Study On Germination Diversity And Salt Resistance Mechanisms Of Melatonin And Ascorbic Acid In Alfalfa

Alfalfa（Medicago sativa L.）is a high-quality forage widely grown for grazing,silage and hay making.Seed germination is the beginning of the life cycle of seed plants,which not only directly affects seedling survival,individual adaptation and plant life history,but also plays a key role in population persistence,population dynamics and community structure,and is an important factor affecting crop yield and quality.High temperature or low temperature inhibits the germination of alfalfa.In addition,there is a genetic diversity in germinability and germination rate at different temperatures.NaCl and nitrate are two common stresses in alfalfa planting,which seriously affect the growth,development,quality and yield of alfalfa.Melatonin（MT）and ascorbic acid（AsA）have excellent resistance to many stresses.Hence,they have great potential to improve plant ability to withstand adversity,promote growth and increase yield.Based on them,the physiological and biochemical pathways such as photosynthetic pigment synthesis,photosynthetic gas parameters,stomatal movement,ion toxicity,osmotic homeostasis,lipid peroxidation,redox homeostasis,cell integrity,nitrogen metabolism,energy metabolism,hormone synthesis and gene expression,as well as microscopic techniques to observe stomatal characteristics and leaf tissue structure,were used to comprehensively investigate the diversity of alfalfa germination response to temperature,and the mechanisms of MT and AsA regulating alfalfa response to NaCl and Nit stresses.The main results are as follows:1.The seed germination test of 391 alfalfa cultivars were selected to investigate the diversity of germination response to different temperatures（5,15 and 34℃）by traits such as germination rate（G）,lag time to start germination（tc）,maximum germination rate（α）and germinability（ymax）.The results indicate that the range of G at 5,15 and 34°C was 34.5-96.8%,54.6-97.0%and 1.5-93.6%,respectively.Both high temperature and low temperature significantly（p<0.05）inhibited G,while high temperature was significantly lower than low temperature（p<0.05）,even the change was greater.The variation ranges of tc＿5 and tc＿15were 96.6-178.4 h and 17.4-21.9 h（p<0.05）,respectively.At 15°C,αvaried greatly（0.91-8.85）with a diversity,and ymax was relatively stable（62.78-100%）.G was positively correlated at different temperatures,but negatively correlated with tc.There were positive correlations among G,αand ymax（R=0.6,R=0.77 and R=0.58）.Dormancy and tc were negatively correlated at low temperature.Dim 1 and Dim 2 explained 75.5%of the total variables,and the variables of PC 1-2 were higher than the average contribution rate were G＿5,G＿15,tc＿15and G＿34,respectively.Therefore,temperature affects the germination of alfalfa,and high temperature has a stronger inhibitory effect,and germination has diversity in response to different temperatures.However,the grouping of genetic markers has no relationship with germination traits.2.Alfalfa under 150 mmol L^-1 NaCl stress was sprayed with exogenous 100μmol L^-1 MT,the results show that MT enhances NaCl resistance of alfalfa through the following ways:（1）MT inhibites chlorophyll degradation and regulates photosynthetic gas parameters and stomatal movement,thus enhancing photosynthetic capacity under NaCl stress.（2）MT regulates osmotic regulatory substances and alleviates osmotic stress caused by NaCl stress.（3）MT maintains ion homeostasis by reducing Na⁺toxicity and promoting mineral nutrition.（4）MT upregulates antioxidant system gene（MsCu/Zn-SOD,MsFe-SOD,MsMn-SOD,MsCAT,MsPOD and MsAPX）expression,activity and content,thus reducing excessive ROS and lipid peroxidation,and maintaining the integrity of the cell membrane.These parameters correlate with each other,and are divided into PC 1（89.2%）and PC 2（4%）to explain 93.2%of total variables,and the top three contributors are MsCAT,MsMn SOD and MsPOD.Hence,they co-regulate and improve the stress tolerance.3.Alfalfa under 200 mmol L^-1 Nit（KNO₃+Ca（NO₃）₂）stress was sprayed with exogenous100μmol L^-1 MT,the results show that MT could promote the growth of alfalfa under both normal condition and Nit stress.Nit stress caused ion toxicity and inhibited growth,while MT improved the growth parameters and leaf tissue structure,increased the contents of leaf cell contents,chloroplasts and starch granules,and reduced intracellular protoplast agglutination,plasmolysis and cell swelling.MT also regulated photosynthesis,stomatal movement,ion balance and osmotic homeostasis,and enhanced antioxidant system,alleviated oxidative damage caused by ROS,and reduced NO₃^-and NH₄⁺ionic toxicity,thus enhancing alfalfa tolerance to Nit stress.These regulatory pathways were correlated,with PC1（66.4%）and PC2（7.4%）explaining 73.8%of the total variables.Moreover,the most important variables were osmoregulatory substances and non-enzymatic antioxidants（GB,GSH,and proline）.Correlation analysis and principal component analysis reveal that these pathways are jointly involved in regulating the growth of alfalfa and its adaptability to Nit stress.4.Alfalfa under 200 mmol L^-1 Nit（KNO₃+Ca（NO₃）₂）stress was sprayed with exogenous100μmol L^-1 AsA.The results show that AsA alleviated the effects of Nit on growth parameters,photosynthetic pigments and photosynthetic gas parameters,decreased soluble sugar,proline and GB,increased antioxidant enzyme activity and non-enzymatic antioxidant content,significantly reduced ROS damage and lipid peroxidation,and maintained cell membrane integrity（p<0.05）.In addition,AsA enhanced the activities of nitrogen metabolizing enzymes（NR,GS,GOGAT and GDH）,and decreased the accumulation of TN,NO₃^--N and NH₄⁺-N under stress.AsA increased the levels of energy co-factors ATP and EC,decreased the levels of ADP and AMP.AsA promoted the synthesis of GA,and inhibited the synthesis of ABA,JA and ETH.Multiple parameters were significantly correlated,and 86.4%of the total variables were explained by PC 1（66.1%）and PC 2（20.3%）,among which APX,MDA,EL,JA and ETH were the most contributing variables.Therefore,AsA enhances photosynthesis,osmotic balance,redox homeostasis,nitrogen metabolism and energy metabolism under Nit stress,and combine with hormone synthesis to jointly reduce NO₃^-and NH₄⁺ion toxicity,osmotic stress and oxidation damage,so as to alleviate the inhibitory effect of Nit on the growth of alfalfa.5.Alfalfa under 200 mmol L^-1 Nit（KNO₃+Ca（NO₃）₂）stress was sprayed with exogenous MT and AsA.The results show that MT and AsA promoted the growth of alfalfa under normal condition and Nit stress,and the effects of MT and AsA had no significant difference（p>0.05）.MT and AsA improved some nutritional quality parameters.Relative feed value was the nutritional quality parameter that contributed the most.MT and AsA increased relative feed value by 1.3%and 0.8%,respectively;and under Nit stress,MT and AsA increased the relative feed value by 4.7%and 4.8%,respectively.Therefore,MT and AsA can improve nutritional quality under normal condition and Nit stress.In summary,the germination of alfalfa seed has diversity in response to different temperatures,which is of great significance to the breeding,introduction and domestication,and protection of genetic diversity of alfalfa.The mechanisms of MT and AsA in relieving NaCl and Nit stress,promoting growth and improving nutritional quality not only enrich the theoretical basis of alfalfa response to stress,but also provide a reference for practical application.