Cloning And Heat Tolerance Characterization Of FaHsfC1b From Festuca Arundinacea

Tall fescue(Festuca arundinacea)is one of the cool-season turfgrass which have the best ability to resist heat,so it was widely planted in climate transition zone all around the world,and it’s meaningful to find some genes which are related to heat tolerance in tall fescue.It is widely known that heat transcription factors played important roles in heat tolerance of plants,nevertheless,little information was obtained for C class heat transcription factor,the study for C class Hsfs from would be meaningful.The objectives of this study were to characterize a Class C Hsf,FaHsfC1b,cloned from a perennial grass speciestall fescue(Festuca arundinacea)by expression pattern analysis and subcelluar localization,and to determine the physiological functions of FaHsfC1b in plant tolerance to heat stress by overexpressing it in Arabidopsis and analyzing the expression level of candicdate downstreamHsps conferring heat tolerance due to the overexpression of FaHsfC1b.According to OsHsfC1b(XP 015633152),full length open reading frame(ORF)which was 744 bp nucleotide sequences was obtained.Homology comparison of amino acid sequence and phylogenetic tree showed the sequence obtained was most closely related to HsfC1b clade.Therefore,it was designated as Festuca arundinacea HsfC1b(FaHsfC1b).The sequence of FaHsfC1b had been submitted to NCBI and the accession number is KY475613.Characteristic domains of Class C Hsf,such as a DNA binding domain(DBD),oligomerization domain(OD),also named as HR-A/B region,a nuclear localization signal(NLS)domain were found in FaHsfC1b.FaHsfC1b-eGFP recombinant plasmid was contructed and transformed into the protoplasts of Arabidopsis.The GFP signal was overlapped with the DAPI stained nucleus and same results were obtained in the root tips on transgenic Arabidopsis.This indicated that FaHsfC1b protein was localized in nucleus.For tall fescue,the expression levels of FaHsfC1b in roots and leaves increased during 48 h of exposure to heat,cold,osmotic andsalt stress.For heat treatment,the mRNA levels of FaHsfC1b increased in leaves and reach to the peak value(by 40 fold)within 1 h,while in roots FalHsfC1b increased in 4 h and reach to the peak(by 20 fold)in 48 h.We transformed pEarleyGate103-FaHsfC1b into Arabidopsis,survival rates of the four FaHsfC1b-overexpression transgenic lines(OE lines)under heat stress in MS medium plates were significantly greater than WT.And the transgenic seedlings did not show any different phenotype from the WT under non-stress conditions or under osmotic,salt stress and ABA treatment.Expression level of FaHsfC1b is detected in transgenic lines while none was detected in WT.Under heat treatment,mature transgenic plants had less wilted leaves and less yellow leaves after recovery than that of WT.Leaf photochemical efficiency(Fv/Fm)and chlorophyll content in OE lines were significantly higher than that in WT,which proved that FaHsfC1b helped to maintain the photochemical efficiency,chlorophyll content and membrane stability of Arabidopsis.And leaf relative electrolyte leakage in OE lines was significantly lower than that in WT.These indicated that FalHsfC1b elevated the heat tolerance of transgenic Arabidopsis.After 1 h of heat treatment at 37℃,the gene expression levels of AtHsp18.1-CI,AtHsp22.0-ER,AtHsp26.5-P(r)and AtHsp70b were significantly elevated in two OE lines and WT,while the expression levels of the four HSP genes were significantly higher in OE6 and OE16 than in WT.After heat treatment for 1 h,DAB and NBT stain assay conferred that the production of H2O2 and O2-in leaves of transgenic lines were less than that of WT after heat stress,which indicated that heat treatment was hurt less for transgenic lines.In this study,FaHsfC1b was found to response to heat,cold,osmotic and salt stress.FaHsfC1b enhanced the thermostability of transgenic Arabidopsis by elevated the expression level of HSP genes.The function of FaHsfC1b may provide reference for Molecular breeding in the future.