Kinetic characterization of phosphoenolpyruvate carboxylase extracted from whole-leaf and from guard-cell protoplasts of Vicia faba L. (C3 plant) with respect to tissue pre-illumination

Summary Whole leaves and guard-cell protoplasts of the C₃ plant Vicia faba L. (broad bean) were separately extracted following a period of illumination or following a period of darkness. Kinetic parameters of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), V_max and K_{m (PEP · Mg)}, were determined as a function of assay pH (7.0 or 8.1), the presence of 5 mm glucose-6-P_free (Glc-6-P, an activator), and the presence of 5 mm malate_free (an inhibitor). On the basis of these parameters, guard-cell PEPC was distinguished from that of whole leaf, indicating either that guard cells contain a unique isoenzyme of PEPC or a different complement of isoenzymes or - and less likely - that the obligatorily different methodologies for the leaf (intact organ) and the guard-cell (protoplast) enzymes altered them specifically.The values of V_max were relatively unchanged, regardless of assay conditions or tissue pretreatment. The values obtained for whole-leaf PEPC V_max were restricted to a small range (52.4 ± 5.9 (SD) to 64.4 ± 4.8 (SD) mol · g fresh mass^-1 · h^-1; the high value coincided with the presence of Glc-6-P, and the low value was obtained in the presence of malate. Guard-cell PEPC V_max was also restricted to a small range: 7.48 ± 0.89 (SD) pmol · guard-cell pair^-1 · h^-1 (pH 8.1, light, +Glc-6-P) to 5.79 ± 0.60 (SD) pmol · guard-cell pair^-1 · h^-1 (pH 7.0, dark, +malate). Depending on effectors, and particularly pH, large changes in K_{m (PEP · Mg}) were calculated (whole-leaf PEPC: 0.03 to 3.84 mm; guard-cell PEPC: 0.06 to 3.43 mm). For both extracts, the low values were obtained at pH 8.1, +Glc-6-P, and the high values at pH 7.0, +malate. Although the ranges of K_m values were broadly similar, the PEPCs reacted differently to individual changes in assay components. In very general terms, whole-leaf PEPC was relatively more efficient at pH 8.1, whereas at pH 7.0, the enzymes behaved more similarly.An effect of in vivo pre-illumination on guard-cell PEPC was not detected. A leaf pre-illumination effect on whole-leaf PEPC was highly statistically significant when assayed under control conditions at pH 7.0. The effect was small - typically a 26% decrease in K_{m (PEP · Mg}) this typical decrease was less than the range of values in replicate experiments. Such a small pre-illumination effect (even if real) could, therefore, easily go undetected. Whether such a small change could have physiological relevance is an open question. Neither with the whole-leaf PEPC nor with the guard-cell PEPC was the IC₅₀ (malate) or A_0.5 (Glc-6-P) determined for any condition. These kinetic parameters are a focus of present work.     

Partial purification and characterization of stomatal phosphoenolpyruvate carboxylase from Vicia faba

Stomatal phosphoenolpyruvate carboxylase (PEPCase EC 4.1.1.31), extracted from abaxial epidermal peels of Vicia faba L. cv. Frühe Weiβkeimige, was partially purified by ammoniumsulfate precipitation, and molecular sieve (Sepharosc S-400) and ion exchange (DEAE-Sepharose) chromatography. The partially purified enzyme, essentially free of a PEPCase isoform existing in mesophyll and epidermal cells, had a specific activity of 300 nkat mg-¹ protein at 25°C. Western immunoblot analysis revealed that the stomatal enzyme had two bands (M_: of 110000 and 112000), crossreacting with PEPCase antibodies raised against PEPCase from Ka-lanchoe daigremontiana . The native molecular mass of the enzyme (467000) points to a tetrameric subunit structure. The temperature optimum was found to be 35°C; cold treatments of PEPCase before assaying were accompanied by inactivation. The energy of activation was calculated to 51 kJ mol-¹. The kinetic behaviour of the enzyme at fixed MgCl₂ concentrations is characterized by a pH optimum between pH 8.0–8.2 with or without 1 m M malate or 5 m M glucose-6-phosphate (Glc-6-P), but a combination of both effectors resulted in a shift of the optimum to pH 7.6. The enzyme showed a pH sensitive inhibition by 1 m M malate and an activation by Glc-6-P. At low pH (6–7), Glc-6-P was able to compensate for the malate induced inhibition of the enzyme. Malate and Glc-6-P both affected K_m(PEP), drastically and influenced V_max at pH 7, but not at pH 8.3. The inhibition constant of malate was determined to be 1.2 m M at pH 7. From the Dixon plot, a competitive inhibition of malate was assumed under defined assay conditions.     

Modulation of the activity of phosphoenolypyruvate carboxylase during potassium-induced swelling of guard-cell protoplasts of Vicia faba L. after light and dark treatments

Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) activity was found to be modulated by light and darkness when measured in the presence of K⁺, which had been added to induce swelling of guard-cell protoplasts (GCPs) from Vicia faba L., whereas no modulation was detected in the absence of K⁺ (PEPcase activity remained constant at 1.5±0.15 pmol PEP metabolized · GCP^–1 ·h^–1; subsequently, pmol GCP^–1 ·h^–1 will be used). The activity of PEPCase increased by 100% (from 1.5 to 3 pmol·protoplast^–1·h^–1) in darkness and by 200% (from 1.7 to 5 pmol·protoplast^–1· h^–1) in light and oscillations in activity of these magnitudes were repeated at intervals of 2 min (dark) and 2.5 min (light) for a period of 10 min during K⁺-induced increase in the volume of GCPs. The oscillations were reflected in changes in malate-pool sizes determined in plastids, mitochondria and the supernatant fraction (consisting of the cytosol and the vacuole). Malate probably functioned as a mitochondrial substrate, thus supplying ATP for K⁺ uptake and the swelling of the protoplasts. On the basis of the present paper and previous results (H. Schnabl and B. Michalke 1988, Life Sci. Adv. Plant Physiol. 7, 203–207) involving adenine nucleotidepool sizes in fractionated GCPs, a model is proposed to explain the cause-effect relationship between K⁺, PEPCase, the cytosolic and mitochondrial malate levels and ATP levels during the K⁺-induced increase of GCP volume.Abbreviations GCP dtguard-cell protoplast - PEP phosphoenol-pyruvate - PEPCase PEP carboxylase The authors thank Professor Hermann Schnabl, University of Stuttgart (FRG), for his assistance in applying the graph theory analysis. This work was supported by Deutsche Forschungsgemeinschaft to H.S.     

Thidiazuron-induced plant regeneration from protoplasts of Vicia faba cv. Mythos

Summary Protoplasts of 10 cultivars of V. faba were isolated from etiolated shoot-tips and tested for their regeneration capacity. After purification, protoplasts were embedded in sodium alginate and cultivated in the medium of Kao and Michayluk (1975) containing 0.5 mg·1^–1 of each 2,4-dichlorophenoxyacetic acid, naphthylacetic acid and 6-benzylaminopurine. Depending on cultivar, division frequencies of up to 40% were obtained. Six weeks after embedding, protoplast-derived calluses were transferred to Gelrite-solidified media with different combinations of growth regulators. A two step protocol (auxin high/low) was tested for its ability to induce somatic embryogenesis. The formation of globular structures was observed, but no embryo formation could be achieved. In contrast, cultivation of protocalluses on medium supplemented with thidiazuron resulted in shoot development in cultivar Mythos. To generate mature plants, the shoots were grafted onto young seedlings. In order to optimize the in vitro-conditions, different concentrations of thidiazuron alone or in combination with naphthylacetic acid were tested, showing that an increase of thidiazuron and the addition of naphthylacetic acid positively affects both the viability of protocalluses and the regeneration frequency.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 5-benzylaminopurine - GA₃ gibberellic acid - IBA indole-3-butyric acid - Kin kinetin - KM Kao and Michayluk - MS Murashige and Skoog - NAA naphthylacetic acid - TDZ thidiazuron - Zea zeatin     

A comparison of the kinetic properties of phosphoenolpyruvate carboxylase from guard-cell and mesophyll-cell protoplasts of Commelina communis

Some kinetic properties of partially purified phosphoenolpyruvate carboxylase (PEPCase) from guard-cell and mesophyll-cell protoplasts of Commelina communis are described. The PEPCase activity inherent to each cell type was determined and the apparent K _m (phosphoenolpyruvate) and K _i (malate) were compared. Malate sensitivity was much higher (K _i malate 0.4 mol m^–3) in the extract of guard-cell protoplasts than in that of mesophyllcell protoplasts (K _i malate 4.2 mol m^–3). The stimulation of activity by glucose-6-phosphate in the presence of malate (deinhibition) was also investigated in extracts from both cell types and was found to be similar to previously reported results with epidermal tissue. The effect of contamination of an extract of guard-cell protoplasts with mesophyll-cell protoplasts was measured in the presence and absence of malate. It was found that a small amount to mesophyll-cell contaminant appears to desensitize the malate inhibition of PEPCase from guard-cell protoplasts. It is concluded that experiments which use epidermal tissue to study guardcell PEPCase may give misleading information as a consequence of mesophyll contamination.Abbreviations Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase     

Regulation of blue-light-induced proton pumping by Vicia faba L. guard-cell protoplasts: Energetic contributions by chloroplastic and mitochondrial activities

An initial response during signal transduction in guard cells, following absorption of blue light, is the extrusion of protons. Translocation of protons across the guard-cell plasmalemma is an energy-requiring activity. The present study has investigated the energetic contribution from guard-cell chloroplasts and mitochondria to blue-light-induced proton pumping by Vicia faba guard-cell protoplasts. The addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea to the protoplast suspension had a minimal effect on rates of acidification when oxygen concentrations of the medium were maintained close to near-saturating levels. Under the same conditions, oligomycin reduced both the rates of blue-light-induced acidification and total proton efflux. Lowering the oxygen concentration of the suspending medium to approximately 20 M resulted in complete inhibition of blue-light-induced acidification activity. Swelling of protoplasts induced by blue light was also inhibited by low oxygen levels. Levels of ATP from whole-protoplast extracts were reduced by about 64% when exposed to low levels of oxygen. Increasing oxygen levels to near-saturating levels restored both blue-light-induced acidification rates and swelling of the protoplasts within a 60-min recovery period. Levels of ATP also increased during the recovery period. Addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea or oligomycin to the suspending medium prior to increasing the oxygen concentration caused a reduction in acidification rates after the recovery period by 40 and 80%, respectively. Levels of ATP in guard-cell protoplasts were also reduced by both inhibitors after a 60-min recovery period. The results demonstrate that both guard-cell chloroplasts and mitochondria contribute energetically to blue-light-induced proton pumping by guard-cell protoplasts. Furthermore, both energy sources are inhibited by low oxygen concentrations, suggesting coordinated metabolic regulation between photo- and oxidative phosphorylation in guard cells.Abbreviations BL blue light - Chl chlorophyll - DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - GCPs guard-cell protoplasts This research was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada and a University Research Grant from The University of Calgary. Dr. L. Gedamu (University of Calgary) is thanked for providing access to the bioluminometer. Technical assistance by C. Chmielewski, C. Turnnir, S. Ham and K. Meyer is gratefully acknowledged.     

Kinetic changes with temperature of phosphoenolpyruvate carboxylase from a CAM plant

Abstract. Purified and crude phosphoenolpyruvate carboxylase from the CAM plant Kalanchoë daigremontiana Hamet et Perrier ( Bryophyllum diagremontianum ) was assayed at temperatures between 10 and 45° C. The optimum temperature of the enzyme activity changed with substrate availability and effector concentration in the assay. l -malate inhibited the enzyme activity and lowered the optimum temperature. Glucose-6-phosphate raised the optimum temperature to 43°C. K _m values for phosphoenolpyruvate increased with assay temperature from 0.12 mol m^-3 at 15° C to 0.36 mol^m−3 at 35° C. Inhibition by malate increased with temperature and acidity of the assay. In the crude enzyme 50% of control activity was inhibited by 1.65 mol m^-3 malate at 15° C and by 0.5 mol m^-3 at 35° C (at pH 7.0). With purification malate sensitivity was lost ( K _i values for malate at least 10 times higher). The shift in optimum temperatures for PEP-carboxylase activity thus results from changes in the kinetic parameters with temperature and allosteric effectors. The often low optimum temperatures for CO₂ fixation observed in nature may thus be the result of substrate and effector concentrations in the cytoplasm and the antagonistic effect of temperature on substrate affinity and effector efficiency on phosphoenolpyruvate carboxylase.     

Purification and characterization of trypsin inhibitor from seeds of faba bean (Vicia faba L.)

A trypsin inhibitor from seeds of faba bean (Vicia faba L.) was purified to near homogeneity as judged by native-PAGE with about 11 % recovery using ammonium sulphate fractionation, ion-exchange chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. The inhibitor had a molecular weight of 18 kD as determined by SDS-PAGE and Sephadex G-100. The inhibitor inhibited trypsin and chymotrypsin to the extent of 48 and 12 %, respectively. The inhibtion was of non-competitive type with dissociation constant for the enzyme inhibitor complex in the region of 0.07 mg·ml⁻¹. The inhibtor was stable between pH 4 and 5. It completely lost its activity when heated at 125 °C for 1 h or at 100 °C for 2 h. The inhibitor also lost its activity on exposure to 2-mercaptoethanol. Based on these properties, it could be concluded that Vicia faba trypsin inhibitor belongs to Bowman-Birk type of inhibitors, as it has molecular weight lower than generally observed for Kunitz type inhibitors.     

Intercultivar difference with respect to mutagenicity of fava beans (Vicia faba L.) after incubation with nitrite

The incidence of gastric cancer in some areas in Colombia is positively correlated with the consumption of fava beans. Incubation of fava beans in nitrite-containing acidic solutions (simulated gastric conditions) resulted in the formation of mutagens as detected with the Salmonella/microsome assay. More than 15-fold differences in mutagenic response were observed for the various cultivars tested. In general, brown cooking cultivars exhibited less mutagenic activity than white cooking cultivars. Introduction of cultivars of fava beans with a low mutagenic activity might result in a reduction in the incidence of stomach cancer.     

The effect of red and far-red light on proton secretion from mesophyll-cell protoplasts of Vicia faba L.

The influence of red and far-red irradiation on the transport of H⁺ and ⁸⁶Rb⁺ through the plasmalemma was studied using parenchymal protoplasts isolated from Vicia faba leaves. The results indicate that red light stimulates H⁺ secretion and the uptake of ⁸⁶Rb⁺. Moreover, it has been demonstrated that far-red irradiation acts antagonistically with respect to red light in both these processes.     

Purification and characterization of phosphoenolpyruvate carboxylase from a cyanobacterium.

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified 100-fold from the cyanobacterium Coccochloris peniocystis with a yield of 10%. A single isozyme was found at all stages of purification, and activity of other beta-carboxylase enzymes was not detected. The apparent molecular weight of the native enzyme was 560,000. Optimal activity was observed at pH 8.0 and 40 degrees C, yielding a Vmax of 8.84 mumol/mg of protein per min. The enzyme was not protected from heat inactivation by aspartate, malate, or oxalacetate. Michaelis-Menten reaction kinetics were observed for various concentrations of PEP, Mg2+, and HCO3-, yielding Km values of 0.6, 0.27, and 0.8 mM, respectively. Enzyme activity was inhibited by aspartate and tricarboxylic acid cycle intermediates and noncompetitively inhibited by oxalacetate, while activation by any compound was not observed. However, the enzyme was sensitive to metabolic control at subsaturating substrate concentrations at neutral pH. These data indicate that cyanobacterial PEP carboxylase resembles the enzyme isolated from C3 plants (plants which initially incorporate CO2 into C3 sugars) and suggest that PEP carboxylase functions anapleurotically in cyanobacteria.     

Phosphoenolpyruvate carboxylase in developing seeds of Vicia faba L.: gene expression and metabolic regulation

To analyze the role of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) during seed development, two cDNA clones encoding two isoforms of PEPCase were isolated from a seed-specific library of Vicia faba. The two sequences (VfPEPCase1 and VfPEPCase2) have a sequence identity of 82 and 89% on the nucleotide and amino acid levels. The VfPEPCase1 mRNA was found to be predominantly expressed in roots and developing cotyledons whereas the VfPEPCase2 mRNA was more abundant in green and maternal tissues. In the cotyledons, PEPCase mRNAs accumulated from early to mid cotyledon stage and decreased thereafter. The PEPCase activity increased continuously during cotyledon development. The enzyme was strongly activated by glucose-6-phosphate, but not by glucose, fructose or sucrose. Asparagine was weakly activating whereas malate, aspartate and glutamate were inhibitory. The inhibitors became less effective with increasing pH. Aspartate was a much stronger inhibitor of cotyledonary PEPCase than glutamate at both pH 7.0 and 7.5. The sensitivity of PEPCase to malate inhibition decreased from early to mid cotyledon stage at a time when storage proteins are synthesized. This indicates activation on the protein level, possibly by protein phosphorylation. Nitrogen starvation in the presence of hexoses but not sucrose decreased mRNA levels of VfPEPCase1 and enzyme activity, indicating control on the mRNA level by both carbon and nitrogen. It is concluded that in developing cotyledons PEPCase is probably important for the synthesis of organic acids to provide carbon skeletons for amino acid synthesis. Received: 15 July 1998 / Accepted: 10 October 1998     

The amino acid sequence of plastocyanin from Vicia faba L. (broad bean)

The amino acid sequence of plastocyanin from broad bean was determined. It consists of a single polypeptide chain of 99 residues. The sequence was determined by using a Beckman 890C sequencer and by dansyl-phenyl isothiocyanate analysis of peptides obtained by the enzymic cleavage of purified cyanogen bromide fragments. Some parts of the sequence depend on the results of Edman degradation of peptides for which amino acid analyses were not obtained. The evidence for one overlap is not strong.     

The primary processes of photosystem II in purified guard-cell protoplasts and mesophyll-cell protoplasts from Commelina communis L.

The performances of four gas chromatographic detectors (nitrogen-phosphorus, flamephotometric, electron-capture and flame-ionisation) were compared for the analysis of permethylated cytokinins. The nitrogen-phosphorus and flame-photometric detectors (selective for nitrogen and sulphur-containing compounds respectively) gave greatly enhanced sensitivity and-or specificity compared with the flame-ionisation detector for a wide range of cytokinin types. The response of the electron-capture and flame-ionisation detectors was quantitatively similar. The improved performance of the nitrogen-phosphorus and flame-photometric detectors was used to develop rapid and simple purification techniques for the assay of zeatin and zeatin riboside in coconut milk; dihydrozeatin and dihydrozeatin riboside in bean leaves and cytokinin nucleosides in wheat-germ transfer RNA.     

Rapid method for isolation and purification of protoplasts from epidermal tissue ofVicia faba L. leaves

Summary A method has been developed for isolating and purifying epidermal and guard cell protoplasts (ECPs and GCPs) from leaves ofVicia faba L. This method has three essential characteristics: 1) requires only small quantities of initial plant tissue; 2) is rapid, being based on a two-step enzymatic digestion and purification by discontinuous density gradient centrifugation using Percoll, and 3) gives a high viability of purified protoplasts. The procedure yielded about 6% ECPs and 10% GCPs on the basis of their occurrence on epidermal foliar tissue, the final suspension of protoplasts being 100% pure. Cell viability was assessed by the ability of each protoplast type to accumulate neutral red in their vacuoles. Values of 90% and 95% were obtained for ECPs and GCPs respectively. The complete lack of cell wall after enzymatic treatment was checked at the light microscope level by the absence of Calcofluor fluorescent staining of cellulosic material. Representative counts for purified ECPs and GCPs obtained at the interfaces of 20/30% and 40/80% Percoll gradients were 1.32×10⁵ and 3.7×10⁵ elements/ml, which represents a yield of 930 and 2,200 protoplasts/cm² of epidermal tissue respectively. The integrity of the plasma membrane and organelles after the isolation procedures was confirmed by transmission electron microscopy and by the ability of protoplasts to exclude Evans blue.

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Resumen Se ha desarrollado un método para el aislamiento y purifición de protoplastos de células epidérmicas y células guardianas (PCEs PCGs) de hojas deVicia faba L. Este método posee tres características esenciales: 1) solamente requiere pequeñas cantidades de tejido vegetal inicial; 2) rapidez, en base a una digestión enzimática de sólo dos etapas y centrifugación en gradiente discontinuo de Percoll, y 3) la elevada viabilidad de los protoplastos purificados. Este método permitió obtener ca. 6% de PCEs y 10% de PCGs sobre la base de su ocurrencia en el tejido epidérmico foliar, con una pureza del 100% para las suspensiones finales de protoplastos. Se determinó la viabilidad de cada tipo celular por su habilidad de acumular rojo neutro en sus vacuolas, obteniéndose valores de 90% y 95% para PCEs y PCGs respectivamente. Se determinó la ausencia total de pared celular después del tratamiento enzimático mediante microscopía de fluorescencia con Calcofluor, específico para sustancias celulósicas. El recuento de PCEs y PCGs purificados — obtenidos en las interfases 20/30% y 40/80% del gradiente de Percoll — fue de 1,32×10⁵ y 3,7×10⁵ elementos/ml, lo cual representó un rendimiento de 930 y 2200 protoplastosi cm² de tejido epidérmico respectivamente. La integridad de la membrana plasmática y de las organelas fue confirmada por microscopía electrónica de transmisión y por la habilidad de los protoplastos de excluir azul de Evans.

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Résumé Une méthode a été développée pour l'isolement et la purification de proto-plastes épidermiques (ECPs) et de cellules de garde (GCPs) de feuilles deVicia faba L. Cette méthode présente trois caractéristiques essentielles: 1) Elle ne requiert que de petites quantités du tissu végétal originel; 2) elle est rapide, car elle se base sur une digestion enzymatique en deux étapes et une purification dans un gradient discontinu de densité utilisant le Percoll, et 3) elle fournit une forte proportion de protoplastes prufiés bien vivants. La procédure fournit environ 6% d'ECPs et 10% de GCPs sur la base de leur présence dans le tissue foliaire épidermique et d'une suspension pure à 100% de protoplastes. La viabilité des cellules a été testée par la capacité de chaque type de protoplaste d'accumuler de rouge neutre dans ses vacuoles. On a obtenu des valeurs de 90% et de 95% respectivement pour les ECPs et les GCPs. L'absence totale de paroi cellulaire après le traitement enzymatique a été vérifiée au microscope optique par l'absence de fluorescence après coloration du matériel cellulosique par le calcofluor. Des comptages typiques pour les ECPs et les GCPs purifiés obtenus aux interfaces 20/30% et 40/80% des gradients de Percoll ont donné 1.32×10⁵ et 3.7×10⁵ éléments/ml, ce qui représente des rendements respectifs de 930 et de 2200 protoplastes par cm² de tissue épidermique. L'intégrité de la membrane plasmique et des organites après les procédures d'isolement a été confirmée par microscopie électronique à transmission et par la papacité de protoplastes d'exclure le bleu d'Evans.

     

Comparative studies of phosphoenolpyruvate carboxylase from c(3) and c(4) plants

Phosphoenolpyruvate carboxylase (PEPC) from several C₃ plants was compared to maize PEPC by immunoblotting using an antibody against maize PEPC and by peptide mapping. In C₃ gramineous plants, PEPCs of slightly different monomeric sizes were detected as two bands for wheat and barley leaves, as three bands for etiolated maize leaves and as four bands for rice leaves by SDS-polyacrylamide gel electrophoresis and immunoblotting, whereas only one PEPC band was detected for maize leaves, a C₄ plant, or tobacco leaves, a dicotyledonous C₃ plant. The peptide fragment patterns of the lower molecular weight PEPC (major band in immunoblotting) in wheat leaves was similar to that of maize PEPC in peptide mapping by protein staining or by immunological detection, but the upper one (minor band) had a different pattern from the lower one in peptide mapping by immunological detection and few peptide fragments from this were recognized by the anti-(maize) PEPC antibody. These results suggest that there are multiple forms of PEPC subunits in the gramineous plants tested, and the major PEPC has a primary structure similar to that of maize PEPC. To obtain information about the expression of PEPCs in C₃ plants, changes in the amount of PEPC protein were investigated during the greening of rice and wheat seedlings. Judging from the regulation by light, there were two types of PEPCs in greening rice seedlings, one induced by light and the other reduced by it. Greening wheat seedlings also show a PEPC band induced by light. These findings indicate that some PEPCs in C₃ gramineous plants not only have structures similar to that of maize PEPC, but also are regulated by light in a similar manner.     

Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.     

Generation and characterization of 11 novel est derived microsatellites from Vicia faba (Fabaceae)

? Premise of the study: Simple sequence repeat (SSR) markers were developed for faba bean using expressed sequence tags (ESTs) from the NCBI database to study for genetic diversity. ? Methods and Results: A total of 11 novel EST-SSR loci were generated and characterized when tested on four populations of 29 faba bean individuals from China and Europe. The number of alleles (A) ranged from 1 to 3 in each population, and observed heterozygosity (H(O)) and expected heterozygosity (H(E)) ranged from 0 to 0.5000 and 0.6400, respectively. Furthermore, transferable analysis revealed that eight of these loci (72.73%) amplified in Pisum sativum L., six of which (75.00%) detected polymorphism. ? Conclusions: The developed markers in this study will provide valuable tools for genetic diversity, resource conservation, genetic mapping, and marker-assisted breeding of faba bean in the future.