The Study On The Mechanism Of SHMT And FDH Overexpression-Altered Formaldehyde-Uptake And-Metabolism Ability Of Tobacco

The remediation methods for formaldehyde-pollution have been widely reported.Among the reported methods,phytoremediation have attracted more and more attention owing to its simple,natural and economic advantages.Tobacco and Arabidopsis thaliana are two model plants that are commonly used in botany researches.Previous studies had showed that formaldehyde-absorption and-resistance of tobacco is weaker than those of Arabidopsis.Formate dehydrogenase(FDH)and serine hydroxymethyl transferase(SHMT)are the two key enzymes in one-carbon metabolism.FDH can oxidize formate to CO2.SHMT mediates the interconversion of Gly and Ser.The expression of FDH and SHMT can be induced by various abiotic stresses.Both FDH and SHMT plays an important role in response to abiotic stress.During HCHO metabolism in Arabidopsis,FDH oxidizes formate to CO2 while SHMT catalyzes the reaction of Glycine and 5,10-13CH2-THF to produce Ser.The expression of FDH and SHMT are all induced by HCHO stress.In this study,the correlation between formaldehyde metabolism and formaldehyde uptake by tobacco were firstly analyzed.Arabidopsis FDH and SHMT were subsequently expressed alone or simultaneously in transgenic tobacco.Changes in HCHO-metabolic mechanism,-uptake and-resistance of transgenic tobacco under liquid and gaseous HCHO stress were investigated to clarify the physiological roles and functional mechanisms of FDH and SHMT over-expression in transgenic tobacco.Changes in the expression,phosphorylation level and interaction of proteins in the signaling pathway that regulates the stomatal movement were investigated in transgenic tobacco leaves under gaseous formaldehyde stress to reveal the molecular mechanism of FDH and SHMT overexpression altered-formaldehyde uptake ability in tobacco.The obtained results provide the theory basis and new operation strategies for the plant genetic engineering on enhancement of plant formaldehyde-uptake by utilizing formaldehyde-response genes.The main results are as follows:After treatement in 2 mM liquid formaldehyde solution for 24 h,the wild type(WT)tobacco leaves absorbed approximately 24.1%of the formaldehyde,validating that tobacco leaves has a certain absorption capacity for formaldehyde.13C-NMR analysis showed that[4-13C]Asn,[3-13C]Gln,[U-13C]oxalic acid(OA)and H13COOH are major metabolic intermediates in tobacco leaves treated with 2 mM liquid H13CHO.The pretreatment of cyclosporin A(CSA)and dark almost completely inhibited the generation of[4-13C]Asn,[3-13C]Gln and[U-13C]OA but did not suppressed the production of H13COOH in 2 mM liquid H13CHO-treated tobacco leaves.The analyze of the chlorophyll and Rubisco contents indicated that CSA and dark pretreatments did not severely affect the survival of cells in tobacco leaves but significantly inhibited the formaldehyde absorption by tobacco leaves.The metabolic pathway consisting of[4-13C]Asn,[3-13C]Gln and[U-13C]OA in tobacco leaves had 62.9-69.5%contribution to the formaldehyde absorption.The results suggested that this pathway played a major role in tobacco leaves for formaldehyde absorption,indicating that formaldehyde-metabolism and-absorption has a good correlation in tobacco leaves.Format produced by formaldehyde oxidation can be further oxidized to CO2 during formaldehyde detoxification in the Arabidopsis.Then CO2 enters the Calvin cycle and is used for the synthesis of glucose.However,the carbon flux of the formaldehyde metabolism in tobacco leaves did not enter the Calvin cycle to produce glucose.FDH has dual locations targeting to both chloroplasts and mitochondria in Arabidopsis,but only has a mitochondria location in tobacco.This study attempted to over-express the FDH from Arabidopsis in chloroplasts of transgenic tobacco leaves by using the light inducible promoter(PrbcS)and chloroplast signal peptide.In this way,the carbon flus of formaldehyde metabolism might be introduced into the Calvin cycle to synthesize glucose,which would improve the formaldehyde-resistance and-absorption ability of the FDH transgenic tobacco.13C-NMR was used to analyze the metabolic profiles in transgenic tobacco leaves treated with 2 and 6 mM liquid H13CHO.The results showed that the carbon flux of H13CHO metabolism was not introduced into the Calvin cycle to produce glucose by FDH over-expression in chloroplasts of transgenic tobacco.However,the[4-13C]Asn,[3-13C]Gln,[U-13C]OA and H13COOH production was enhanced in transgenic tobacco leaves.Further investigation showed that over-expression of FDH improved the liquid formaldehyde-resistance and-absorption ability of transgenic tobacco.These results revealed that that over-expression of FDH in chloroplasts improved the formaldehyde-absorption ability of transgenic tobacco by enhancing the formaldehyde-resistance of tobacco leaves and the function of the original formaldehyde-metabolic pathways in leaves.Ser is one of the important metabolites for formaldehyde metabolism in Arabidopsis.However,Ser was not produced during formaldehyde metabolism in tobacco leaves.This study attempted to over-express Arabidopsis SHMT1 of the photorespiration pathway in leaves using the PrbcS promoter to produce SHMT transgenic tobacco.Then the carbon flux of formaldehyde metabolism can be introduced into the Ser synthesis to improve the HCHO-resistance and-absorption ability of tobacco leaves.13C-NMR was conduct to analyze the formaldehyde metabolism in transgenic tobacco leaves treated with 2 and 6 mM liquid H13CHO.The results showed that SHMT over-expression in transgenic tobacco leaves did not introduce the carbon flux of formaldehyde metabolism into the Ser synthesis.However,the generation of[4-13C]Asn,[3-13C]Gln,[U-13C]OA and H13COOH increased.Further investigation indicated that the over-expression of SHMT also improved liquid HCHO-resistance and-absorption ability of transgenic tobacco.Thus it can be concluded that the role of of SHMT is similar to that of FDH in transgenic tobacco leaves,both of which increased the HCHO-absorption ability of tobacco leaves through augmentation of formaldehyde-resistance and the function of the original HCHO-metabolic pathways.The Arabidopsis FDH was overexpressed in SHMT transgenic tobacco using PrbcS promoter to produce SHMT/FDH double transgenic tobacco.13C-NMR was performed to analyzed the H13CHO metabolism in the SHMT/FDH transgenic tobacco leaves treated with 2mM and 6mM liquid H13CHO.The results showed that the carbon flux of HCHO-metabolism could not be used for Ser synthesis but was introduced into the glucose production in SHMT/FDH double transgenic tobacco leaves.Simultaneously,the generation of[4-13C]Asn,[3-13C]Gln,[U-13C]OA and H13COOH in the SHMT/FDH double transgenic tobacco leaves was greater than that in the FDH and SHMT single transgenic tobacco leaves.These results indicated that simultaneous over-expression of FDH and SHMT in transgenic tobacco leaves strongly enhanced the role of the original formaldehyde metabolism pathways,which conferred the double transgenic tobacco leaves a stronger HCHO-resistance and-absorption capacity than FDH and SHMT single transgenic tobacco.The main limiting factors of phytoremediation for the formaldehyde-polluted air are physiological characteristics of leaf stomata.The results of this study showed that stomatal conductance and aperture of the three transgenic tobacco were significantly larger than those of the WT under low concentrations(3,5 and 7 ppm)of gaseous formaldehyde stress.These factors of SHMT/FDH double transgenic tobacco were larger than those in FDH and SHMT single transgenic tobacco.Under 5 ppm gaseous formaldehyde stress,formaldehyde-absorption by the three transgenic tobacco were better than that of WT.Moreover,gaseous HCHO-absorption of SHMT/FDH double transgenic tobacco was significantly greater than that of FDH and SHMT single transgenic tobacco.The oxidative stress-related indicators including H2O2 content in the three transgenic tobacco leaves were lower than those in WT.These results indicated that over-expression of Arabidopsis SHMT and FDH in tobacco leaves with light-inducible promoter could improve stomata resistance to gaseous formaldehyde and alleviated the oxidative damage caused by gaseous formaldehyde stress in tobacco leaves.As a result,gaseous formaldehyde absorption capacity of transgenic tobacco increased.The effects of simultaneous over-expression of SHMT and FDH are better than those of individual overexpression of SHMT or FDH.Western blot analysis showed that the phosphorylation level of stomatal regulation key factors,phototropin and plasma membrane(PM)H+-ATPase in FDH and SHMT transgenic tobacco leaves were higher than that in WT under 5 ppm gaseous formaldehyde stress.Furthermore,the phosphorylation level of phototropin and PM H+-ATPase in the SHMT/FDH double transgenic tobacco leaves was greater than that in the FDH and SHMT single transgenic tobacco.The co-immunoprecipitation analysis suggested that the interactions between 14-3-3 protein and phototropin as well as PM H+-ATPase in the FDH and SHMT transgenic tobacco leaves was less inhibited than those in leaves of WT by 5 ppm gaseous formaldehyde stress.The inhibition on the interaction of these proteins in the SHMT/FDH double transgenic tobacco leaves was less than those in the FDH and SHMT single transgenic tobacco leaves.Consequently,PM H+-ATPase and H+pump activity in FDH and SHMT transgenic tobacco leaves were greater than those in WT leaves,and these activity in the FDH/SHMT double transgenic tobacco leaves was higher than those in the FDH and SHMT single transgenic tobacco leaves under 5 ppm gaseous formaldehyde stress.These results indicated that over-expression of SHMT and FDH elevated the phosphorylation levels of phototropin and PM H+-ATPase and thus enhanced the interactions of 14-3-3 protein with phototropin and PM H+-ATPase,which increased the PM H+-ATPase and H+ pump activity in transgenic tobacco leaves under gaseous formaldehyde stress.As a result,the stomatal conductance and aperture increased,the absorption capacity of gaseous formaldehyde was augmented.The role of simultaneous over-expression of FDH and SHMT is better than that of individual overexpression FDH or SHMT.