| Atropa belladonna L.is an important herb in the Atropa of the solanaceae.It is the only source of Tropinge Alkaloids?TAs?prescribed by the Chinese Pharmacopoeia Committee?2011 edition?.The major secondary metabolites hyoscyamine and scopolamine are widely used in clinical practice as an anticholinergic drug for anesthesia,pain relief,control of motion sickness,improve microcirculation,treatment of Parkinson's disease and detoxification etc.The market demand is huge.The degree of soil salinization in China is becoming more and more serious.NaCl stress threatens the growth and development of plants and is one of the key factors limiting plant cultivation.The addition of sodium nitroprusside?SNP?to provide exogenous NO enhances plant resistance ability to stress and regulates secondary metabolite content has been extensively studied.NO acts as a signal molecule to mediate plant signal transduction under adversity.In order to clarify the effects of different concentrations of SNP on the physiological characteristics and secondary metabolism regulation of Atropa belladonna L.seedlings under NaCl stress,we used Atropa belladonna L.seedlings as materials.100 mmol·L-1NaCl solution was used to simulate salt stress,and different concentrations of SNP treatment group were set:0 mmol·L-1,0.05 mmol·L-1,0.1mmol·L-1,0.2 mmol·L-1,0.5 mmol·L-1.The effects of different concentrations of SNP on the photosynthetic characteristics,physiological indexes of adversity,nitrogen metabolism,secondary metabolite content,precursor substances and key genes expression of tropane alkaloids metabolic Synthetic pathway were studied under different treatment time?4d,8d,12d,16d?.The main results are as follows:1.Investigating the effects of different concentrations of SNP on Photosynthetic Characteristics and physiological indexes of adversity under NaCl stress,were found NaCl stress significantly inhibited the photosynthetic characteristics,decreased the activity of SOD,POD,CAT,increased the content of MDA,and intensified the membrane lipid peroxidation;The total chlorophyll content,carotenoid content,Fv/Fm,ETR,qP,Yield and antioxidant enzyme activity gradually increased,but the MDA content,F0 gradually decreased under SNP treatment.It shows that SNP is propitious to relieve the damage caused by NaCl stress,protect photosynthetic apparatus,improve photosynthesis,enhance the ability of antioxidant enzyme system,and improve resistance to NaCl stress.Overall,0.2 mmol·L-1SNP had the most alleviation effect.2.Investigating the effects of different concentrations of SNP on the variation of the Nitrogen metabolism under NaCl stress,were found GDH activity first increased and then decreased,the ammonium nitrogen content continued to rise,but the nitrate nitrogen content,free amino acid content,soluble protein content,GS activity and NR activity had been steadily dropping with the increase of the number of NaCl stress days.It indicates that NaCl stress reducing the absorption of nitrate nitrogen,accumulating a large amount of ammonium nitrogen toxic cells,and reducing key enzymes activity,thus causing nitrogen metabolism disorder;Under different SNP treatment,0.1 mmol L-11 and 0.2 mmol L-11 SNP both had better effects on increasing nitrate nitrogen content,free amino acid content,soluble protein content,NR activity,GDH activity and GS activity,and reducing ammonium nitrogen content.The results showed that the appropriate concentration of SNP could effectively alleviate the inhibition of NaCl stress on nitrogen metabolism in Atropa belladonna L.,accelerate the reduction and assimilation of nitrogen,accumulate more nitrogenous organic compounds,and provide a material basis for the synthesis of secondary metabolites.3.Investigating the effects of different concentrations of SNP on the metabolic regulation of tropane alkaloids under NaCl stress,were found the content of hyoscyamine first increased and then decreased,but the scopolamine content continued to decrease under NaCl stress.After SNP treatment,the content of hyoscyamine and scopolamine increased in varying degrees in each treatment group,which showed that the concentration of 0.2 mmol.L-11 SNP was more conducive to the accumulation of hyoscyamine and scopolamine.To further study its regulation mechanism,the content of substrates amino acids?ornithine and arginine?,Put,Spd,Spm and the relative expression of key genes?PMT,TRI,H6H,PPAR?in tropane alkaloids synthesis pathway were detected.We found that NaCl stress inhibited the accumulation of ornithine and arginine and significantly reduced the content of polyamines,especially putrescine.Moreover,the relative expression of TRI was slightly increased,but the expression of PMT and PPAR were significantly inhibited under NaCl stress.After treatment with 0.2 mmol.L-1SNP,the ornithine content,arginine content,polyamines content and?Spm+Spd?/Put value increased in varying degrees,which could effectively stimulate the high expression of PMT,TRI and H6H.It is suggested that SNP can promote the accumulation of hyoscyamine and scopolamine by enhancing the substrates and precursors in the tropane alkaloid synthesis pathway and increasing the relative expression of key genes.In conclusion,NaCl stress inhibited photosynthesis of Atropa belladonna L.,destroyed the antioxidant enzyme system,disturbed the balance of nitrogen metabolism and hindered the accumulation of tropane alkaloids.The appropriate concentration of SNP to provide exogenous NO can effectively alleviate the inhibition of NaCl stress on physiological characteristics,nitrogen metabolism and tropane alkaloid metabolism and improve the resistance of Atropa belladonna L.to salt damage.In this study,the physiological regulation mechanism of NO alleviating NaCl stress in Atropa belladonna L.was preliminarily explored,which provided a theoretical basis for solving the salt damage problems encountered in production process,and provided a reference for improving the effective medicinal ingredients. |
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