液体甲醛胁迫下天竺葵叶片甲醛代谢途径对甲醛吸收的贡献作用

Contribution of the major HCHO metabolic pathways to HCHO-uptake by geranium leaves under liquid HCHO stress

通过2mmol/L H13 CHO溶液处理天竺葵叶片4、24和48h,以及用2、4和6mmol/L HCHO溶液处理天竺葵叶片4h,13 C-NMR分析H13 CHO在天竺葵叶片内的具体代谢途径及主要代谢途径对甲醛吸收的贡献.在时间梯度处理中,天竺葵叶片枸橼酸（citric acid,Cit）的含量一直处于上升趋势,在处理48h后,其相对信号积分达到未处理天竺葵叶片（control,CK）的4.54倍.13C-糖类物质[U-13C]葡萄糖（glucose,Gluc）和[U-13C]果糖（fructose,Fruc）的含量在处理的前4h下降再上升,最后为CK的1.72和1.94倍.在浓度梯度处理中,Cit的含量随HCHO浓度增大而明显上升,最后为CK的7.58倍,13C-糖类物质[U-13C]Gluc和[U-13C]Fruc的含量随HCHO浓度增大先大幅下降后稍有上升,最后为CK的0.15和0.2倍.结合天竺葵的HCHO吸收曲线,表明在甲醛胁迫的早期（0-24h）,叶片中起作用的主要甲醛代谢途径是从甲醛产生Cit的途径;在胁迫后期（24-48h）,叶片中产生Cit的途径和13C-糖类物质[U-13C]Gluc和[U-13C]Fruc合成途径同时起作用,使这个时期内叶片从溶液中吸收甲醛量显著增加.在这一时期可能还有部分[U-13 C]Gluc和[U-13 C]Fruc流入糖酵解或三羧酸（tricarboxylic acid,TCA）循环使有机酸的信号峰增强.综上可知,天竺葵主要通过Cit和糖类物质（[U-13C]Gluc和[U-13C]Fruc）合成途径来代谢液体甲醛.

Summary Geranium（Pelargoniumsp.Frensham）is an ornamental plant cultivated in the worldwide.In this study,wild type（WT）geranium leaves were first treated with H13 CHO solutions in time and concentration-gradient manner to clarify the detailed H13 CHO-metabolic pathways in WT geranium leaves and to quantitatively analyze the roles of the major HCHO-metabolic pathways in HCHO-absorption by WT geranium leaves,and then understand the metabolic mechanism of geranium response to liquid HCHO stress.Geranium was used as the experimental material.In the H13 CHO treatment,2g geranium fresh leaves were soaked in different concentrations of H13 CHO [including 0.1% 2-（N-morpholino）ethanesulfonic acid,MES]solution（100mL）,and then incubated under constant light[100μmol/（m2爛s）]at 25 ℃ for different time with shaking（100r/min）.After incubation,the leaves were washed and used to grind.The extract was transferred intoa 5 mm NMR tube and subjected to 13 C-NMR analysis.Resonance peaks were assigned by comparison with authentic compounds chemical shifts and confirmed by spiking the kalium phosphate buffer（KPB）extracts with authentic reference standards.For comparison of the relative contents of the metabolites,the target peaks were integrated relatively to the reference.Geranium leaves were treated in H13 CHO solution to analyze the detailed HCHO metabolic pathways and the contribution of the major metabolic pathway to HCHO-uptake by geranium leaves. In the time gradient,the content of citric acid（Cit）was on an upward trend,after treatment 48 h,its relative signal integral reached 4.54-fold of the unprocessed geranium leaves（control,CK）.The signal integration of13 C-carbohydrate [U-13 C]glucose（Gluc）and [U-13 C]fructose（Fruc）decreased in the first 4htreatment and then increased,and finally achieved 1.72 and 1.94-fold of CK.In the concentration gradient,the content of citric acid increased obviously with the increase of HCHO concentration,and finally reached 7.58-fold of CK.The signal integration of 13 C-carbohydrate [U-13 C]Gluc and [U-13 C]Fruc slightly increased after decreased dramatically with the increase of HCHO concentration,and finally achieved 0.15 and 0.2-fold of CK.Results suggested that the HCHO-absorption by geranium leaves was a power function in relation with the treatment time.In 2 mmol/L HCHO treatment,during the early stage（0-24h）,the primary functioned metabolic pathway was the Cit produced pathway in geranium leaves.In this period,the geranium leaves absorbed 25% HCHO of total absorption.At the late stage（24-48h）of HCHO treatment,two major metabolic pathways,the Cit produced pathway and 13 C-labelled glucide（[U-13 C]Gluc and [U-13 C]Fruc）generation pathway,functioned simultaneously in geranium leaves.Some13 C-labelled glucide might also enter the glycolysis pathway or the tricarboxylate（TCA）cycle,which thereby allowed an enhancement in many organic acid peaks.In this time,the geranium leaves absorbed 50% HCHO of all uptake.It is concluded that HCHO is eventually converted into glyoxylate which enters into the glyoxylate cycle to produce[3-13 C]Cit,and carbohydrates are synthesized via gluconeogenesis pathway,then isocitrate enters into the TCA cycle to produce amino acids,and these metabolic pathways produce various organic acids.Apparently,geranium leaves metabolize liquid formaldehyde mainly through Cit and glucide（[U-13 C]Gluc and [U-13 C]Fruc）synthesis pathway.