Functional Analysis And Application Of LmHsfs Genes To High Temperature Stress In Ryegrass

Italian ryegrass(Lolium multiflorum Lamk.)is a species of genus Lolium L.(Poaceae).It has the advantages of strong regeneration ability,fast growth rate and high yield after castration.As a typical cold-season forage and turfgrass,high temperature stress is the key factor restricting the growth and development of L.multiflorum.The temperature of southern China in summer is high and lasts for a long time,affecting the forage and lawn quality of ryegrass and causes death.Therefore,it is imperative to investigate the molecular mechanism of ryegrass in response to high temperature stress,and to improve the heat resistance of ryegrass.Heat shock transcription factors(Heat shock transcription factor,Hsf)are an important class of high temperature response transcription factors discovered in recent years.They can activate the expression of downstream heat-resistant genes under heat shock conditions,and play a vital role in the response of plants to heat stress.This study first analyzed the basic physicochemical properties and phylogenetic relationship of the ryegrass Hsf family,as well as the expression patterns of Hsf members under high temperature and drought stress conditions.Furthermore,three Hsf genes(LmHsfA2,LmHsfA4 and LmHsfA5)that were significantly induced by high temperature and drought were cloned,and then were transformed into Arabidopsis and rice to explore their high temperature stress tolerance function.The main results are as follows:1.According to the gene annotation by the genomic data of L.perenne,the Hsf genes of ryegrass were screened for non-redundancy.A total of 16 Hsf gene families from the ryegrass genome member were identified.After analyzing and comparing the structure of each Hsf protein sequence,16 LmHsfs were identified.There were eight,five and three belonged to category A,B,and C respectively.2.The heat-resistant variety ’bond’ of ryegrass was treated with high temperature and drought at 38℃.The results showed that LmHsfA2,LmHsfA4,LmHsfA5 and LmHsfB2 were significantly induced by high temperature and had the highest expression level at 1 h;LmHsfA2,LmHsfA4,LmHsfA5,LmHsfA8,LmHsfB1,LmHsfB2 and LmHsfC3 increased with increasing time.We combined the expression patterns under two stress conditions and previous study and selected LmHsfA2,LmHsfA4 and LmHsfA5 genes for further functional analyses.3.LmHsfA2,LmHsfA4 and LmHsfA5 genes were cloned using the ’Bond’ mixed cDNA as a template,and their ORFs contained 1212,1659 and 1215 nucleotides,respectively.Subsequently,the fusion expression vectors pBI121-GFP-LmHsfA2,pBI121-GFP-LmHsfA4 and pBI121-GFPLmHsfA5 were constructed.It was found that LmHsfA2 and LmHsfA4 proteins were located in the nucleus,and LmHsfA5 was located in nucleus and cell membrane.4.The overexpression vectors pCAMBIA1301-LmHsfA2,pCAMBIA1301-LmHsfA4 and pCAMBIA1301-LmHsfA5 were constructed.For each gene,two highly expressed transgenic lines were selected for heat-resistant phenotype analysis.The WT,Vector-control and overexpressing A.thaliana plants at 5 days of age were heat shocked at 46℃ for 60 min,and after 4 days of recovery at 22℃.The survival rate of LmHsfA2,LmHsfA4 and LmHsfA5 overexpressing lines was significantly higher compared to WT and Vector-control,suggesting that heterologous overexpression of LmHsfA2,LmHsfA4 and LmHsfA5 improves the ability of A.thaliana to endure high temperature.Meanwhile,the 3-week-old WT,Vector-control and overexpression lines were treated at high tempreture.The wilted leaves were significantly lower than the control,indicating that overexpression of LmHsfA2 and LmHsfA4 increased the heat resistance of transgenic A.thaliana plants,while LmHsfA5 plants had no significant difference compared with WT.In addition,on the 1/2 MS medium containing 300 mM mannitol,the germination rate of A.thaliana in overexpressed LmHsfA2 and LmHsfA5 plants were significantly higher than that of WT and Vector-control.This indicats that overexpression of LmHsfA2 and LmHsfA5 enhanced the drought tolerance of transgenic plants.5.In order to further explore the regulatory network of LmHsfA2,LmHsfA4 and LmHsfA5 in the heat resistance mechanism of L.multiflorum,the expressions of Hsp101,Hsp90,Hsp70,Hsp18.2,Hsp25.3,GOS1 and HSFA2 were detected using qRT-PCR in 35S::LmHsfA2,35S::LmHsfA4 and 35S::LmHsfA5 overexpressing plants.Under normal conditions,the expression levels of Hsp101,Hsp70 and Hsp18.2 in LmHsfA2 transgenic plants were significantly higher than those in wild type;the expression of Hsp101 was significantly higher than that of wild type in LmHsfA4 transgenic plants;the expression of Hsp101,Hsp70,Hsp18.2 and GOSL1 was significantly higher than that of WT in LmHsfA5 transgenic plants.Moreover,the expression levels of the above 7 genes were all up-regulated in LmHsfA2,LmHsfA4 and LmHsfA5 transgenic plants and WT after 0.5 h heat treatment.These results indicate that Hsp101,Hsp70 and Hsp18.2 may be the target genes of LmHsfA2,Hsp101 may be the target genes of LmHsfA4,and Hsp101,Hsp70,Hsp18.2 and GOSL1 may be the target genes of LmHsfA5,while suggesting that LmHsfA2,LmHsfA4 and LmHsfA5 are regulated by different downstream target genes to improve plant heat resistance.6.In the experiment of heterologous transformation of rice,PHGW-35S-LmHsfA2-4HA,PHGW-35S-LmHsfA4-4HA and PHGW-35S-LmHsfA5-4HA fusion expression vectors have been successfully constructed.The T0 generation transgenic rice plants of LmHsfA2,LmHsfA4 and LmHsfA5 were obtanined for further research by callus induction,subculture,screening,differentiation and rooting steps.In summary,three LmHsfs significantly induced by high temperature and drought were indentidied in this study.Through heterologous transformation model plant A.thaliana,it was found that overexpression of LmHsfA2,LmHsfA4 and LmHsfA5 improved the ability of A.thaliana to resist high temperature.Then,by detecting the expression of heat-responsive genes in overexpressed plants,it was initially revealed that LmHsfA2,LmHsfA4 and LmHsfA5 could improve the heat resistance of plants by regulating different downstream target genes.This study lays the foundation for the investigation of the molecular mechanism of heat resistance of ryegrass,and is helpful to enrich the genetic resources of L.multiflorum through gene editing,while adding in solving the problem of the popularization of ryegrass in high-temperature areas.