Comparative Analyses Of Bermudagrass In Response To Drought, Salt And Submergence Stresses

In recent years, loss from natural disasters increased obviously. Drought, salt and flooding are major abiotic factors limiting plant growth and development which happened from time to time worldwide. Under drought stress condition, limited water supply greatly decreases leaf water content and causes tissue dehydration which is characterized by fundamental changes at physiological, biochemical, molecular and cellular levels. Salt stress condition caused mainly by NaCl resulted in both ionic and osmotic stresses in plants. Flooding is another form of water stress that results from excess water in the root environment, which affects about 10% of the global land area. Flooding, including waterlogging and submergence, has a severe negative influence on the productivity of crops. As one of the most important warm-season turfgrasses, bermudagrass (Cynodon dactylon) is widely used in the construction of golf courses, sport fields, lawns, and wetland vegetation restoration. Previous studies indicated that the bermudagrass exhibited tolerance to several abiotic stresses including drought, salt and submergence. However, limited information is available for stress tolerance mechanisms at proteomic and molecular levels.In this study, comparative physiological, proteomic, and metabolomic analyses were performed to characterize mechanisms of bermudagrass(Cynodon dactylon) in response to abiotic stresses. Firstly, we screened drought tolerance of different bermudagrass cultivars; Second, we compared common and contrasting responses of bermudagrass to drought and salt stresses; Third, we characterized contrasting changes caused by drought and submergence in bermudagrass; Finally, we performed transcriptomic analysis of bermudagrass in response to drought, salt and submergence stresses. Through the analysis of differentially expressed genes and proteins, we tried to reveal molecular mechanisms of bermudagrass responses to drought, salt and submergence stresses. The main contents and results were as following:(1) Identification of drought-responsive proteins in both leaves and stems of two bermudagrass varieties contrasting in drought stress tolerance, including drought sensitive variety (’Yukon’) and drought tolerant variety (’Tifgreen’). Through comparative proteomic analysis,39 proteins with significantly changed abundance were identified, including 3 commonly increased and 2 decreased proteins by drought stress in leaves and stems of ’Yukon’ and ’Tifgreen’ varieties,2 differentially regulated proteins in leaves and stems of two varieties after drought treatment,23 proteins increased by drought stress in ’Yukon’ variety and constitutively expressed in ’Tifgreen’ variety, and other 3 differentially expressed proteins under control and drought stress conditions. Among them, proteins involved in photosynthesis (PS), glycolysis, N-metabolism, tricarboxylicacid (TCA) and redox pathways were largely enriched, which might be contributed to the natural variation of drought resistance between ’Yukon’ and ’Tifgreen’ varieties.(2) Contrasting proteomic and metabolomic responses of bermudagrass to drought and salt stresses:The physiological results indicated that osmolytes accumulations, ROS level and antioxidant enzyme activities were extensive changed by drought and salt stresses. Through comparative proteomic analyses, we successfully identified a total of 77 proteins involved in photosynthesis, oxidative pentose phosphate, glycolysis, and redox metabolic pathways when exposed to drought and salt stresses. Among them,36 proteins were commonly regulated by both treatments and 12 proteins were specifically regulated by drought or salt. Totally 15 proteins were involved in carbon metabolic pathway. Moreover, contents of 37 metabolites including amino acids, organic acids, sugars, and sugar alcohols were regulated by drought and salt treatments. Among them,18 commonly modulated metabolites were involved in carbon and amino acid metabolic pathways. Drought treatment caused less accumulation of sugars and sugar alcohols and increased ROS level in bermudagrass which led to relatively more severe cell membrane and lower survival rate when compared to salt treatment.(3) Contrasting changes of bermudagrass in response to drought and submergence stresses:The results showed that significant physiological changes were observed after drought treatment, while only slight changes after submergence treatment, including compatible solute contents, ROS levels and antioxidant enzyme activities. Proteomics results showed that 81 proteins regulated by drought or submergence treatment were identified by MALDI-TOF-MS. Among them,76 proteins were modulated by drought stress with 46 increased abundance and 30 decreased abundance. Forty-five showed abundance changes after submergence treatment with 10 increased and 35 decreased. Pathway enrichment analysis revealed that pathways of amino acid metabolism and mitochondrial electron transport/ATP synthesis were only enriched by drought treatment, while other pathways including photosynthesis, biodegradation of xenobiotics, oxidative pentose phosphate, glycolysis and redox were commonly over-represented after both drought and submergence treatments. Metabolomic analysis indicated that most of the metabolites were up-regulated by drought stress, while 34 of 40 metabolites contents exhibited down-regulation or no significant changes when exposed to submergence stress, including sugars and sugar alcohols. These data indicated that drought stress extensively promoted photosynthesis and redox metabolisms while submergence stress caused declined metabolisms and dormancy in Cynodon dactylon. Taken together, the quiescence strategy with retarded growth might allow bermudagrass to be adaptive to long-term submerged environment, while activation of photosynthesis and redox, and accumulation of compatible solutes and molecular chaperones increased bermudagrass tolerance to drought stress.(4) Transcriptomic analysis and gene function analysis in bermudagrass:Based on RNA seq analysis, we identified thousands of genes which showed differential expression level changes after drought, salt and submergence treatments. CdPYL8 gene, the Arabidopsis thaliana AtPYL8 homologous gene, was selected from transcriptomic data and successful cloned. Then we did functional analysis for the gene. First, subcellular localization of CdPYL8 was determined by transient expression of CdPYL8::GFP fusion in transformed onion epidermal cells. CdPYL8::GFP fusion protein fluorescence was localized at plasma membrane, nucleus and cytoplasm. Second, to dissect the role of AtPYL8 in ABA signaling pathway, protoplast transformation assay was performed to reconstruct the ABA signaling pathway. The result showed that CdPYL8 was involved in ABA signaling, and exhibited similar function to Arabidopsis ABA receptors. In.order to test whether CdPYL8 functioned as an ABA receptor in the ABA signaling pathway, yeast two hybrid assay was used to identify the interaction between CdPYL8 and AtABIl. The result showed CdPYL8 interacted with AtABIl, further confirming role of the CdPYL8 in ABA signaling pathway.