Mechanisms Of Cold Stress Response And Functional Analysis Of DREB1 Genes In Bermudagrass And Tall Fescue

Turfgrass plays important roles in landscaping,urban construction and ecological protection.Low temperature stress is the main factor affecting the quality and green period of turfgrass.Bermudagrass(Cynodon dactylon)is a typical warm-season turfgrass which is relatively sensitive to low temperature,while tall fescue(Festuca arundinacea)is a cool season turfgras,which is more tolerant to low temperature.Characterization of cold tolerance mechanism of bermudagrass and tall fescue is of great significance for screening and breeding of turfgrass germplasms with excellent cold-tolerant traits.In this study,the cold tolerance of tall fescue varieties was screened.Cold stress responses of bermudagrass and tall fescue at physiological level were compared.The RNA-seq was used to analyze transcriptomic changes of bermudagrass and tall fescue after low temperature treatment.The cold-responsive gene DREB1 A was cloned and functionally characterized.The main results of this study are as follows:1.Freeze tolerance of 6 species of tall fescue varieties was screened.The results indicated that the tall fescue varieties showed different tolerances,and ‘Essential' is most tolerant while the ‘SlopProtect' is most sensitive to low temperature.After the freeze treatment,freezing tolerant cultivar ‘Essential' exhibited lower EL,accumulated higher soluble sugar and proline,and showed higher CAT and SOD activities when compared with freezing intolerant ‘SlopProtect'.Expression profiles showed that freezing tolerant cultivar only activated specific FaDREB1 unigenes,while freezing intolerant cultivar activated all six FaDREB1 s during cold acclimation period,indicating freezing tolerant and intolerant tall fescue cultivars developed different approaches to cope with low temperature through modulating different subsets of FaDREB1 unigenes.2.The physiological changes of bermudagrass and tall fescue under cold stress condition were studied.The freeze treatment significantly inhibited the growth of bermudagrass,but had less inhibitory effect on growth of tall fescue.Tall fescue accumulated higher amount of osmoprotectants such as soluble sugar,proline and MDA after freeze treatment when compared to bermudagrass.Moreover,the activity of POD and CAT in tall fescue was higher,while SOD active in tall fescue is lower than that in bermudagrass.3.Transcriptomic changes of bermudagrass and tall fescue in response to low temperature for 6h and 72 h was analyzed using RNA-seq method.After 6h cold treatment,expression level of 2424 and 2626 unigene in the bermudagrass and tall fescue were significantly changed,respectively.Meanwhile,7431 and 4450 unigene in the bermudagrass and tall fescue were modulated by cold treatment,respectively.Among them,486 and 1595 unigenes were co-regulated after 6h and72 h cold treatments.GO term analysis showed that sugar,cellulose,embryonic development carbohydrate,ethylene signaling pathway GOs were enriched in bermudagrass,while cold,heat,salt,amino acid,lipid and carbohydrate metabolism related GOs were enriched in tall fescue after 6h cold treatment.Additionally,cellular process metabolism,DNA metabolism,carbohydrate,sugar,pigment metabolism and hormone related GOs were enriched in bermudagrass,while GOs including nuclear and protein pathways,organic acid and vitamin biosynthesis process were enriched in tall fescue after cold treatment for 72h.4.According to the transcriptome data,the DREB1 gene was cloned and the detailed functions were characrterized.Phylogenetic analysis indicated that DREB1 genes were highly homologus to OsDREB1 A genes.FaDREB1 A and CdDREB1 A transgenic Arabidopsis were created and the results showed that both genes were highly expressed in transgenic Arabidopsis.After low temperature treatment,the transgenic lines showed improved cold tolerance as evidenced by less leaf wilting and yellowing,lower EL,higher proline and soluble sugar contents.In addition,the promoter regions of FaDREB1 A and CdDREB1 A genes were cloned and motif was analyzed.Four promoter elements related to abiotic stress were found in the promoter region of FaDREB1 A,including ABRE,methyl jasmonate response elements(CGTCA,TGACG)and LTR element,while promoter of CdDREB1 A contains only jasmonate response elements(CGTCA,TGACG)and LTR elements.Interestingly,promoter of FaDREB1 A contains 3 ABRE and 2 LTR elements,while those of CdDREB1 A are 0 and 1,respectively.