| Tall fescue(Festuca arundinacea Schreb.)is one of the most widely used coldseason turf grasses with outstanding resistance to stress,long green life,and resistance to trampling.As a cold-season lawn grass,tall fescue is sensitive to heat stress;the optimum growth temperature is 18-25℃.In subtropical and tropical areas of China,heat stress leads to severe retardation in growth and development,the decline of lawn quality and even death.Therefore,the widespread use of tall fescue is limited because of its weak heat tolerance.NAC proteins belong to plant-specific transcription factor,whose members are widely distributed in terrestrial plants.NAC family proteins are widely involved in many biological processes such as plant growth and development,biological and abiotic stress response and aging regulation.The discovery and functional analysis of NAC transcriptional factors in response to high temperature stress in tall fescue can provide scientific basis for improving the high temperature resistance of tall fescue,which has important theoretical significance and application value.In this study,germplasm resources collection,heat tolerance evaluation,heatresponsive gene mining and functional analysis were carried out,and the main results were as follows:1.Selection of high temperature resistant genotypes of tall fescue.A total of 188 tall fescue genotypes were collected from different countries and regions,64 of them were selected to conduct outdoor summer-crossing experiments by taking advantage of the summer high temperature in Wuhan.The heat tolerance of the tall fescue was measured,and 10 heat-sensitive and 10 heat-tolerant genotypes were obtained,which will provide the parental materials for cultivating new heat-tolerant varieties.2.Discovery and identification of FaNAC74 gene in tall fescue.Based on the database of tall fescue transcriptome in laboratory,a heat-responsive transcription factor FaNAC74 was isolated.The expression patterns of FaNAC74 among the heat-sensitive and heat-tolerant genotypes were detected under normal and heat treatment conditions.The FaNAC74 gene expression was significantly induced by heat stress,and the expression level was higher in the heat-tolerant genotype than that of heat-sensitive genotype,indicating its involvement in the heat stress response in tall fescue,indicating that FaNAC74 gene may be related to the heat tolerance of tall fescue.The coding region of FaNAC74 was cloned from the heat-tolerant genotype treated by heat stress.The multiple protein sequence alignment and the phylogenetic analysis revealed that FaNAC74 had high amino similarity with BdNAC74 from Brachypodium distachyon.A transmembrane region was predicted in the C-terminal region of FaNAC74.Furthermore,transient expression in tobacco epidermis showed that FaNAC74 protein was localized in the cell membrane.3.Functional analysis of FaNAC74.In order to research the function of FaNAC74 transcription factor,we constructed overexpression vector and transformed Arabidopsis model to obtain transgenic plants.The results showed that overexpression of FaNAC74 gene enhanced heat tolerance as indicated by a higher survival rate of the transgenic plants after 2 hours’ acclimatization at 42℃ and 3 days’ recovery compared to control plants,indicating that FaNAC74 positively regulated the heat tolerance of plants.4.Discovery of downstream genes of FaNAC74 transcription factor.In order to reveal the mechanism of heat tolerance at the molecular level due to the overexpression of FaNAC74,heat stress responsive genes were comprehensively investigated by RNA-seq.Through the analysis of the gene expression patterns,2043 differentially expressed genes,including 1261 up-regulated genes and 782 downregulated genes were found in control and heat-treated groups.Through GO analysis of DEGs,in biological process and molecular function,differential genes were mainly enriched in response to heat stress and the binding of unfolded proteins.In addition,we have identified several genes were stress-related,such as HSP70-16,HSP18.1,ANAC13,and DREB2 C were significantly higher in the overexpressed plants than the control plants.Its response to high temperature stress was most significantly different from the binding of intracellular unfolded proteins.The above results indicate that FaNAC74 may respond to high temperature stress by regulating the expression of downstream stress response genes.In conclusion,the heat tolerance of the tall fescue materials was comprehensively evaluated and a heat-stress related transcription factor FaNAC74 was identified.Furthermore,our study revealed that FaNAC74 gene is a positive regulator mediating plants response to heat stress.We carried out the research on the characteristic identification and functional analysis of this transcription factor,which provided scientific basis for the breeding of new high temperature resistant and tall fescue germplasm. |
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