Study On Mechanism And Application Of Absorption And Metabolism Of Formaldehyde In Black Soybean Roots

Formaldehyde?HCHO?is widely used in chemical synthesis,industrial manufacturing,pharmaceutical synthesis and other industrial fields.Formaldehyde plays an important role in the field of chemical pesticides and intermediates synthesis,so it is inevitable to produce a large number of production wastewater containing liquid formaldehyde in the modern chemical and chemical pesticide production process.On the other hand,with the improvement of the quality of life the decoration has become part of modern life.Formaldehyde is widely used in a lot of decoration materials as adhesives.Non-crosslinked free formaldehyde in the adhesive is continuously released to cause severe indoor air formaldehyde contamination.Many studies have observed that different plant varietles have different formaldehyde uptake capacity.Due to the hazards of formaldehyde pollution is very extensive,so there are many studies committed to the development of formaldehyde pollution control technology and methods.In many governance methods,based on the governance method of microbial and plant absorb formaldehyde capacity is an effective and cost-effective way.Most studies suggest that the absorption of formaldehyde in the air was completed by the blade,some studies have illustrated formaldehyde metabolism in the leaves of plants.Recent studies have shown that when air or wastewater contaminating formaldehyde flows through the bioreactor of plant roots and soil substrates,formaldehyde in air or waste water can be absorbed into the plant roots through soil,and was degradation under the action of metabolic enzymes in the root cells.Plant roots can effectively remove the formaldehyde during the day and night.In the night the removal of formaldehyde by roots is 10 times the aerial parts.In this study,used the larger seed cultivra Tamba black soybean?referred to as RB?and smaller seed cultivar black soybean?referred to as SB?roots as materials.Analysis and comparison of RB and SB root absorption dynamic model of formaldehyde and the mechanism of formaldehyde metabolism in two black soybean roots was investigated by 13C-NMR analysis combined with physiological and biochemical methods,and provide the theoretical basis for the scientific use of plant roots to repair formaldehyde pollution.Treated RB and SB roots with 2-6mM HCHO,measured the absorption of HCHO in the period of 0.5-48h.The results show that both RB and SB roots can effectively absorb formaldehyde in the treatment solution.The kinetics of formaldehyde absorption in the two black soybean roots during the 48 h treatment period was similar,the absorption mode is slowly at first and then faster.The relationship between the absorption of formaldehyde and the time is consistent with the power function model.13C-NMR analysis showed that in the early of H13CHO stress?0-4h?,H13CHO absorbed by roots is first oxidized to formic acid by an oxidation pathway that depends on FALDH,and formic acid may be condensed to glyoxylic acid by the catalytic of GXS.In the subsequent metabolism,some glyoxylic acid may enter the glyoxylic acid cycle to produce Cit and Icit.A portion of glyoxylic acid is converted to Gly by the action of transaminase?GAT?or metabolized to Asn and Gln by unknown pathway.In addition,some glyoxylic acid is oxidized to oxalic acid?OA?.At the late stage of H13CHO treatment?24-48 h?,Asn and Gln,OA are converted to Cit and Icit by TCA cycle;Cit and Icit enter the glyoxylic acid cycle to produce Mal.The Mal output from glyoxolic acid enter into the gluconeogenesis pathway then converted to[U-13C]Gluc.It can be seen that glyoxylic acid cycle may be the center of formaldehyde metabolism.In the late formaldehyde treatment,due to glyoxylate pathway in the soybean root is fully effective,leads to the root of formaldehyde absorption capacity increased rapidly,thus,the contribution of formaldehyde absorbed by roots towards for formaldehyde removal during this period is significantly increased.The differences in the metabolic mechanism of formaldehyde in RB and SB roots were carefully compared.It was found that the effect of formaldehyde metabolism on RB and Gly pathway in RB roots was stronger than SB.Besides,13C-NMR analysis confirmed that root-absorbed formaldehyde was not transferred to the leaves for metabolic transformation.The accumulation of Pro in RB leaves was significantly higher than that in SB under 2 mM formaldehyde stress,Therefore,the leaf wilting of SB plant was more serious than that of RB.However,the accumulation of Pro in the roots of SB was significantly higher than that of RB,and the oxidative stress was lower than that of RB,so most of the subsequent experimental materials were treated with SB roots.CSA is a specific mitochondrial permeability transporter inhibitor.Analysis the Subcellular localization of formaldehyde metabolic pathway in RB and SB roots via CSA pre-treatment,comparison of the difference between the pretreatment with CSA pretreatment and the non-pretreated roots at 2 mM H13CHO for 4 h and 24h.Analysis of 4h formaldehyde metabolic profile,the results show that CSA completely inhibit the role of formaldehyde metabolism in the production of Cit pathway,the formation of Icit partial inhibition,suggesting that Cit and Icit production pathway located in the role of mitochondria or glyoxysome.The yields of OA,Gly2,Gln,Asn and Ser3 were not inhibited,indicating that the metabolic pathways that produced these organic acids played a role in the cytoplasm of the roots.CSA pretreatment resulted in the 13C metabolic flux of Cit and Ici entering the metabolic pathway of these organic acids.The formation of glucose was also significantly affected by the 24-hour formaldehyde metabolic profile,which was another result of the inhibition of the glyoxylic acid pathway.13C-NMR analysis confirmed that CSA pretreatment also affected the role of the Calvin cycle in the chloroplast of the leaves,and the leaf glucose content was significantly decreased.The results of formaldehyde absorption curve showed that CSA pretreatment had only slight inhibitory effect on formaldehyde absorption in RB and SB roots,which was due to CSA inhibition of glyoxylic acid pathway without affecting OA,Gly2,Gln,Asn and Ser3,and the two pathways are independent.The effects of various metabolic pathways on the absorption of formaldehyde in black soybean roots were investigated by using the inhibitory effect of formaldehyde metabolites on the metabolic pathway and the compounds that affected the expression of key enzymes in the acetaldehyde pathway,the results showed that GX,Gly,Ser and OA pretreatment significantly inhibited formaldehyde uptake in SB roots.It was confirmed that GX,Gly,Ser and OA production pathways play the role in the formaldehyde absorption process of SB.Cit pretreatment also has a similar effect,confirming the role of glyoxylic acid pathway in the formaldehyde absorption process of SB roots.The pretreatment of Glucose,Sucrose and Mannose can inhibit the formaldehyde absorption in SB roots,These sugars could regulate the expression of ICL and MS,which are the key enzymes of glyoxylate cycle in other plants.The results showed that 10 mM Glucose,5 mM Sucrose and 10 mM Mannose produced the strongest inhibit effect.In addition,the analysis of antioxidant ASA and GSH pretreatment results showed that 2mM ASA pretreatment effect of the best can make SB root formaldehyde absorption increased by 6 times.4mM ASA pretreatment effect is the best,can make SB root formaldehyde absorption increased by 5 times.The results showed that the pretreatment of ASA and GSH could alleviate the oxidative stress of formaldehyde,and the absorption efficiency of formaldehyde in SB was improved greatly.Methanol?CH₃OH?can stimulate plant growth and increase plant biomass in higher plants and crops have been confirmed.The addition of the appropriate concentration of methanol to the solid medium promoted the effect of SB on rooting.Methanol foliage spraying can promote the elongation of SB stem.The results of 13C-NMR analysis showed that 13CH₃OH was very slow in the roots of SB,and its metabolites were only Ser2 and Cit?2,4?.Most of the 13CH₃OH absorbed by roots after 24 hours was still in the free state.The mathematical model was designed by Central Composite Design,and the optimum condition of formaldehyde absorption was obtained by the response surface optimization method,i.e.methanol pretreatment concentration 0.73 mM,formaldehyde treatment concentration 1.3mM and formaldehyde treatment time 22h,under this condition SB plant on the absorption of formaldehyde model is 85.86%.And the real value verified by the experiment is 84.72%,which indicates that the model is well fitted.The results showed that the pretreatment of methanol promoted the formation of FA,Gly2,Ser3,Cit,Icit,Asn,Gln and Glucose,and the results showed that the pretreatment of FA was higher than that of the control group?P<0.05?.The yield of these metabolites did not increase during the methanol pretreatment stage,which ruled out the possibility of increasing these metabolites by the metabolism of methanol.Therefore,it is speculated that methanol pretreatment may change the mechanism of formaldehyde metabolism by affecting the expression of formaldehyde metabolism related enzymes in SB roots and promote the absorption of formaldehyde.In order to better understand the mechanism of the application of methanol to promote the growth of SB plants at the molecular level,we used cDNA microarray to identify the methanol response genes in SB leaves after 5%methanol spraying.Results 731 possible methanol response genes were identified,and the number of up-regulated and down-regulated genes were 522 and 209,respectively.