| Alfalfa is a forage crop with high yield,good quality,high protein content and strong resistance,and plays an important role in the development of the global livestock husbandry.Drought is the main limiting factor which affects the yield affecting the high yield of alfalfa.Alfalfa seeds and seeding leaves were used as experimental materials,they were pretreated with nitric oxide donor sodium nitroprusside(SNP)and nitric oxide scavenger c-PTIO,the methods of spectrophotometry and SDS-PAGE electrophoresis were used to analyze the antioxidative enzymes and their isoenzymes,nitrogenous substances and nitrogen metabolism-related enzymes during seed germination and seedling growth stage of alfalfa to explain the physiological mechanism of alfalfa’s drought resistance.The results showed that:1.Exogenous NO promoted seed germination and aboveground biomass of alfalfa under PEG stress.Under the stress of 15% PEG,the seed germination of alfalfa was significantly inhibited.The seed germination rate,germination energy,germination index and vital index of alfalfa increased by 8.34%,14.63%,28.77% and 34.06% with 0.1 mmol/L SNP,respectively.Under the stress of PEG the external application of 0.1 mmol/L SNP on seeding leaves,the fresh weight and dry weight of shoots,fresh weight and dry weight of roots,relative water content of leaves were significantly increased by 63.3%,100%,33.3%,61.5% and 27.0%(P<0.05),respectively.2.Exogenous NO showed different results on different antioxidant enzymes and isoenzymes of alfalfa under the stress of PEG.The activities of SOD and CAT were promoted,but the activity of POD was inhibited.NO mainly effected expressed content of isoenzymes.Compared with PEG treatment on alfalfa seeds,the external application of 0.1 mmol/L SNP,in seeds the POD activity was decreased by 32.72%,the SOD and CAT activities of seeds were increased by 10.48% and 23.60%.In seedling leaves,the POD activity was decreased by 32.18%,SOD and CAT activities increased by 9.81% and 43.37%.These showed that exogenous NO can effectively alleviate the oxidative damage of PEG.Under the stress of PEG,the external application of c-PTIO,it inhibited the activity of antioxidant system.According to the number and intensity of isozymes,with prolonging the PEG stress time,the bands of POD isoenzymes activity increased,the expression of isoenzymes of SOD and CAT were not significantly,but the strength of the enzyme band changed to some extent;CAT isoenzyme band had been kept at 2 bands all through,showed no significantly changes in strength.3.Under the stress of PEG,the external application of 0.1 mmol/L SNP,it significantly promoted the protein content and soluble protein content,but it reduced the accumulation of free amino acids,and increased the activities of NR,GS and GOGAT,GDH activity is inhibited.The content of protein and soluble protein in seed germination increased by 33.60% and 59.36%,and the free amino acid content decreased by 26.66%;the protein and soluble protein content increased by 32.23% and 16.48% in seedling leaves,the free amino acid content decreased by 11.36%.The addition of nitric oxide scavenger c-PTIO inhibited protein content and soluble protein content,but it increased the accumulation of free amino acids.Under the stress of PEG,the external application of 0.1 mmol/L SNP,the activities of NR,GS and GOGAT in seed germination increased by 23.85%,25.38% and 42.12%,but the GDH activity was decreased by 21.48%;the activities of NR,GS and GOGAT in the seedlings leaves increased by 13.97%,23.56% and 72.88%,but the GDH activity decreased by 35.90%.However,application of c-PTIO,the activities of NR,GS and GOGAT reduced,but GDH activity increased.These results showed that endogenous NO was involved in the regulation of nitrogen metabolism in alfalfa under drought stress. |
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