The Physiological And Molecular Mechanisms Of Tibetan Wild Elymus Nutans Responses To Cold Stress

Low temperatures are major environmental fators that limit plant productivity in northern and alpine regions and affect the geographical distribution of plants in nature.According to statistics,the global annual losses of crop production caused by cold stress is up to several hundred billion dollars.So an investigation of low temperature adaptation mechanism in plant and improvement of its adaptability has important scientific value and practical significance.Elymus nutans Griseb.is widely distributed in Tibet under extreme conditions,which acts as an ideal forage to study the adpative mechanism of cold stress.To understand the mechanism of how E.nutans resists low temperature limitations,an investigation of the physiological responses and gene expression profile of E.nutans under cold stress is imperative.In this study,Tibetan wild E.nutans(Damxung,DX)and Gannan E.nutans cultivar(Gannan,GN)were exposed to cold stress,the photosynthetic responses,endogenous hormone levels,carbohydrate metabolism and transcriptomic characteristics were investigated.The main findings were summarized as follows:(1)Eight natural accessions of E.nutans that originate from a wide range of altitudes show clinal variation in cold tolerance.Physiological adaptations and stimulation of key genes occur over an altitudinal gradient in this plant.Natural variations in the accumulation of sugars and dynamic changes in phytohormone levels under cold stress showed different acclimation mechanisms to that stress.The induction of EnCBFs and EnCOR14 a genes may exert crucial roles in the regulation of the early cold stress response and protect cell membrane of high-altitude accessions against oxidative damage.(2)Maintenance of higher Pn and Pmax under cold stress and recovery in cold-tolerant DX could be attributed to lower diffusion limitations and the ability to rapidly recover from metabolic limitations.Three photosynthetic enzymes,in addition to carbohydrate accumulation,may contribute to higher Pn maintenance in cold-stressed DX compared to cold-sensitive GN during cold stress and recover.The lack of full recovery of Pn,gs,gm,and Tr after removal from cold stress in both genotypes indicated permanent cold induced damage in E.nutans stomata.(3)Both exogenous melatonin and ABA have the ability to alleviate cold stress inducedoxidative damage.Exogenous melatonin improved cold tolerance via induction of endogenous melatonin production,which might serve as a second messenger activating downstream cold-responsive genes such as EnCBF9,EnCBF14,and EnCOR14 a,thus stimulating antioxidant defense systems alleviating ROS accumulation-induced oxidative damage.Further investigations revealed that this melatonin-induced antioxidant defense may function via ABA-dependent and ABA-independent signaling pathways.(4)To comprehensively survey the genes associated with cold response in E.nutans,48 cDNA libraries were constructed from leaves and crowns in DX and GN with 0 h,3 h,24 h,and 5 d of cold treatments.A total of 317.64 Gb data were obtained from 48 libraries.In total5600 DEGs were detected in at least one sample of the leaves and crowns of both genotypes during the cold-stress course,when compared with their respective controls.Hierarchical clustering revealed that the DEGs clustered separately in leaves and crowns of DX and GN,indicating changes in genotype-and-tissue specific gene expression under cold stress.(5)Based on GO and KEGG enrichment analysis,we found that BR/JA mediated signaling pathway in DX were detected in the early cold response,while those genes were inhibited in GN.Phenylpropanoid biosynthesis,fructan biosynthesis,and alpha-linolenic acid metabolism in DX leaves and crowns may contribute to better adaptation to cold stress.(8)Co-expression network analysis showed that twenty-six of co-expressed genes(22 in DX,4 in GN)were strongly interconnected,with each gene having more than 50 edges.These genes were therefore defined as hub genes.These hub genes were directly connected to each other via 428 edges in DX and 12 edges in GN,forming a highly interconnected subnetwork.84.6 % of hub genes were annotated as COR(COR413PM1,COR410,DHN5,etc.),reflecting their central roles in the acquisition of cold tolerance.Taken together,we conducted the first deep-sequencing transcriptome profiling and cold stress regulatory network analysis of E.nutans,an important alpine plant with inherited cold tolerance.The results presented in this paper will be informative for gene discovery and the molecular mechanisms related to plant cold tolerance.