To Heat-resistance And Molecular Cloning Of HSP17.8 And HSP17.9 In Tall Fescue

Tall fescue(Festuca arundinacea Schreb.)is generally cultivated as a major turfgrass species in transition zone and warm climatic regions.High temperature is regarded as one of critical stress factors threatening turfgrass quality inferiority and even causing widespread death.Thermal acclimation of plants can transiently induce the expression of small heat shock proteins,which have close correlation with the acquisition of thermo tolerance.Thus it is of the utmost importance to improve our understanding of the physiological and molecular basis of plant responding to heat stress.One of the objectives in this study was to investigate the variation acclimation of enzymatic antioxidant metabolism and photosystem II photochemistry in response to drought,heat,and the combined stress for tall fescue genotypes differing in high-temperature tolerance.On the other hand,we aim to find out two trainable inducible sHSPs genes(FaHSP17.8 and FaHSP17.9)in respond to heat stress,which may provide the basis on further insight into the molecular mechanism of tall fescue on thermo tolerance.The results are listed as follows:1.Investigation of physiological characteristics under heat and drought stressWide variations were found between heat-tolerant and heat-sensitive tall fescue for malondialdehyde(MDA)and electrolyte leakage(EL)under drought,heat or a combination of both stresses.The heat-sensitive tall fescue exposed to combined stresses was associated with lower superoxide dismutase(SOD)activity and higher H2O2 accumulation.Tall fescue leaf cells excessively produced SOD,peroxidase(POD)and catalase(CAT)to protect itself from adverse stress.Drought,heat or the combined stress can exert an effect on varying degrees on photosystem II in chloroplast.The value of fluorescence parameters was analyzed by the JIP-test quantifying the photosynthetic behavior of the tall fescue leaves.Treatment with combination stress in the two kinds of leaves significantly decreased the values of PIABS,DFABS,?Po as compared with the control group.Heat-tolerant tall fescue had a higher potential to protect the PSII system under drought and high temperature stress.2.qRT-PCR analysis of FaHSPsThe expression patterns of four sHSP genes(HSP17.8,HSP17.9,HSP18.6 and HSP21.9)under heat acclimation were investigated.HSP18.6 and HSP21.9,maintained nearly same level or exhibited a sharp decline in expression during heat acclimation and so they are identified as non-trainable genes.HSP17.8 and HSP17.9 genes displayed considerably higher expression levels during one or more sub-sequent stresses compared to the initial stress,and thus they belonged to the trainable genes.3.Cloning and sequence analysis of FaHSPl 7.8 and FaHSP17.9 genesBased on the research of the transcriptome collection in Unigene database,the full length CDS of the two small trainable heat shock protein genes-FaHSP17.8 and FaHSP17.9-were acquired for the first time from tall fescue.The FaHSP17.8,FaHSP17.9 cDNA contained 489-and 459-bp open reading frames with molecular weight of 17.8K,17K Da,respectively.The genomic organizations of FaHSP17.8,FaHSP17.9 were also analyzed.Interestingly,we found that both genes had no introns.Multiple sequence alignment and phylogenetic analysis indicated their proximity with the class II and I sHSP members and shared 94%,91%sequence identity with triticum HSP17.8 and oryza HSP17.9,respectively.The fluorescent signal of both FaHSPl 7.8-GFP and FaHSP17.9-GFP were detected in cytoplasm and nuleus.The gene expression levels of FaHSP17.8 and FaHSPl 7.9 at 40 ? after 2 hours in leaves were the highest compared with stems and roots.4.Cloning and sequence traits of the FaHSP17.9 promoter regionA genome walking kit was used to clone the promoter of FaHSP17.9 gene.Here,a fragment of 917 bp upstream of the initiator codon was isolated from tall fescue genomic DNA.Analysis by PlantCARE database suggested several putative stress cis-regulatory elements.Importantly,HSE-motif(CCAAT)involved in high-temperature responsiveness was found and CCAAT box sequences acted cooperatively with HSEs to increase the heat shock promoter activity.In conclusion,the various adaptations of physiological traits in tall fescue under drought and heat and the combined stresses were researched,and then we further excavated FaHSP17.8 and FaHSP17.9 genes and analyzed the characteristics and expression profile.Our data may build a theoretical basis for further research of epigenetic regulation and their function in plant stress response,and assist in the selection and breeding of tolerant grass varieties.