Phosphoenolpyruvate Carboxylase and CarbonEconomy of Apple Seedlings

Seeds of apple cv. Golden Delicious were germinated and cultivatedin the greenhouse until the third leaf emerged. Respirationofgerminating seeds or photosynthesis of the first leaves wasmeasured by infra-red gas analysis and porometry, respectively.To study the role of phosphoenolpyruvate carboxylase (PEPC),the dominant carboxylase in the carbon economy, its CO₂ refixationpotentialwas related to the amount of CO₂ lost in respiration. With arange of 0.2 (dry seeds) to 18 (cotyledons) µmol CO₂ h^–1g^–1 PEPC activity resembled or exceeded the amount ofC0₂ lost in respiration before the third leaf developed. Itis concludedthat PEPC largely contributes to economize the carbonmetabolism of apple seedlings before they become photosyntheticallycompetent. Key words: Apple (Malus pumila Mill.) seedling, carbon economy, phosphoenolpyruvate carboxylase, photosynthesis, respiration     

活性氧对苋菜磷酸烯醇式丙酮酸羧化酶活性的影响

用活性氧H     

低温对玉米叶片PEP羧化酶及其调节特性的影响

低温贮存期间,玉米叶片PEP羧化酶活性随贮存时间的延长而明显降低,对效应剂Gly的敏感性也减弱。多羟基醇(甘油和山梨醇)以及PEP羧化酶的正效应剂G-6-P在与Gly和甘油同时作用时,对PEP羧化酶在低温贮存期间的活性和对Gly的敏感性均有保护效应,且对两者的保护程度相一致,表明低温贮存期间PEP羧化酶对效应剂敏感性减弱与其低温失活有直接关系。     

脱落酸和己糖激酶抑制剂对高表达C4-PEPC转基因稻苗耐旱性的影响

为了研究高表达转玉米C₄-磷酸烯醇式丙酮酸羧化酶（phosphoenolpyruvate carboxylase,PEPC）基因水稻（PC）的耐旱性机制,本研究以PC和未转基因野生型原种kitaake为材料,分别在光照和黑暗预处理24 h,其中光照处理中光强为600 μmol·m^-2·s^-1,预处理后稻苗再在15％聚乙二醇-6000模拟干旱胁迫下,同时使用药理学的方法,通过加入脱落酸和己糖激酶的专一性抑制剂100 μmol·L^-1去甲二氢愈创木酸和10 mmol·L^-1葡萄糖胺,观察两种水稻4~5叶期稻苗耐旱表现。结果发现,与WT水稻相比,在PEG-6000处理后,经过光预处理的PC水稻叶片相对含水量下降较少,始终比WT的高,而且丙二醛含量则较少,脯氨酸诱导增加,表现耐旱;而经过暗预处理后PC植株显著降低这个优势,表明光预处理有利于PC耐旱性的表现;黑暗预处理均显著下调了2供试材料植株叶片中可溶性糖的含量,而对可溶性蛋白的含量影响不显著;而光预处理后PC水稻叶片内可溶性糖含量比WT增加,而在黑暗预处理则PC的显著低于WT的,其中葡萄糖胺对光预处理下PC的可溶性糖含量的下调作用最显著;暗预处理逆转或消除了NO,H₂O₂和钙离子含量变化趋势,这些变化与暗预处理减少了两材料叶片蔗糖和葡萄糖含量变化同步;光暗预处理对两材料的PEPC酶活性的差异影响不大,表明外源玉米C₄-PEPC在水稻中是组成型表达。可见PC叶片可部分通过糖组分,参与内源糖介导ABA和HXK信号途径,缓解干旱处理对叶片的伤害,稳定光合能力。     

Correlation of the Activities of Phosphoenolpyruvate Carboxylase and Pyruvate,Orthophosphate Dikinase with Biomass in Maize Seedlings

The relations of three carbon-assimilating enzymes in maizeto biomass productivity were studied. There was no significantcorrelation between biomass and the amount of fraction I protein(RuBP carboxylase/oxygenase protein). In contrast, both theactivities of phosphoenolpyruvate carboxylase and pyruvate,P₁dikinase were highly correlated to the biomass. (Received February 7, 1983; Accepted March 26, 1983)     

Oligomerization and the Affinity of Maize Phosphoenolpyruvate Carboxylase for Its Substrate

When two different forms of phosphoenolpyruvate carboxylase (PEPC) from maize (Zea mays L.) leaves are present in an assay it is possible to estimate the ratio of Vmax to Km (V/K) for the two forms separately. This measure of the binding of the substrate by the enzyme permits evaluation of the effects of various treatments on the relative substrate-binding velocity of the enzyme. PEPC diluted 1/20 is present in a mixture of a tetrameric form with a high affinity for phosphoenolpyruvate and a dimeric form with a low affinity (M.-X. Wu, C.R. Meyer, K.O. Willeford, R.T. Wedding [1990] Arch Biochem Biophys 281: 324-329). Malate at 5 mM reduced (V/K)1,[mdash]the V/K of the probable tetrameric form[mdash]almost to zero, but reduced (V/K)2[mdash]the V/K of the probable dimer[mdash]by only about 80%. Glucose-6-phosphate (Glc-6-P) at 5 mM increased (V/K)1 to 155% of the control but had no effect on (V/K)2. Glycerol (20%) alone increased both V/Ks, and its effects are additive to the Glc-6-P effects, implying different mechanisms for activation by Glc-6-P and glycerol.     

Effect of Cadmium on gamma-Glutamylcysteine Synthesis in Maize Seedlings

Cysteine, γ-glutamylcysteine, and glutathione and the extractable activity of the enzymes of glutathione biosynthesis, γ-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3), were measured in roots and leaves of maize seedlings (Zea mays L. cv LG 9) exposed to CdCl₂ concentrations up to 200 micromolar. At 50 micromolar Cd²⁺, γ-glutamylcysteine contents increased continuously during 4 days up to 21-fold and eightfold of the control in roots and leaves, respectively. Even at 0.5 micromolar Cd²⁺, the concentration of γ-glutamylcysteine in the roots was significantly higher than in the control. At 5 micromolar and higher Cd²⁺ concentrations, a significant increase in γ-glutamylcysteine synthetase activity was measured in the roots, whereas in the leaves this enzyme activity was enhanced only at 200 micromolar Cd²⁺. Labeling of isolated roots with [³⁵S]sulfate showed that both sulfate assimilation and glutathione synthesis were increased by Cd. The accumulation of γ-glutamylcysteine in the roots did not affect the root exudation rate of this compound. Our results indicate that maize roots are at least in part autonomous in providing the additional thiols required for phytochelatin synthesis induced by Cd.     

In Situ C4 Phosphoenolpyruvate Carboxylase Activity and Kinetic Properties in Isolated Digitaria Sanguinalis Mesophyll Cells

Isolated mesophyll cells from darkened leaves of the C(4) plant Digitaria sanguinalis keep functional plasmodesmata that allow the free exchange of low molecular mass compounds with the surrounding medium. This cell suspension system has been used to measure C(4) PEPC activity in situ using a spectrophotometric assay. Compared to the extracted enzyme assayed in vitro, the essentially non-phosphorylated 'in-cell' C(4) PEPC showed altered functional and regulatory properties. While the S (0.5) for PEP at pH 7.3 was only modestly changed (0.4-0.6 mM), the response to pH was shifted towards the acidic range, being close to the maximal value at pH 7.3. Using expected physiological concentrations of the metabolites, at pH 7.3, the IC(50) for malate showed a five-fold increase, from 1.5 to 8 mM, and was increased further to 22 mM in the presence of the allosteric activator glucose-6-phosphate (4 mM). Thiol compounds like DTT, mercaptoethanol and reduced glutathione weakened the in-situ sensitivity of C(4) PEPC to malate. However, none of them had any effect on this process in vitro. This was not due to thioredoxin-mediated or phoshorylation-dependent processes. Since glutathione is a physiological compound that is present mostly in the reduced state in the cell cytosol, a possible contribution of this thiol to the protection of the enzyme against malate in situ is proposed.     

Cooperative Effects of Light and Temperature on the Activity of Phosphoenolpyruvate Carboxylase from Amaranthus paniculatus L

The phosphoenolpyruvate carboxylase of Amaranthus paniculatus shows in vitro optimum affinity (S_0.5) to phosphoenolpyruvate at a relatively high temperature (about 35°C); even in the presence of activators, it functions efficiently only above 25 to 27°C. At lower temperatures, a steep increase of activity with temperature is observed, due to the high activation energy for the catalyzed reaction. The same behavior in vivo could amplify the photoactivation of the enzyme to a large extent, since the night/day transition is soon followed by a considerable rise in leaf temperature.     

Effect of Diethylpyrocarbonate on the Allosteric Properties of Phosphoenolpyruvate Carboxylase from Crassula argentea

Phosphoenolpyruvate carboxylase from the Crassulacean acid metabolism plant Crassula argentea was substantially desensitized to the effects of regulatory ligands by treatment with diethylpyrocarbonate, a reagent which selectively modifies histidyl residues. Desensitization of the enzyme to the inhibitor malate and the activator glucose 6-phosphate was accompanied by the appearance of a peak in the ultraviolet difference spectrum at 240 nanometers, indicating the formation of ethoxyformylhistidyl derivatives. Hydroxylamine reversed part of the spectral change under native conditions, and almost all of the change under denaturing conditions, but failed to restore sensitivity to effectors. The pH profiles of desensitization to malate and glucose 6-phosphate indicated the involvement of groups on the enzyme with pK, values of 6.8 and 6.4, respectively. Under denaturing conditions, a total of 15 histidine residues per subunit were modified by diethylpyrocarbonate, whereas for the native enzyme nine histidines were modified per subunit. Effector desensitization occurs after the modification of two to three histidyl residues per subunit. The presence of malate reduced the apparent rate constant for desensitization by 60%, suggesting that the modification occurred at the malate binding site. Diethylpyrocarbonate treatment also eliminated the kinetic lag caused by malate. Glucose 6-phosphate did not protect the enzyme against diethylpyrocarbonate-induced desensitization.     

Effect of Increasing Concentrations of Lead and Cadmium on Cucumber Seedlings

The effect of lead and cadmium on biomass accumulation of cucumber seedlings (Cucumis sativus L.) as well as the contents of abscisic acid (ABA), free proline and soluble proteins in leaves were studied. Seedlings were subjected to lead nitrate or cadmium bromide in low concentrations (1 – 5 µM) for 1, 4 or 7 d, and then to the action of the same substances in high concentrations (500 – 1000 µM). The pretreatments of the seedlings with heavy metals in low concentrations enabled them to tolerate the subsequent high concentrations of cadmium and lead without injury. The plant responses to heavy metal treatment were accompanied by the accumulation of ABA, free proline and soluble proteins in leaf tissues.     

Multi-Wall Carbon Nanotubes Effects on Plant Seedlings Growth and Cadmium/Lead Uptake In Vitro

The effects of multi-wall carbon nanotubes (MWCNTs) on plant growth and Cd/Pb accumulation was investigated on seedlings of three plant species including Brassica napus L., Helianthus annus L. and Cannabis sativa L. The experiment consisted of MWCNTs on three concentration levels (0, 10, 50 mg/L) and 200 μM CdCl₂ or 500 μM Pb(NO₃)₂. MWCNTs application effectively improved root and shoot growth inhibited by Cd and Pb salts. In B. napus, total chlorophyll (Chl) content increased by both MWCNTs 10 and 50 mg/L exposure under cadmium or lead stress. MWCNT 10 mg/L mitigated the deleterious effects of Cd ions on total chlorophyll content of H. annus and C. sativa. Wherease higher concentration of MWCNTs decreased Chl content under either Cd or Pb treatments on sunflower seedlings. MWCNT10 effectivly raised cadmium accumulation in seedlings of all three species. MWCNT10 and 50 mg/L also caused higher Pb accumulation in canola and cannabis seedlings, respectively. Based on the results, it seems that the effects of MWCNTs on growth parameters and heavy metal accumulation in plant seedlings is strongly depends on heavy metal type, MWCNTs concentration and plant species.     

温度、pH及效应剂对高粱PEP羧化酶活性的协同作用

纯化的高粱PEP羧化酶活性随pH升高(pH6.6～8.0)而增大。在G6P和Mal存在下,酶活性仍有随pH升高而增大的趋势,但G6P对酶的激活百分率和Mal的抑制百分率随pH升高而降低。高粱PEP羧化酶活性的最适温度高于40℃、酶的催化效率(V_(max)/K_m)随温度升高而增大。高温下,反应激活能降低,Mal对酶活性抑制百分率亦随温度升高而下降,I_(0.5)值增大,Mal增大K_m(PEP)的效应变小。     

从铅锌矿渣中分离的微生物对重金属吸附特性的研究

从铅锌矿渣中分离到 16种菌 (包括 7株细菌和 9株真菌 ) ,并研究了它们对Zn2 + ,Pb2 + ,Cu2 + 的吸附特性。发现大多数菌株对Pb2 + 与Zn2 + 有不同程度的吸附 ,但对Cu2 + 的吸附能力较小。菌株对Zn2 + 的吸附率大于对Pb2 + 的吸附 ,能吸附Pb2 + 的菌株也能吸附Zn2 + 。pH 4～ 6是真菌吸附金属离子的较好范围 ,细菌仅在pH =5 .0条件下 ,对Pb2 + 与Zn2 + 有吸附。在测试的不同金属离子浓度范围内 (5 0mg/L 相似文献   

Partitioning of Nitrogen among Ribulose-1,5-bisphosphate Carboxylase/Oxygenase, Phosphoenolpyruvate Carboxylase, and Pyruvate Orthophosphate Dikinase as Related to Biomass Productivity in Maize Seedlings

Maize (Zea mays L. cv Golden Cross Bantam T51) seedlings were grown under full sunlight or 50% sunlight in a temperature-controlled glasshouse at the temperatures of near optimum (30/25°C) and suboptimum (17/13°C) with seven levels of nitrate-N (0.4 to 12 millimolars). The contents of phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPD), and ribulose-1,5-P₂ carboxylase/oxygenase (RuBisCO) were immunochemically determined for each treatment with rabbit antibodies raised against the respective maize leaf proteins (anti-PEPC and anti-PPD) or spinach leaf protein (anti-RuBisCO). The content of each enzymic protein increased with increasing N and raised under reduced temperature. The positive effect of light intensity on their contents was evident only at near optimal temperature. The relative increase in PEPC and PPD content with increasing N was significantly greater than that of RuBisCO irrespective of growth conditions. These enzymic proteins comprised about 8, 6, and 35% of total soluble protein, respectively, at near optimal growth condition. In contrast to significant increase in the proportion of soluble protein allocated to PEPC and PPD seen under certain conditions, the proportion allocated to RuBisCO decreased reciprocally with an increased biomass yield by N supply.

These results indicated that the levels of PEPC and PPD parallel to maize biomass more tightly than that of RuBisCO at least under near optimal growth condition.

     

Induction of the Inactivation and Degradation of Phosphoenolpyruvate Carboxylase and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in Maize Leaves by Freezing and Thawing

When frozen leaves of 24-day-old maize (Zea mays L.) plant werethawed on moist filter paper at 26°C (freeze-thaw treatment)several enzymes, including phosphoenolpyruvate carboxylase (PEPC)and ribulose-1,5-bisphosphate carboxylase (RuBPC), were rapidlyinactivated and degraded. The kinetics of the inactivation anddegradation were pseudo first-order, and the halftimes for inactivationof PEPC and RuBPC were 3.2 and 2.4 min, respectively. The effectof the freeze-thaw treatment on the inactivation and degradationdiffered among various enzymes: the residual activities of RuBPC,PEPC, hydroxypyruvate reductase, Cyt c oxidase, NADP-malic enzymeand a-mannosidase 10 min after the start of the thawing treatmentwere 7, 16, 54, 64, 97 and 98% of the initial respective levels.Thirty min after the starting of thawing treatment, the amountsof total soluble protein, the large subunit of RuBPC, the smallsubunit of RuBPC, the PEPC subunit and the NADP-malic enzymesubunit had fallen to 61, 2, 16, 8, and 66% of the initial respectiveamounts. The effect of freeze-thaw treatment on PEPC was greater in oldleaves than in young leaves. There was a steady increase ofthe rate of degradation of PEPC by freeze-thaw treatment asplants aged from 6 to 24 days. These results are discussed inthe context of protein degradation in plant cells. (Received August 9, 1993; Accepted January 10, 1994)     

Structure and Regulation of Phosphoenolpyruvate Carboxylase from Sorghum Leaves

Phosphoenolpyruvate carboxylase (ortho-phosphate: oxaloacetate carboxylase, EC 4.11.31, PEPCase), an enzyme widely occurringin bacteria, algae and plants, is an importantcarboxylating enzyme serving a variety of func-tions ranging from photosynthetic carbon dioxidefixation to nitrogen assimilation (Latzko andKelly 1983, O'Leary 1982). It is a key regula-tory enzyme in both C_4 and CAM photosyn-thesis. In C_4 plants, PEPCase is localized inthe mesophyll-cell cytoplasm and catalyzesthe conversion of PEP and bicarbonate to