Glyphosine Inhibits Maize Leaf Phosphoenolpyruvate Carboxylase

Glyphosine [N, N-bis-(phosphonomethyl) glycine] inhibited maizeleaf P-enolpyruvate carboxylase competitively with respect toP-enolpyruvate. The inhibition was dependent on glyphosine concentrationand pH. Glycine, but not glucose-6-phosphate, protected theenzyme from the effect of glyphosine. A related compound, glyphosate[N-(phosphonomethyl) glycine], produced little or no inhibition.P-enolpyruvate carboxylase could be one of the targets of glyphosineaction, causing growth inhibition as reported (Croft, S. M.,C. J. Arntzen, L. N. Vanderhoef and C. S. Zettinger (1974) Biochim.Biophys. Acta 335: 211-217). (Received July 10, 1986; Accepted December 4, 1986)     

deltaC Values in Maize Leaf Correlate with Phosphoenolpyruvate Carboxylase Levels

Values of δ¹³C and levels of phosphoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/oxygenase were analyzed in segments from the fourth leaf of young maize (Zea mays L.) plants. The δ¹³C values became significantly more negative from the base to the tip of the leaves. Phosphoenolpyruvate carboxylase levels and ribulose bisphosphate carboxylase levels both increased from the base to the tip. The principal effect of phosphoenolpyruvate carboxylase levels or δ¹³C should arise through its effect on the carboxylation/diffusion balance in the mesophyll. In this case, δ¹³C values should become more negative as phosphoenolpyruvate carboxylase levels increase, unless there are offsetting changes in stomatal aperture. The principal effect of ribulose bisphosphate carboxylase/oxygenase on δ¹³C should occur through its effect on the extent of leakage of CO₂ from the bundle sheath cells. In this case, δ¹³C values should become more positive as ribulose bisphosphate carboxylase levels increase. Accordingly, the variation in δ¹³C values seen in maize leaves appears to be the result of variations in the level of phosphoenolpyruvate carboxylase.     

Change in Properties of Phosphoenolpyruvate Carboxylase in Kalanchoe daigremontiana with Leaf Age

The activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31 [EC] )increased with leaf age in Kalanchoe daigremontiana. Polyacrylamidegel electrophoresis showed 3 bands of PEP carboxylase activity,one predominant in young leaves, another predominant in olderleaves. PEP carboxylase activities in desalted extract differedaccording to pH, at acidic pH the enzyme activity of young leavesdecreased drastically, whereas that of older leaves remainednearly constant. (Received August 2, 1982; Accepted September 27, 1982)     

Glutamine Induces the N-Dependent Accumulation of mRNAs Encoding Phosphoenolpyruvate Carboxylase and Carbonic Anhydrase in Detached Maize Leaf Tissue

We have used detached leaves to study the N-dependent control of expression of phosphoenolpyruvate carboxylase (PEPC) and carbonic anhydrase (CA) genes in maize (Zea mays L. cv Golden Cross Bantam T51). Following supplementation with an N-source and zeatin, PEPC and CA mRNA levels increased in leaves detached from N-deficient maize plants. Addition of methionine sulfoximine (MSX), a specific inhibitor of glutamine synthetase, inhibited the nitrate-dependent increase of PEPC and CA mRNA but did not affect the glutamine-dependent increase of PEPC and CA mRNA levels. Glutamine levels in detached maize leaves treated with various N sources in the presence or absence of MSX correlated with the levels of PEPC and CA mRNA. We conclude that glutamine is the most likely effector for controlling the N-dependent expression of PEPC and CA in maize plants.     

Fluorescence Study of Chemical Modification of Phosphoenolpyruvate Carboxylase from Crassula argentea

The chemical modification of phosphoenolpyruvate carboxylase purified from Crassula argentea leaves was studied using the fluorescence of the extrinsic probe 8-anilino-1-naphalenesulfonate. The effects of ligands on kinetic parameters of phosphoenolpyruvate carboxylase activity, and its response to pH and metal cations, were associated with the binding of the ligands to the enzyme as measured by fluorescence. Binding of the ligands phosphoenolpyruvate, malate, and glucose-6-phosphate revealed by fluorescence measurements corresponds to competitive phenomena observed in kinetic studies. The fluorescence measurements also suggest the involvement of specific amino acids in the binding of a given ligand. Arginyl residues modified by 2,3-butanedione appear to be directly involved in the binding of phosphoenolpyruvate and malate to the active and the inhibition sites, respectively. A histidyl residue was involved in the binding of malate, accounting for the lack of inhibition by malate in kinetic studies of the enzyme treated with diethylpyrocarbonate. Although activity was lost, there was no decrease in the ability of the treated enzyme to bind phosphoenolpyruvate, suggesting that additional histidyl residues are essential for activity although not directly involved in the binding of phosphoenolpyruvate. The lysine reagent trinitrobenzenesulfonate caused a loss of activity and a reduction in malate inhibition and glucose-6-phosphate activation, but these modifications were not related to changes in the ability of the enzyme to bind any of the three ligands. This suggests that lysine residues were not directly involved in the binding of these ligands.     

Evidence for Light-stimulated Synthesis of Phosphoenolpyruvate Carboxylase in Leaves of Maize

Illumination (22,000 lumens per meter²) of etiolated maize plants for 80 hours brings about a 5-fold increase in phosphoenolpyruvate carboxylase activity per unit of protein. An increase in carboxylase protein and incorporation of [³⁵S]methionine into the protein occurs simultaneously with the activity increase. In green plants, the level of phosphoenolpyruvate carboxylase protein and enzyme activity is dependent on the intensity of light during growth. These results are consistent with the conclusion that the activity increase results from light-stimulated de novo synthesis of phosphoenolypyruvate carboxylase protein.     

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.     

转玉米pepc基因水稻的某些光合特性

测定转玉米pepc基因水稻和未转化粳稻品种Kitaake在分蘖初期、分蘖盛期、拔节期、始穗期、齐穗期、成熟期和剑叶不同生长时期的光合特性动态变化的结果表明:转玉米pepc基因水稻的磷酸烯醇式丙酮酸羧化酶(PEPCase)活性和净光合速率(Pn)在不同时期各不相同,相对于Kitaake而言,均有不同程度的提高;剑叶完全展开时的PEPCase活性和Pn提高最明显.     

Characterization of a Maize Ca2+-Dependent Protein Kinase Phosphorylating Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31 [EC] ] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca²⁺-requiring protein kinase from Ca²⁺-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa²⁺-dependent but calmodulin-independent protein kinase (CDPK).Although the M_r, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an M_r of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan. ²Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan. ⁴N.O. and N.Y. contributed equally to this work.     

Salt-stimulated Phosphoenolpyruvate Carboxylase in Cakile maritima

The effects of NaCl and other salts, in vivo and in vitro, on the activity of phosphoenolpyruvate carboxylase from the coastal C₃ halophyte Cakile maritima Scop, were investigated. Plants grown with 100 mM NaCl in their growth medium yielded some 30% higher rates of phosphoenolpyruvate carboxylase activity than did salt-depleted plants. Activity of the enzyme was stimulated when NaCl was added to the reaction mixture in concentrations of up to 200 mM. The magnitude of this in vitro stimulation was similar for plants grown in the presence or absence of NaCl. The effect seems to be caused by chloride rather than by sodium ions.     

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)     

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     

Involvement of Ubiquitin in Phosphoenolpyruvate Carboxylase Degradation

Western immunoblot analysis of protein extracts prepared from epidermal peels, whole leaves, and mesophyll protoplasts with ubiquitin and PEPCase antibodies indicated ubiquitinated PEPCase bands and degradation products only in crude extracts which have been obtained in the presence of the proteolysis inhibitors leupeptin and hemin. After ammonium sulfate precipitation and further purification, PEPCase forms were stable and not ubiquitinated. It is assumed, that only a certain part of PEPCase is degraded via the ubiquitin-dependent proteolysis.     

Expression Profiles and Post-Translational Modifications of Phosphoenolpyruvate Carboxylase Isozymes of Bienertia sinuspersici during Leaf Development

Russian Journal of Plant Physiology - Phosphoenolpyruvate carboxylase (PEPC) is the primary acceptor of carbon dioxide in C4 photosynthesis. Three isozymes of PEPC: PEPC1, PEPC2, and PEPC3, in the...     

Effects of Nitrate and Ammonium on Gene Expression of Phosphoenolpyruvate Carboxylase and Nitrogen Metabolism in Maize Leaf Tissue during Recovery from Nitrogen Stress

We previously showed that the selective accumulation of phosphoenolpyruvate carboxylase (PEPC) in photosynthetically maturing maize (Zea mays L.) leaf cells induced by nitrate supply to nitrogen-starved plants was primarily a consequence of the level of its mRNA (B Sugiharto, K Miyata, H Nakamoto, H Sasakawa, T Sugiyama [1990] Plant Physiol 92: 963-969). To determine the specificity of inorganic nitrogen sources for the regulation of PEPC gene expression, nitrate (16 millimolar) or ammonium (6 millimolar) was supplied to plants grown previously in low nitrate (0.8 millimolar), and changes in the level of PEPC and its mRNA were measured in the basal region of the youngest, fully developed leaves of plants during recovery from nitrogen stress. The exogenous supply of nitrogen selectively increased the levels of protein and mRNA for PEPC. This increase was more pronounced in plants supplemented with ammonium than with nitrate. The accumulation of PEPC during nitrogen recovery increased in parallel with the increase in the activity of glutamine synthetase and/or ferredoxin-dependent glutamate synthase. Among the major amino acids, glutamine was the most influenced during recovery, and its level increased in parallel with the steady-state level of PEPC mRNA for 7 hours after nitrogen supply. The administration of glutamine (12 millimolar) to nitrogen-starved plants increased the steady-state level of PEPC mRNA 7 hours after administration, whereas 12 millimolar glutamate decreased the level of PEPC mRNA. The results indicate that glutamine and/or its metabolite(s) can be a positive control on the nitrogen-dependent regulation of PEPC gene expression in maize leaf cells.     

Heterokaryon Identification Through Simultaneous Fluorescence of Tetramethylrhodamine Isothiocyanate and Fluorescein Isothiocyanate Labelled Protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein iso-thiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emisson spectrum, but not both, was possible for any single filter set.