The Study On The Mechanism And Application Of Formaldehyde Absorption And Metabolism In Vicia Faba L.

The technology of using plant metabolism to assimilate HCHO to purify environmental HCHO pollution has attracted more and more attention because of its simplicity,low cost and remarkable effect.With the deepening of the research on the mechanism of plant purification of HCHO,besides screening plants which can purify air efficiently,we can understand the metabolic pathway of HCHO in plants and further reveal the plant biochemical response to HCHO stress at high concentrations,so that we can use genetic engineering technology to improve plant exogenous HCHO.Purification capacity,which is internationally recognized as a new way to purify polluted gases.At present,studies on plant assimilation pathway of HCHO mainly focus on photosynthetic tissues,and it was found that plant roots produced glyoxylic acid cycle-related products during HCHO metabolism,so glyoxylic acid cycle may play an important role in the process of HCHO detoxification.Further study on the role of glyoxylic acid cycle in plant HCHO metabolism will help us understand the mechanism of plant metabolic response to HCHO stress more comprehensively,and provide theoretical basis for developing more phytoremediation techniques for HCHO pollution.In view of more reports on the biochemical processes of model plant Vicia faba,this study focused on the effects of glyoxylic acid pathway on the detoxification process of HCHO,using Vicia faba plants,isolated leaves and roots as experimental materials and drawing on the existing research results of glyoxylic acid cycle of plants.In this study,stable isotope tracer was used to analyze the metabolites of different tissues treated with H¹³CHO by nuclear magnetic resonance(¹³C-NMR)to clarify the role of glyoxylic acid pathway in the process of HCHO detoxification in Vicia faba cells.At the same time,we added carbohydrates and methanol to inhibit or induce the expression of key enzymes in glyoxylic acid pathway under HCHO stress,changed the key enzymes in glyoxylic acid pathway,analyzed the changes of HCHO absorption capacity and HCHO metabolic capacity of Vicia faba,and qualitatively evaluated the contribution of glyoxylic acid pathway to HCHO detoxification in Vicia faba tissues,thus revealed the metabolic response of glyoxylic acid pathway in Vicia faba to liquid HCHO stress.Finally,the mathematical model was used to optimize the optimum conditions for Vicia faba plants to absorb and metabolize HCHO.On this basis,the bioreactor was developed to explore the feasibility of the reactor to purify HCHO pollution in the actual environment.The research results were as follows.The kinetics curve of HCHO absorption in vitro roots of Vicia faba was slow in the early stage and fast in the late stage.The main driving force was root HCHO metabolism.The mechanism of HCHO assimilation in vitro roots of Vicia faba was analyzed by ¹³C-NMR.The results showed that H¹³CHO was oxidized to H¹³COOH by2mM H¹³CHO time gradient,and then a very strong[2,4-¹³C]Citric acid?Cit?signal peak appeared.The signals of[2,3-¹³C]Succinic acid?Su?and[3-¹³C]Cit were also observed.Peak enhancement indicated that glyoxylic acid and tricarboxylic acid?TCA?cycle played a role in the assimilation of HCOOH.The yield of[2,4-¹³C]Cit was more than 85%of the total metabolites,and it was a marker metabolite.Isocitrate lyase?ICL?was a key enzyme in glyoxylic acid pathway.Its activity was induced by HCHO.Therefore,high concentration H¹³CHO?4mM and 6mM?treatment significantly increased the yield of[2,4-¹³C]Cit.In addition,the yield of[2,4-¹³C]Cit and[3-¹³C]Cit could be increased by treating roots with 2mM H¹³CHO after induction of ICL enzyme activity by high concentration?10 mM?methanol.Pretreatment of isolated roots with cyclosporine A?CSA?prevented ¹³C-rich metabolites from entering mitochondria,resulting in a significant decrease in[2,4-¹³C]Cit yield in H¹³CHO treated roots.At the same time,the accumulation of[2,3-¹³C]Su and the production of HCO₃^-produced by the oxidation of HCOOH increased.These evidences suggested that the assimilation of HCHO in the isolated root of Vicia faba was accomplished through the synergistic action of glyoxylic acid pathway and TCA cycle.Based on the above experimental results,the metabolic pathway of HCHO in the isolated root of Vicia faba was revealed:H¹³CHO was absorbed into root cells and oxidized to formic acid first,then most formic acid was condensed to glyoxylic acid.A small amount of formic acid was oxidized to CO₂ in the form of HCO₃^-.Most glyoxylic acid entered the glyoxylic acid pathway and was assimilated into[3,6-¹³C]Cit and[2-¹³C]Su.[2-¹³C]Su entered the TCA cycle and further assimilated into[2,4-¹³C]Cit.The metabolism of[2-¹³C]Su and[2,4-¹³C]Cit in TCA cycle produced oxaloacetic acid?OAA?and ketoglutarate?KGA?.These metabolites were then transaminated to produce[3,4-¹³C]Glutamic acid?Glu?,[2-¹³C]Aspartic acid?Asp?and[2-¹³C]Asparagine?Asn?.The absorption curve of HCHO in excised leaves of Vicia faba was similar to that in roots of Vicia faba.The absorption pattern was slow in early stage and fast in late stage.The main driving force was HCHO metabolism in leaves.The main products of HCHO metabolism in Vicia faba leaves in vitro included FA,CO₂,[2,4-¹³C]Cit,[2,3-¹³C]Su,[2-¹³C]Mal,[3-¹³C]Serine?Ser?and[2-¹³C]Glycine?Gly?.The yield of[2,4-¹³C]Cit accounted for 51%of the total metabolites.The activity of ICL was induced by high concentration methanol,and the change pattern was consistent with that of[2,4-¹³C]Cit.CSA pretreatment enhanced the signal peaks of[2,4-¹³C]Cit,[2-¹³C]Mal and[2,3-¹³C]Su.These evidences suggested that the synergistic effect of glyoxylic acid pathway and TCA cycle played an important role in the process of CHO assimilation in isolated Vicia faba leaves.Based on the above experimental results,the metabolic pathway of HCHO in isolated Vicia faba leaves was elucidated:H¹³CHO first produced[3-¹³C]Ser through Serine pathway.Then[¹³C]Ser was deaminated by serine dehydratase to produce[¹³C]Pyruvic acid,and[¹³C]Pyruvic acid was oxidized by pyruvate dehydrogenase?PDH?to decarboxylate[¹³C]Acetyl coenzyme A into glyoxylic acid cycle metabolism.In the glyoxylic acid cycle,[¹³C]Acetyl coenzyme A reacted with oxaloacetic acid?OAA?to form[2,4-¹³C]Cit by nucleophilic addition reaction.[2,4-¹³C]Cit was isomerized to[2-¹³C]Icit by aconitase.[2,3-¹³C]Su and[2-¹³C]Glyoxalic acid were synthesized by decarboxylation,reduction and pyrolysis of[2-¹³C]Icit.[2,3-¹³C]Su entered mitochondria and participated in the metabolism of TCA to produce OAA and ketoglutarate?KGA?.The similarities of HCHO metabolic mechanism between isolated roots and leaves of Vicia faba lied in:?1?HCHO can be oxidized to HCOOH in both leaves and roots,and HCOOH can continue to oxidize to CO₂ or condensate to glyoxylic acid.Some glyoxylic acid entered the glyoxylic acid pathway,others entered the peroxisome reaction to produce Gly,Gly entered the mitochondria to produce Ser.?2?In vitro roots and leaves,[2,4-¹³C]Cit was a marker product of HCHO metabolism.?3?Glycolysis occured in both leaves and roots,and free sugar concentration continued to decrease.The differences of HCHO metabolic mechanism between isolated roots and leaves of Vicia faba lied in:?1?HCHO can be assimilated by Serine pathway in isolated leaves.?2?HCOOOH can produce OAA in isolated leaves,and OAA entered TCA cycle to participate in metabolism.?3?CSA pretreatment experiments showed that[2,4-¹³C]Cit production in isolated roots was localized in TCA cycle,while[2,4-¹³C]Cit production in leaves was localized in glyoxylic acid cycle.?4?Cys production increased in isolated leaves and decreased in isolated roots.The dynamic curve of absorption of HCHO solution by Vicia faba plants was similar to that of in vitro roots and leaves,both of which were slow in the early stage and fast in the later stage.Vicia faba had a"water-plant-air"circulation system,which can transfer liquid HCHO from root to air near leaves.The concentration of HCHO in leaf was highly correlated with the transpiration of leaves.After absorbing liquid or gas HCHO,Vicia faba plants accumulated in all parts of rhizome and leaf,with the highest cumulative concentration in root,the second in leaf and the lowest in stem.The metabolites of HCHO assimilated by Vicia faba plants included Cit,Glu,FA,Asn,Asp and Su.The metabolic pathway of HCHO in Vicia faba leaves and roots was similar to that in vitro roots and leaves.Glyoxylic acid pathway and TCA cycle played a major role,and[2,4-¹³C]Cit was a marker metabolite.The mechanism of HCHO metabolism in Vicia faba plants was different from that in vitro roots and leaves:?1?HCO₃^-production occured in the process of HCHO metabolism in vitro roots and leaves,but no HCO₃^-production occured in the process of HCHO metabolism in Vicia faba plants.?2?The production of[2-¹³C]Cys in the process of HCHO metabolism in vitro roots decreases,while the production of[2-¹³C]Cys in the process of HCHO metabolism in Vicia faba plants.?3?The yield of free sugar decreased continuously in the process of HCHO metabolism in vitro roots and leaves,while the yield of free sugar increased in the process of HCHO metabolism in Vicia faba plants.The stem of Vicia faba plants can also metabolize HCHO,and its metabolic pathway was similar to that of leaves.HCHO metabolic capacity of Vicia faba root was the strongest,followed by leaves and stems.The factors affecting the absorption efficiency of HCHO by Vicia faba plants included plant weight,concentration of treatment solution,volume of treatment liquid and treatment time.Single factor experiments showed that the total absorption rate decreased with the increase of volume of treatment liquid,while the total absorption rate increased with the increase of fresh weight.The central combination model was used to optimize the concentration,volume and fresh weight of HCHO absorbed by Vicia faba plants.It was found that there was a significant interaction between the volume and concentration of the treated liquids.Fixed concentration of the treated liquids increased the absorption of HCHO by increasing the volume of the treated liquids and decreased the interaction between the fresh weight of the plants and the volume of the treated liquids.Significantly,the fresh weight of fixed plant and the volume of treated liquid increased,and the absorption rate of HCHO decreased.This study explored the optimal factors:the concentration of treatment solution was 0.099mM,the volume of treatment liquid was 0.248 L and the fresh weight of plant was 85.5g.The actual absorption rate of HCHO reached 88.91%,which was 21.11%higher than that before optimization,and was close to the highest value predicted by the model.According to the experimental results of surface response,a suspension reactor of practical application level was designed.The effects of fresh weight of Vicia faba plant,water volume,air velocity and pollutant concentration on the HCHO pollution efficiency of the reactor were investigated.It was found that the HCHO purification efficiency increased with the increase of fresh weight of plant.The HCHO purification rate decreased with the increase of water volume in the reactor.And the HCHO purification rate increased with the increase of air velocity.In the experimental space?53m ³?with HCHO concentration of 1.1mg/m³,the reactor operated continuously for7 days.The average purification rate of HCHO reached 41.6%,and the purification capacity was 0.15 mg.m^-3.h^-1.