Exploration On Abnormal Metabolism And The Mechanism Of Red-skin Substance Formation In Red Skin Ginseng

Panax ginseng C.A.Meyer,known as"the king of all herbs",plays an important role in maintaining the health of the people and the development of Chinese medicine industry.Ginseng red-skin（GRS）symptom is one common disease in ginseng production,with a general incidence of about 25%and serious cases up to 80%or even 100%.The content of various components of diseased ginseng decreases,which seriously affects the quality of ginseng and limits the development of ginseng industry.Previous research on the formation mechanism of Ginseng red-skin symptom has focused on the metal ion content,polyphenol content changes and microbial changes etc.The causes of GRS included microbial infestation,metal ion deposition,etc.With little research on its red-skin substance,the formation mechanism has not been reported yetIn this study,4-year-old ginseng was used as the study subject,and TMT were used to quantify the proteome of red-skin ginseng（RSG）and healthy ginseng.Abnormal C/N metabolism was found to be associated with the occurrence of GRS.The detection of substances related to C/N metabolism and their enzymatic activities in RSG confirmed this inference.Meanwhile,nitrite,the nitrogen metabolite,its content was increased in the ginseng tissues of RSG,and nitrate reductase activity was also increased,suggesting that nitrite may be involved in the formation of red-skin substance in RSG.In vitro simulations of red-skin substances production using ion complexation have confirmed that nitrite（NO₂^-）plays an important role in red-skin coloration formation.Then two models of potted ginseng and hydroponic ginseng were used to verify this result.And the mechanisms were investigated by integrating morphological observation,substance content determination,transcriptome and metabolome techniques.In addition,the effect of melatonin（MT）,one can improve plant resistance,on GRS and its molecular mechanism was also investigated.In addition,melatonin（MT）,a hormone that enhances plant stress tolerance,was used to study the effect on GRS and its molecular mechanism was also investigated.The main research results were as follows:（1）Quantification of the proteome of RSG by TMTPrevious studies on red-skin ginseng have mostly focused on changes in their secondary metabolites,with less qualitative and quantitative analysis of proteins.Previous literature also implies a trend of abnormal C/N metabolism involvement in RSG,but few studies have been conducted.In this study,four-year-old RSG was used as the test material,and healthy ginseng in the same growth environment was used as the control material for proteome detection by LC-MS/MS with TMT.GO enrichment analysis showed that these differentially expressed proteins were significantly up-regulated in oxidation-reduction process,fatty acid metabolic process and peroxisome,and significantly down-regulated in carbohydrate metabolism and calmodulin binding.KEGG enrichment analysis showed that the differentially expressed proteins were mainly enriched in the pathways of peroxisome,carbon metabolism,and Metabolism of xenobiotics by cytochrome P450.The differentially expressed proteins associated with plant stress resistance were mainly enriched in peroxisomal and ascorbate and aldehyde metabolism pathways,and differentially expressed proteins associated with C/N metabolism were also enriched,indicating that the occurrence of GRS was associated with C/N metabolism.（2）Measurement of substances content and related enzyme activities to C/N metabolism in RSGProteomic assays suggest abnormalities in C/N metabolism in diseased tissues of RSG,but there are few reports on the determination of substances related to C/N metabolism and enzyme activity,so this experiment was conducted.The results of C/N metabolism related substances of RSG showed that the contents of soluble sugars and starch decreased significantly compared with healthy ginseng tissues.The contents of soluble protein,free amino acid,free proline and total nitrogen increased significantly compared with healthy ginseng tissues.In addition,the content of ammonium nitrogen increased significantly and the content of nitrate nitrogen decreased significantly.The enzymes activities related to C/N metabolism,including amylase,starch synthase,nitrate reductase,and nitrite reductase,all had significantly increased,indicating that GRS were in dynamic development of injury and repair,and various substance contents and enzyme activities were in the process of change.In conclusion,the experiment confirmed that the occurrence of GRS was related to the change of C/N metabolism at the level of substance content and enzyme activity.（3）Ionic complexation method simulates the formation of red substances in RSGThe composition of the red-skin substances in RSG is complex,and previous studies on their composition and conditions of production have been unsuccessful.The study of metabolomics in the diseased part of RSG has provided assistance in this research.This experiment builds on the research of our group.Ten compounds significantly enriched in diseased tissues of RSG were selected to complete complexation tests with nine metal salts commonly found in the laboratory.Four tests were designed for direct complexation,aluminum trichloride colorimetric method,Na NO₂^--Al₂（SO₄）₃-Na OH colorimetric method,and complexation reactions involving sodium nitrite.The results showed that the first three complexation tests did not meet expectations,and only the fourth complexation reaction produced the expected red substance that met the test requirements.In the presence of nitrite,the metal ions that produced the red substances in complexation with the compounds were Fe³⁺,Fe²⁺,and Al³⁺in order,while Ca²⁺,Mn²⁺,and Zn²⁺did not produce red substances with any of the compounds.In this study,the nitrite,total flavonoid and total polyphenol contents in the diseased tissues of RSG were also examined,and the results showed that the contents were significantly increased,which provided the material basis for the formation of red substances.The experimental results provided theoretical support for the next step of validation,and the three elements Mn²⁺,Ca²⁺,and Zn²⁺were expected to be potential mineral element drugs to alleviate the grade of GRS.In conclusion,due to the involvement of nitrite,the simulation assay in vitro obtained red skin substance.（4）Transcriptomic analysis of potted RSG induced by NO₂^-/Fe³⁺systemIn vitro simulation tests confirmed that nitrite can promote the complexation of metal ions with compounds to form red skin substances,so this paper designed a validation experiment for ginseng organisms.Healthy ginseng with the same growth potential for 2 years were used as the test material,and NO₂^-/Fe³⁺system solution was used as the inducer to establish the potted RSG model.Melatonin was foliar sprayed along with induction to investigate the effect of melatonin on GRS.The results showed that red spots appeared on the surface of the induced ginseng,and the model was established successfully as observed in paraffin sections with RSG characteristics.The photosynthetic parameters,osmoregulatory substance content,total flavonoid and total polyphenol content of the induced ginseng were measured,showing that the net photosynthetic rate and transpiration of ginseng were significantly inhibited by NO₂^-/Fe³⁺treatment,while the intercellular CO₂concentration increased significantly.The osmoregulatory substance content,total flavonoid and total polyphenol content increased significantly,showing the growth of ginseng was significantly inhibited.After MT intervention,these indicators changed significantly,suggesting that MT mitigated the effects of stress on ginseng.The transcriptomic results showed that phenylpropanoid biosynthesis,flavonoid biosynthesis,plant hormone signal transduction,and plant-pathogenic pathways were significantly enriched,which were related to plant resistance to stress and that was the response mechanism of ginseng to induced conditions.The intervention of MT resulted in significant changes in the expression levels of related genes,elucidating the molecular mechanism of action of MT as a candidate drug to reduce the pathogenic grade of GRS.In conclusion,the pot experiment confirmed that nitrite could induce GRS and affected the physiological and biochemical functions of ginseng.（5）Combined transcriptomic and metabolomic analysis of ginseng induced by NO₂^-/Fe³⁺system in hydroponic GRSThe pot test ginseng is affected by more factors and cannot visually reflect the formation of the red substance of GRS.Based on the validation experiments in pots,the induction of GRS under hydroponic conditions was established,and combined transcriptomic and metabolomic analysis was performed to the induced ginseng.The results showed that after induction by NO₂^-/Fe³⁺system solution,ginseng leaves showed obvious crinkling and red substance appeared on the roots.Pathological sections showed basic consistency with RSG characteristics,and the model induction was successful.The transcriptomic results revealed that the molecular mechanism of red substance formation was related to the energy and substances provided by the pathways of photosynthesis,phenylpropanoid biosynthesis,flavonoid biosynthesis,and phytohormone signaling.Metabolomics analysis showed that the differential metabolites were mainly involved in the enrichment of mineral absorption,amino acid biosynthesis and other pathways,showing the molecular regulatory mechanism of ginseng under the influence of NO₂^-/Fe³⁺solution.The combined transcriptomic and metabolomic analysis showed that the formation of red substances was associated with glutamine,Sinapyl alcohol,phenylalanine,choline and their corresponding genes.In addition,MT showed good potential as a resistance drug on GRS in alleviating the morphological characteristics and reducing the accumulation of red-skin substances in hydroponically grown ginseng.Based on the results of this experiment,combined with the previous research of our team,the following mechanism was proposed for the formation of red-skin substances in GRS.Abnormal C/N metabolism in ginseng under stress resulted in the increase of nitrite content,altered secondary metabolism,and massive accumulation of polyphenols and flavonoids in ginseng tissues.Nitrite in ginseng promoted the complexation of Fe³⁺,Fe²⁺and Al³⁺ion with these compounds to form red-skin substances.This conclusion was verified in vitro complexation as well as in the pot test and hydroponic test,which confirmed the important role of nitrite in the formation of red-skin substances and provided new ideas for the control of GRS.