Study On Heat Tolerance Of CtHsfA2b From African Bermudagrass

African bermudagrass(Cynodon transvaalensis Burtt-Davy)is warm season turfgrass,which is heat-tolerant and widely used for breeding of hybrid bermudagrass.As sessile organisms,plants had to construct specialized defense systems to maintain growth and propagation under HS.As the terminal components of the heat signal transduction chain,HSFs bind to the heat stress elements(HSEs)present in the promoter of downstream heat-inducible genes and play a central role in the HSR.Heat stress transcription factor A2(HsfA2)is a key regulator of plant responses to high temperature.The objectives of this study were to isolate CtHsfA2b gene from African bermudagrass,perform ectopic expression in Arabidopsis and investigate the physiological functions of CtHsfA2b conferring heat tolerance.To determine heat tolerance mediated by the A2 type HSF before we apply it to heat tolerance molecular breeding of cool-season turfgrass.1 We obtained cDNA sequence of HsfA2 gene consisted of 1386 bp nucleotides with a 1095 bp ORF encoding a 365-residue polypeptide from African bermudagrass.Homologous alignment of the deduced amino acid sequences revealed that CtHsfA2b had high sequence identity to HsfA2b of Setaria italica,Oryza sativa and Brachypodium distachyon.The gene was classified into A2b subgroup of HSF by the cluster analysis and named African bermudagrass HsfA2b(CtHsfA2b,GenBank accession number:KU156630).2 CtHsfA2b-eGFP was constructed and transformed into Arabidopsis mesophyll protoplasts.The completely overlapped fluorescence signal of DAPI and GFP demonstrated that CtHsfA2b was localized in nucleus.After obtaining CtHsfA2b transgenic Arabidopsis with the plant expression vector pEarleyGate 103 which also has a GFP encoding region,we further confirmed that CtHsfA2b protein was localized in nucleus by scanning root tips of transgenic plants.3 qRT-PCR analysis demonstrated that CtHsfA2b exhibited rapid response to heat stress,as the expression level increased within 30 min and reached the highest level by 1h of 42℃ heat treatment.The expression level of CtHsfA2b in leaves was higher and response earlier than that in roots.After reaching to the highest expression at 1h,the gene expression attenuated and return to the initiate level after recovery to 30℃ normal growth temperature.4 CtHsfA2b expression vector was transformed into Arabidopsis for ectopic expression analysis.Results of qRT-PCR and GFP observation showed that CtHsfA2b gene and its GFP-fused protein were expressed in 35S:CtHsfA2b and 35S:CtHsfA2b/hsfa2 but not in hsfa2 mutant and WT.Net photosynthetic rate,photochemical efficiency and chlorophyll content of 35S:CtHsfA2b transgenic lines were significantly higher than that of WT after heat treatment,which demonstrated ectopic expression of CtHsfA2b improved heat tolerance of Arabidopsis seedlings.5 Recovery from heat stress was also examined,which confirmed that the heat treatment was lethal to the hsfa2 mutant plants,but had less damage to WT and 35S:CtHsfA2b/hsfa2.Ectopic expression of CtHsfA2b can rescue the heat-sensitive phenotype of hsfa2 mutant to the wild type level at the adult seedling stage.6 Transgenic plants up-regulated a series of downstream genes,include antioxid ant gene(AtApx2)and HSP genes[AtHsp18.1-CI,AtHsp22.0-ER,AtHsp25.3-P and A tHsp26.5-P(r),AtHsp70b and AtHsp101-3].All the genes exhibited typical heat respo nse characteristics that induced by high temperature.7 The accumulation of Apx-related superoxide radical and hydrogen peroxide in the WT was higher than that in transgenic plants after 1 h of heat stress,indicate d that AtApx2 may play important role in enhancing oxidation resistance of transge nic plants.Y1H and luciferase(LUC)assay in Arabidopsis protoplasts showed that CtHsfA2b could bind to the HSE on ProAtApx2,and then trigger the activation of report er gene expression.These results suggested that CthsfA2b was heat-inducible and exhibited rapid response to heat stress.CtHsfA2b could play roles in heat protection by up-regulating antioxidant and chaperoning defense mechanisms.CtHsfA2b has potential to be useful as a candidate gene to genetically modify cool-season species for improving heat stress tolerance.