Physiological And Molecular Mechanisms Of Response To Drought Stress In Different Drought-resistant Alfalfa (Medicago Sativa L. ) Varieties

Drought is a global problem restricting ecological construction,plant distribution and productivity.The arid and semi-arid regions account for almost 35%of the total land area,and drought trends increased significantly year by year.Drought-induced loss in crop yield probabaly exceeds the losses caused by all other environmental factors.Therefore,it is imperative to under-stand the morphological,physio-biochemical and molecular mechanisms of plant response to drought stress,and to explore the regulatory/functional genes involved in plant drought stress re-sponse and clarify their functionary mechanism,which will provide theoretical and practical guid-ance for enhancing plant drought resistance,breeding of drought-resistant varieties and develop-ing agriculture with drought-resistant and high-yield.Alfalfa?Medicago sativa L.?is a well-known perennial leguminous forage that is grown worldwide for its natural advantages in the improvement of ecological environment and soil and water conservation.However,the increasing drought poses a serious threat to the planted acreage and yield of alfalfa.The introgression of drought-resistant germplasm into breeding programmes is an effective approach to counterbalance the anticipated losses in yield.By comparing the differ-ential morphological,physiological and molecular responses of different drought-resistant alfalfa varieties to drought stress will contribute to identify the key adaptive mechanisms of alfalfa,thus providing a theoretical basis for enhancing the drought resistance of alfalfa.In this study,firstly,the differential morphological and physiological responses of three different drought-resistant al-falfa varieties at the seedling stage were compared under different degrees and durations of drought stress induced by polyethylene glycol 6000?PEG-6000?with strong drought-resistant Longzhong as test material as well as moderate drought-resistant Longdong and drought-sensitive Gannong No.3 as reference materials.Then,the multi-omics analysis with integration of tran-scriptomics,proteomics and metabolomics was used to understand the molecular mechanisms in the roots of drought-resistant Longzhong and drought-sensitive Gannong No.3 seedlings and to identify several key stress-responsive candidate genes,proteins and metabolites.Finally,the key adapation mechanisms of strong drought-resistant Longzhong in response to PEG-induced drought stress were comprehensively analyzed at morphological,physiological and molecular lev-els,the main results are summarized as follow:?1?Drought stress simulated by-1.2 MPa PEG-6000 for nine days was a sensitive stress treatment condition for distinguishing the growth and physiological responses of alfalfa varieties with differing drought-resistance.The root system was the key organ system that might contribute to the drought resistance of alfalfa seedlings.The drought resistance of alfalfa was closely associ-ated with the enhanced antioxidative protection and declined lipid peroxidation,meanwhile,ascorbate-glutathione?AsA-GSH?cycle was an important mechanism to improve the drought re-sistance of alfalfa.Under long-term drought stress,Longzhong showed the highest leaf water re-tention capacity,photosynthetic performance and osmoregulation capacity,the lowest lipid perox-idation and highest activities of antioxidase?GPX,MDAR,DHAR and GR?and increased expres-sion levels of antioxidase?MsGPX,MsMDAR,MsDHAR and MsGR?,which were mainly involved in the AsA-GSH cycle to maintain a balance between intracellular ROS production and scaveng-ing.Gannong No.3 showed the highest lipid peroxidation and the lowest antioxidant enzyme ac-tivities and gene expression patterns.Longdong had a moderate ability to maintain photosynthesis performance and coordinate enzymatic and non-enzymatic antioxidant systems.?2?After-1.2 MPa PEG-simulated drought stress for 9 days,RNA sequencing-based tran-scriptomics analysis was performed to compare the global transcriptional profiles in the roots of different drought-resistant alfalfa varieties.A total of 18,973 differentially expressed genes?DEGs?involved in drought stress response were identified from two alfalfa varieties.Among these DEGs,6,605 up-and 5,980 down-regulated genes were present in Longzhong,and 8,049 up-and 6,675down-regulated genes were present in Gannong No.3,while both alfalfa varieties shared 4,013up-and 4,323 down-regulated genes.Most of these DEGs were involved in 7 functional pathways,including carbohydrate metabolism,lipid metabolism,amino acid metabolism and secondary me-tabolism,signal transduction,cell defense and intracellular transport,transcription and translation-al regulation and other unknown functional pathway.Overall,drought stress significantly induced the up-regulation of genes implicated in structural carbonhydrate metabolism,lipid metabolism?glycerolipid metabolism,cutin,suberine and wax biosynthesis?,amino acid metabolism,second-ary metabolism,signal transduction(Ca²⁺signaling,ethylene and jasmonic acid biosynthesis),cell defense?tubulin and peroxisome?and aminoacyl-tRNA biosynthesis in the roots of Longzhong compared with those in roots of Gannong No.3.In contrast,the expressions of transcription fac-tors and genes involved in transcriptional and translational regulation were more susceptible to drought stress in Gannong No.3.?3?After-1.2 MPa PEG-simulated drought stress for 9 days,using isobaric tags for relative and absolute quantification?iTRAQ?,142 differentially accumulated proteins?DAPs?were identi-fied from two alfalfa varieties,including 47 up-and 24 down-regulated proteins in Longzhong,41up-and 49 down-regulated proteins in Gannong No.3,and 19 proteins?13 up-and 6 down-regulated?shared by both varieties.Most of these DAPs were mainly involved in carbohydrate and energy metabolism,stress and defense,protein metabolism,cell membrane and transport,sig-nal transduction,as well as cell wall and cytoskeleton metabolism.Drought stress significantly increased the abundance of proteins associated with reactive oxygen species?ROS?detoxification,secondary metabolism,protein processing,transmembrane transport,cell wall and cytoskeleton metabolism in the roots of Longzhong,and significantly altered the signal transduction-related proteins in roots of Gannong No.3.?4?After-1.2 MPa PEG-simulated drought stress for 9 days,liquid chromatography tandem mass spectrometry?LC-MS/MS?-based non-targeted metabolomics analysis was used to compare the global metabolic profiles of roots of different drought-resistant alfalfa varieties.A total of 79differentially expressed metabolites?DEMs?were identified,including 38 up-and 21 down-regulated metabolites in Longzhong,39 up-and 27 down-regulated metabolites in Gannong No.3,46 DEMs?30 up-and 16 down-regulated?simultaneously expressed in both varieties.The altera-tions in the contents of amino acids and their derivatives,lipids?glycerolipids and glycerophos-pholipids?,secondary metabolites?mainly including organic acids,isoflavones,and flavonoids?,nucleotides and their derivatives and other metabolites were the common metabolic responses of alfalfa to drought stress.Furthermore,metabolites in roots of Gannong No.3 were significantly reduced by drought stress,while most of metabolites related to amino acid metabolism,phe-nylpropanoid synthesis,purine and pyrimidine metabolism were significantly accumulated in the roots of Longzhong compared with Gannong No.3.?5?At the morphological and physiological levels,strong drought resistance of Longzhong seedlings was associated with its effective mechanism of biomass allocation,stronger leaf water retention capacity and osmotic adjustment ability,lower lipid peroxidation and ROS accumulation,as well as stronger enzymatic and non-enzymatic antioxidant defense systems.At the molecular level,Longzhong could dramatically induce the expressions of stress-responsive genes related to carbohydrate metabolism,lipid metabolism,amino acid metabolism and phenylpropanoid biosyn-thesis,signal transduction,cell defense and aminoacyl-tRNA biosynthesis;effectively activate the stress-responsive proteins involved in stress defense and detoxification,transmembrane transport and cell wall modification,maintain the balance of protein processing and degradation.Further-more,Longzhong could effectively improve the abilities of osmotic adjustment and ROS detoxifi-cation and maintain cell membrane stability by promoting the anabolic pathways of key metabo-lites,and ultimately enhance its drought resistance.The isoflavone biosynthesis pathway was a key metabolic pathway of Longzhong in response to drought stress.The present study preliminarily clarified the major morphological,physiological and molecu-lar adaptation mechanisms of drought-resistant alfalfa variety in responses to drought stress.Fur-thermore,the integrated analysis of transcriptome,proteome and metabolome substantially ex-panded the understanding of drought-resistant molecular mechanism of alfalfa and identify several key metabolic pathways and critical drought-responsive genes,proteins and metabolites involved in the responses of alfalfa to drought stress.However,the complex regulation mechanisms under-lying drought-resistant candidate genes,proeteins and metabolites remain to be further studied.