The Mechanism Of Nitrogen Metabolism Disorder Induced By Aluminum Stress In Red Skin Disease Of Panax Ginseng

Objective:By analyzing the effects of red skin disease of Panax ginseng（RSD）on absorption and transport of nitrogen in ginseng.Regulation of nitrogen metabolism in ginseng cells by aluminum.To elucidate the regulatory mechanism of the disturbance of nitrogen metabolism in RSD induced by aluminum stress.Methods:（1）The content of aluminum,iron and manganese in soil and ginseng plants was determined by ICP-OES.（2）The contents of amino acids in ginseng plants and cells were determined by automatic amino acid analyzer.（3）The effect of different aluminum concentrations on the survival rate of ginseng cells was determined by CCK-8 colorimetry.（4）Protein content in plants and cells was determined by Bradford method.（5）The contents of nitrate nitrogen（NO₃^-）and ammonium nitrogen（NH₄⁺）in ginseng plants and cells were determined by colorimetry.（6）Using the method of spectrophotometry and enzyme-linked immunosorbent（ELISA）determination of nitrate reductase in ginseng plant and cell（NR）,glutamine synthetase（GS）,glutamate synthetase（GOGAT）,glutamate dehydrogenase（GDH）and proton pump（H⁺-ATPase）of activity,and metabolites of alpha ketone glutaric acid（α-KA）and glutamine（Gln）content.（7）The expressions of Ammonium transporter（AMT）,Aluminum malate transporter（ALMT）,Multidrug and toxic extrusion transporter（MATE）in ginseng cells were determined by PCR method.Results:（1）By analyzing the changes of metal elements in RSD,it was found that the content of aluminum was significantly higher than that of healthy ginseng,indicating that aluminum stress may be one of the main inducement of RSD.（2）The content of nitrogenous end product,protein and amino acid decreased and the content of glutamate increased.It shows that the effective use of nitrogen in RSD is affected.（3）The increased contents of nitrogen source,NO₃^-and NH₄⁺suggest that the absorption of nitrogen is blocked in RSD.（4）The key enzyme of nitrogen absorption and assimilation,NR activity decreased,and the activity of GS,GOGAT,GDH,and H⁺-ATPase increased in RSD.The contents of the intermediate products of nitrogen metabolismα-KA and Gln increased,indicating that GS/GOGAT circulation was abnormal and nitrogen metabolism was disorder.（5）Different concentrations of aluminum ions act on ginseng cells,10mM Al³⁺stimulate ginseng cells for 3 days,ginseng cell survival rate decreased to 62.30%,establish aluminum induced ginseng cell model in RSD.（6）The contents of NO₃^-and NH₄⁺in ginseng cells were increased by Al³⁺stress.（7）Al³⁺stress reduced the activity of NR and increased the activity of GS,GOGAT,GDH,H⁺-ATPase in ginseng cells.（8）Al³⁺stress increased the contents ofα-KA and Gln in ginseng cells,while decreased the contents of protein and amino acid indicating that aluminum induced GS/GOGAT circulation disorder in ginseng cells.The disturbance of nitrogen absorption and assimilation was consistent with the change of nitrogen metabolism in RSD.（9）The increased expression of AMT in RSD cell model may be related to the excessive accumulation of ammonium.（10）Al³⁺stress induced increased expression levels of ALMT and MATE transporters,thereby alleviating toxic effects of aluminum.Conclusion:Aluminum stress is one of the main inducers of RSD,which leads to the disturbance of nitrogen absorption,assimilation and utilization of ginseng by blocking GS/GOGAT cycle,resulting in excessive accumulation of ammonium and the generation of ammonium toxicity.RSD can increase intracellular ammonium transport by increasing the expression of AMT.At the same time,the expression of ALMT and MATE was promoted and the intracellular transport of aluminum was increased to alleviate the toxic effect of aluminum.