Phosphoenolpyruvate carboxylase in root nodules of Vicia faba: Partial purification and properties

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) was purified 56-fold from Vicia faba root nodules to a specific activity of 24.8 units mg^-1 protein. Native molecular mass was determined to be 443 kDa by gel permeation chromatography, whereas a molecular mass of 113 kDa was obtained for the subunit by means of SDS-PAGE, indicating that the enzyme is a homotetramer. One peak of activity was obtained by ion-exchange chromatography or gel filtration, and thus there was no evidence of isoenzymes. The effect of pH on PEPC activity was studied, the pH optimum found at 8.25. The effect of substrate (phosphoenolpyruvate, PEP) on the enzyme activity was studied at five different pH values from 6.5 to 9.5. The K_m(PEP) at pH 8.25 proved to be 0.064 m M. Inhibition by malate or activation by glucose-6-phosphate was dependent on the pH of the reaction mixture. Malate behaved as a non-competitive mixed-type inhibitor with a K_i of 0.76 m M , a K_i(s) of 1.15 m M and a K_i(i) of 0.72 m M , at pH 7.0 while at pH 8.25 K_i was about 140 m M. Activation by glucose-6-P was 70% with 4 m M PEP at pH 7, whereas no effect was found at pH 8.25. Experiments with mixed effectors at pH 7 and 1 m M PEP, showed that glucose-6-P can reverse the inhibition caused by L-malate on the PEPC activity.     

Multiple forms of phosphoenolpyruvate carboxylase in mesophyll, epidermal and guard cells of Vicia faba

The distribution of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in different leaf‐cell‐types and tissues of Vicia faba L. cv. 3‐fach Weiße was studied. The highest specific PEPCase activity was found in guard cell protoplasts (16.3 µmol mg⁻¹ protein h⁻¹) whereas for epidermal and mesophyll protoplasts remarkably lower specific activities were found (1.6 and 1.0 µmol mg⁻¹ protein h⁻¹, respectively). On chlorophyll and protoplast basis, a similar distribution of enzyme activity was observed. Compared with epidermal extracts, the specific PEPCase activity of mesophyll tissue was 17‐fold lower. Immunological studies with polyclonal antibodies to PEPCase indicated 3 immunoreactive proteins in epidermal tissue and guard cell protoplasts with molecular masses of 107 000, 110 000, and 112 000. Only the M_r 107 000 protein was found in extracts of mesophyll and epidermis protoplasts. Western immunoblots after native electrophoresis of epidermal and mesophyll proteins showed a significant difference in PEPCase mobility. It is assumed, that the immunostained proteins of M_r 110 000 and 112 000 represent isoforms or subunits of the PEPCase and that they are involved in stomatal movements.     

Expression and manipulation of phosphoenolpyruvate carboxykinase 1 identifies a role for malate metabolism in stomatal closure

Malate, along with potassium and chloride ions, is an important solute for maintaining turgor pressure during stomatal opening. Although malate is exported from guard cells during stomatal closure, there is controversy as to whether malate is also metabolised. We provide evidence that phosphoenolpyruvate carboxykinase (PEPCK), an enzyme involved in malate metabolism and gluconeogenesis, is necessary for full stomatal closure in the dark. Analysis of the Arabidopsis PCK1 gene promoter indicated that this PEPCK isoform is specifically expressed in guard cells and trichomes of the leaf. Spatially distinct promoter elements were found to be required for post-germinative, vascular expression and guard cell/trichome expression of PCK1. We show that pck1 mutant plants have reduced drought tolerance, and show increased stomatal conductance and wider stomatal apertures compared with the wild type. During light-dark transients the PEPCK mutant plants show both increased overall stomatal conductance and less responsiveness of the stomata to darkness than the wild type, indicating that stomata get 'jammed' in the open position. These results show that malate metabolism is important during dark-induced stomatal closure and that PEPCK is involved in this process.     

Dynamics of ionic activities in the apoplast of the sub-stomatal cavity of intact Vicia faba leaves during stomatal closure evoked by ABA and darkness

Stomatal movement is accomplished by changes in the ionic content within guard cells as well as in the cell wall of the surrounding stomatal pore. In this study, the sub-stomatal apoplastic activities of K+, Cl-, Ca2+ and H+ were continuously monitored by inserting ion-selective micro-electrodes through the open stomata of intact Vicia faba leaves. In light-adapted leaves, the mean activities were 2.59 mM (K+), 1.26 mM (Cl-), 64 microM (Ca2+) and 89 microM (H+). Stomatal closure was investigated through exposure to abscisic acid (ABA), sudden darkness or both. Feeding the leaves with ABA through the cut petiole initially resulted in peaks after 9-10 min, in which Ca2+ and H+ activities transiently decreased, and Cl- and K+ activities transiently increased. Thereafter, Ca2+, H+ and Cl- activities completely recovered, while K+ activity approached an elevated level of around 10 mM within 20 min. Similar responses were observed following sudden darkness, with the difference that Cl- and Ca2+ activities recovered more slowly. Addition of ABA to dark-adapted leaves evoked responses of Cl- and Ca2+ similar to those observed in the light. K+ activity, starting from its elevated level, responded to ABA with a transient increase peaking around 16 mM, but then returned to its dark level. During stomatal closure, membrane potential changes in mesophyll cells showed no correlation with the K+ kinetics in the sub-stomatal cavity. We thus conclude that the increase in K+ activity mainly resulted from K+ release by the guard cells, indicating apoplastic compartmentation. Based on the close correlation between Cl- and Ca2+ changes, we suggest that anion channels are activated by a rise in cytosolic free Ca2+, a process which activates depolarization-activated K+ release channels.     

The influence of exogenous proline on the stomatal resistance in Vicia faba

Both the upper and lower stomata of Vicia faba responded to different concentrations of proline supplied either to the 'detached' leaves or sprayed to 'intact' leaves. The stomatal resistance of abaxial surfaces was increased more than that of the adaxial surfaces. A 1-h treatment with 5 m M proline had greater influence than a prolonged treatment. The 'young' leaves responded more than the 'mature' ones to the exogenous proline. A relationship could be demonstrated between endogenous proline, which increased markedly due to exogenous supply, and stomatal resistance.     

Effect of sulphite on phosphoenolpyruvate carboxylase and malate formation in extracts of Zea mays

The effect of SO₃^2? on the activity of PEP-carboxylase and on subsequent malate formation has been studied in leaf extracts of Zea mays. PEP-carboxylase was assayed by incorporation of H¹⁴CO₃ - into oxaloacetate dinitrophenylhydrazone and by a spectrophotometric method. In contrast to ribulose diphosphate carboxylase, PEP-carboxylase was not inhibited by 10 mM SO₃^2? with respect to PEP. As was the case with ribulose diphosphate carboxylase, the activity of PEP-carboxylase was inhibited non-competitively with respect to Mg²⁺. However, the K_i value (84.5 mM) was found to be very high. With respect to HCO₃^?, like ribulose diphosphate carboxylase, PEP-carboxylase was inhibited competitively, but the K_i value (27 mM SO₃^2?) increased by about the same factor (× 9) as the K_m, (0·5 mM HCO₃^?) is decreased. This indicates that the replacement of HCO₃^? by SO₃^2?, common to both enzymes, is facilitated by decreasing the affinity of the enzyme for HCO₃^?. At substrate saturating conditions malate formation by the combined action of PEP-carboxylase and endogenous NADH-dependent malate dehydrogenase in leaf extracts was not inhibited by 10 mM SO₃^2?. Although the malate dehydrogenase is inhibited at this SO₃^2? concentration to about 85%, malate formation is unaffected, as PEP-carboxylase is the rate limiting step its turnover rate being only about 8% of NADH-dependent malate dehydrogenase.     

The kinetics of stomatal responses to VPD in Vicia faba: electrophysiological and water relations models

Abstract. An Ohm's law analogy is frequently employed to calculate parameters of leaf gas exchange. For example, resistance to water vapour loss is calculated as the quotient of vapour pressure difference (VPD) and vapour loss by transpiration. In the present research, this electrical analogy was extended. Steady-state transpiration as a function of VPD, assayed in leaflets of Vicia faba using gas exchange techniques, was compared with steady-state K⁺ current magnitude as a function of voltage in isolated guard cell protoplasts of Vicia faba, assayed using the patch clamping technique in the whole cell configuration. An electrophysiological model originally developed to explain the kinetics of current changes following step changes in voltage across a cell membrane was used to fit the kinetics of transpiration changes following step changes in VPD applied to leaflets of Vicia faba. Following step increases in VPD, transpiration exhibited an initial increase, reflecting the increased driving force for water loss and, for large step increases in VPD, a transient decrease in stomatal resistance. Transpiration subsequently declined, reflecting stomatal closure. By analogy to electrophysiological responses, it is hypothesized that the humidity parameter that is sensed by guard cells is VPD. Two models based on epidermal water relations were also applied to transpiration kinetics. In the first model, the transient increase in transpiration following a step increase in VPD was attributed partially to an increase in the Physical driving force (VPD) and partially to a transient decrease in stomatal resistance resulting from reduced epidermal backpressure. In the second model, the transient decrease in stomatal resistance was attributed to a direct response of the guard cells to VPD. Both models based on water relations gave good fits of the data, emphasizing the need for further study regarding the metabolic nature of the guard cell response to humidity.     

Mutual stimulation of temperature and light effects on C(4) phosphoenolpyruvate carboxylase in leaf discs and leaves of Amaranthus hypochondriacus

This article reports marked modulation of the activity and regulatory properties of phosphoenolpyruvate carboxylase (PEPC) by temperature and light in leaf discs as well as leaves of Amaranthus hypochondriacus. The activity of PEPC increased by 1.7-fold at 45 degrees C over 25 degrees C. Warm temperature also stimulated the photoactivation of PEPC. The activation by light of PEPC was 1.9-fold at 25 degrees C and increased to 2.2-fold at 45 degrees C. The sensitivity of PEPC to its inhibitor malate was less and the activation by glucose-6-phosphate (G-6-P) or inorganic phosphate (Pi) was more at 45 degrees C than that at 25 degrees C. These effects of temperature were quite pronounced in light. Similar responses were observed when detached leaves were exposed to varying ambient temperature (dry heat). The activity of PEPC increased by 1.6-fold at 45 degrees C over 25 degrees C in the dark. The activation of PEPC by light was 2.1-fold at 25 degrees C and increased to 2.6-fold at 45 degrees C. Inhibition by malate was less and activation by G-6-P or Pi was more at 45 degrees C than that at 25 degrees C. Thus, there was a marked modulation of not only the activity but also the regulatory properties of the enzyme by temperature and light, independently as well as cooperatively with each other. Further experiments suggested that PEPC was able to memorize to a significant extent the changes induced by warm temperature and that these changes were complemented by subsequent illumination. These effects were not due to changes in PEPC protein levels. We conclude that temperature and light can modulate PEPC activity and regulatory properties not only individually but also in a significantly cooperative manner with each other. As significant increases in temperature are common during daytime in tropical or subtropical conditions, we suggest that the synergistic effects of temperature and light are quite relevant in optimizing the activity of PEPC in leaves of C(4) plants.     

Effects of turgor potentials of epidermal cells neighbouring guard cells on stomatal opening in detached leaf epidermis and intact leaflets of Vicia faba L. (faba bean)

Leaflets of Vicia faba L. (faba bean) were used to determine whether the mechanical forces resulting from the turgor potentials (Φ_p) of the larger epidermal cells neighbouring guard cells play a significant role in regulating stomatal aperture. When Φ_p, of epidermis and Φ_p of bulk leaflet tissue were compared at midday, Φ_p of epidermis were only 15–25% those of bulk leaflet tissue at all but the most negative leaflet water potentials (Φ). When plants were bagged to increase Φ by reducing vapour pressure differences between leaflets and air, Φ_p of bulk leaflet tissue increased to predawn values, but Φ_p, of epidermis increased to only = 20% of predawn values and stomata opened to their widest apertures. Stomatal apertures were positively correlated with Φ_p of bulk leaflet tissue but they were not correlated with Φ_p of epidermis. Reductions in epidermal Φ_p, began predawn, before stomata were open, and reached minimum values at midday, when stomata were open. We conclude that, in Vicia faba, (1) reduction of Φ_p of epidermal cells begins predawn, reducing the counterforce to stomatal opening that would exist if full epidermal turgor were maintained throughout the day, and (2) changes in Φ_p, of leaf epidermal cells do not play a significant role in regulating stomatal aperture.     

Comparative studies of coupled assays for phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) from higher plants is usually assayed by using malate dehydrogenase (MDH) as a coupling enzyme. To avoid erroneous readings caused by metal ions, which convert oxaloacetate (OAA) to pyruvate, lactic dehydrogenase can be included. Reporting the total NADH used by both coupling enzymes gives the total OAA production. Microbial PEPC has been assayed by employing citrate synthase (CS) as a coupling enzyme which detects the reaction of CoA with Ellman's reagent. Comparable K_m values for MgPEP are found with the two assays. When MDH alone is used as the coupling system, the V_max value is about 60% larger than the one found with the CS assay. However, when MDH is added to the CS assay without the NADH cofactor, V_max is brought back to the same level as that with the NADH-coupled enzyme. Malate inhibition of PEPC assayed with the CS coupling system is blocked by low concentrations of citrate in the range produced in the assay. High concentrations of citrate inhibit PEPC. Glucose-6-phosphate in concentrations higher than 1 m M blocks the response of PEPC to added MDH in the CS assay.     

Partial purification and characterization of NADH-glutamate synthase from faba bean (Vicia faba) root nodules

The NADH-dependent glutamate synthase (EC 1.4.1.14) from the plant fraction of N₂-fixing faba bean (Vicia faba) nodules has been purified 74-fold to a specific activity of about 3 μmol min⁻¹ mg protein⁻¹ with a final yield of 32%. The NADH-GOGAT activity was associated with a single form of the enzyme that behaved as a monomeric protein with a subunit molecular weight of 195 kDa and a native molecular weight from 222 to 236 kDa estimated by gel filtration or PAGE, respectively. The NADH-GOGAT band on SDS-PAGE was cut out and used to produce antibodies. Western blots of SDS-PAGE of crude nodule proteins revealed a 195 kDa polypeptide in root extracts but not in those of leaves or bacteroids. The antiserum also cross-reacted with a polypeptide of camparable molecular weight (195 kDa) from both amide and ureide transporting species legume nodules, indicating that some antigenic epitopes have been conserved between nodule NADH-GOGAT of different species.     

NADPH氧化酶参与水杨酸诱导的蚕豆气孔关闭过程

水杨酸(SA)可以浓度依赖的方式诱导蚕豆叶片的气孔关闭,1～1000μmol·L~(-1)SA所诱导的气孔关闭可以再开放,而10~(-2)mol·L~(-1)的SA导致的气孔关闭则否。质膜NADPH氧化酶抑制剂二亚苯基碘(DPI)可削弱SA作用的45%～60%。表明SA诱导的气孔关闭可能与H_2O_2的产生有关。以H_2O_2荧光探针H_2DCFDA结合显微注射技术直接检测保卫细胞内产生H_2O_2的结果显示,100μmol·L~(-1)SA可引起保卫细胞内荧光素(DCF)荧光快速增强。在DPI存在的情况下,经SA处理的保卫细胞,仅在其叶绿体部位产生H_2O_2,而质膜附近的DCF荧光增强则受到抑制。表明叶绿体可能是保卫细胞内产生H_2O_2的主要部位,质膜NADPH氧化酶也可能参与SA诱导H_2O_2的产生。     

蚕豆未成熟子叶原生质体再生植株

近年来,豆科植物原生质体诱导再生植株的研究越来越受到国内外学者关注。但在籽粒型食用豆科植物中,迄今成功的种类仍然不多,在文献记载中仅见有大豆、赤豆、豇豆、豌豆和刀豆,说明籽粒型食用豆科植物原生质体再生植株仍有较大困难,要取得禾谷类作物那样的重大进展,尚需作出巨大努力。蚕豆原生质体培养仅见有从预培养的叶肉原生质体再生细胞团、从茎尖和叶肉原生质体再生愈伤组织和从     

Metabolic regulation and gene expression of root phosphoenolpyruvate carboxylase by different nitrogen sources

Alfalfa (Medicago sativa L.) N-sufficient plants were fed 1·5 mM N in the form of NO₃⁻, NH₄⁺ or NO₃⁻ in conjunction with NH₄⁺, or were N-deprived for 2 weeks. The specific activity of phosphoenolpyruvate carboxylase (PEPC) from the non-nodulated roots of N-sufficient plants was increased in comparison with that of N-deprived plants. The PEPC value was highest with NO₃⁻ nutrition, lowest with NH₄⁺ and intermediate in plants that were fed mixed salts. The protein was more abundant in NO₃⁻-fed plants than in either NH₄⁺- or N mixed-fed plants. Nitrogen starvation decreased the level of PEPC mRNA, and nitrate was the N form that most stimulated PEPC gene expression. The malate content was significantly lower in NO₃⁻-deprived than in NO₃⁻-sufficient plants. Root malate accumulation was high in NO₃⁻-fed plants, but decreased significantly in plants that were fed with NH₄⁺. The effect of malate on the desalted enzyme was also investigated. Root PEPC was not very sensitive to malate and PEPC activity was inhibited only by very high concentrations of malate. Asparagine and glutamine enhanced PEPC activity markedly in NO₃⁻-fed plants, but failed to affect plants that were either treated with other N types or N starved. Glutamate and citrate inhibited PEPC activity only at optimal pH. N-nutrition also influenced root nitrate and ammonium accumulation. Nitrate accumulated in the roots of NO₃⁻- and (NO₃⁻ + NH₄⁺)-fed plants, but was undetectable in those administered NH₄⁺. Both the nitrate and the ammonium contents were significantly reduced in NO₃⁻- and (NO₃⁻ + NH₄⁺)-starved plants. Root accumulation of free amino acids was strongly influenced by the type of N administered. It was highest in NH₄⁺-fed plants and the most abundant amides were asparagine and glutamine. It was concluded that root PEPC from alfalfa plants is N regulated and that nitrate exerts a strong influence on the PEPC enzyme by enhancing both PEPC gene expression and activity.     

Effects of low-level ozone exposure under ambient conditions on photosynthesis and stomatal control of Vicia faba L.

Abstract. Gas exchange rates of 4-week-old faba bean plants were measured after exposure to ozonc (120 μg m³) in an open top chamber for 8 h per day over a period of 2 weeks. The exchange rates were compared with those of control plants. Plants exposed between mid-May and the end of July 1987 showed a minor negative effect on stomatal conductance, while there was no effect on photosynthesis and respiration. Plants exposed between the end of August and early October showed a negative effect on both stomatal conductance and photosynthesis. In addition, the dark respiration rate was slightly increased. It is concluded that ozone can have a direct effect on the stomata as well as on the photosynthetic system and that the stomata are more sensitive than the photosynthetic system.     

Inhibition of maize leaf phosphoenolpyruvate carboxylase by diethyl oxaloacetate

Diethyl oxaloacetate was found to be a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase activity with respect to the substrate phosphoenolpyruvate. The K_i values, based on total diethyl oxaloacetate, decreased with increasing pH, while the K_i values, based on the enol tautomer (average of 4 M), were similar and independent of pH. The results suggest that inhibition is dependent on the enol tautomer. Diethyl oxaloacetate was a weak inhibitor following treatment of the enzyme with dithiothreitol; inhibition could be restored by treatment with diamide, indicating inhibition depends on the reduction state of thiol groups on the enzyme.Abbreviations DTT dithiothreitol - HPLC high performance liquid chromatography - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - Tricine N-tris(hydroxymethyl)methylglycine     

Sulfate Ion Effect on Stability and Regulatory Properties of PEP Carboxylase from the C4Plant Cynodon dactylon

When Tris–SO₄was used as an extraction buffer for phosphoenolpyruvate carboxylase (PEPC) from leaves of the C₄plant Cynodon dactylon(L.) Pers., a higher extractable activity was obtained as compared to Tris–HCl, especially at low phosphoenolpyruvate concentrations and an assay pH of 7.2. The Tris–SO₄-extracted PEPC activity was stable under dilution and remained unchanged for at least 24 h at 22°C. This enzyme was less sensitive to both activation by glucose-6-phosphate and inhibition by L-malate. The effects of Tris–SO₄could be attributed to its preferential exclusion from the enzymic protein domain and, therefore, to a shifting of this oligomeric enzyme to a more aggregable form that is more stable and active.     

植物细胞壁伸展测定仪在蚕豆扩张蛋白特性研究中的应用

扩张蛋白(expansin)在细胞扩张和果实成熟中起着极为重要的作用.植物细胞壁伸展测定仪是研究扩张蛋白必不可少的仪器.为此以电涡流传感器为核心部件装配了一种具有结构简单、操作方便和测量准确等优点的新型测定仪,并利用该仪器研究了蚕豆(Vicia faba)扩张蛋白的特性.结果表明蚕豆根、茎、上胚轴和成熟叶片中均存在扩张蛋白,而且叶片和幼根的扩张蛋白活性最强;免疫印迹证实在蚕豆根、茎、上胚轴和成熟叶片中确实存在扩张蛋白.以上结果说明本仪器灵敏且可靠,用此仪器首次发现在成熟叶片中存在扩张蛋白.     

Kinetic properties of phosphoenolpyruvate carboxylase from lupin nodules and roots

The kinetic properties of two forms of phosphoenolpyruvate carboxylase (PEPC I and PEPC II, EC 4.1, 1.31) from lupin ( Lupinus luteus L. cv. Ventus) nodules and one enzyme form (PEPC III) from roots were studied. The Michaelis constant (K_m) values for PEP, Mg²⁺ and especially HCO₃⁻were lower for PEPC I. Kinetic studies showed that aspartate is a competitive inhibitor at pH 7.2 and inhibitor constant (K_i) values are different for the three forms of PEPC. Malate is a competitive inhibitor for PEPC I and PEPC III and shows mixed-type inhibition for PEPC II. Malate inhibition is dependent upon the pH of the assay. Different effect of several metabolites was also observed. The temperature optimum was near 39°C for PEPC I and around 43°C for PEPC II and PEPC III. PEPC I appeared to be the most thermolabile. It is suggested that PEPC I from lupin nodules is closely associated with N₂ fixation.     

Effects of sulfite on phosphoenolpyruvate carboxylase and nicotinamide adenine dinucleotide phosphate-dependent malate dehydrogenase in epidermal peels in two cultivars of pea

We have identified a differential response of stomatal conductance to sulfur dioxide in two cultivars of pea ( Pisum sativum L. cvs P715 and Nugget). The response to sulfite exposure of PEPC activities present in epidermal peels obtained from the two cultivars was qualitatively in agreement with the results obtained for stomatal conductance. With epidermal tissue isolated from the more sensitive cultivar, we have investigated the effect of light and sulfite on guard cell phosphoenolpyruvate carboxylase (E.E. 4.1.1.31.) and NADP-dependent malate dehydrogenase (E.C. 1.1.1.82), two enzymes of the malate biosynthetic pathway. No difference was found between the substrate-saturated activity of phosphoenolpyruvate carboxylase in epidermal tissue incubated in the light or in the dark under the same conditions. Substratesaturated NADP-dependent malate dehydrogenase activity increased nearly 3-fold during a 60 min incubation in the light. Incubations of epidermal tissue in the light in the presence of sulfite resulted in a decrease in the activity of both enzymes. Our results suggest that the inhibition of these two enzymes of the malate biosynthetic pathway may be one cause of sulfur dioxide-mediated stomatal closure.