Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase (A Cardinal Event Influencing the Photosynthesis Rate in Sorghum and Maize)

C4 leaf phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is subject to a day/night regulatory phosphorylation cycle. By using the cytoplasmic protein synthesis inhibitor cycloheximide (CHX), we previously reported that the reversible in vivo light activation of the C4 PEPC protein-serine kinase requires protein synthesis. In the present leaf gas-exchange study, we have examined how and to what extent the CHX-induced inhibition of PEPC protein kinase activity/PEPC phosphorylation in the light influences C4 photosynthesis. Detached Sorghum vulgare and maize (Zea mays) leaves fed 10 [mu]M CHX showed a gradual but marked decrease in photosynthetic CO2 assimilation capacity. A series of control experiments designed to assess deleterious secondary effects of the inhibitor established that this reduction in C4 leaf CO2 assimilation was not due to (a) an increased stomatal resistance to CO2 diffusion, (b) a decrease in the activation state of other photoactivated C4 cycle enzymes, and (c) a perturbation of the Benson-Calvin C3 cycle, as evidenced by the absence of an inhibitory effect of CHX on leaf photosynthesis by a C3 grass (Triticum aestivum). It is notable that the CHX-induced decrease in CO2 assimilation by illuminated Sorghum leaves was highly correlated with a decrease in the apparent phosphorylation status of PEPC and a concomitant change in carbon isotope discrimination consistent with a shift from a C4 to a C3 mode of leaf CO2 fixation. These collective findings indicate that the light-dependent activation of the PEPC protein-serine kinase and the resulting phosphorylation of serine-8 or serine-15 in Sorghum or maize PEPC, respectively, are fundamental regulatory events that influence leaf C4 photosynthesis in vivo.     

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。     

Structure,Regulation and Biosynthesis of Phosphoenolpyruvate Carboxylase from C4 Plants

This review attempts to summarize the large body of information on the structure, regulation and biosynthesis of the enzyme phosphoenolpyruvate carboxylase in C₄ plants which has accumulated particularly since the appearance of the last review in 1987. Among the major discoveries are the involvement of protein phosphorylation-dephosphorylation cascade in the light activation of the enzyme, extraction and characteristics of PEPC-protein serine kinase, dynamic changes in oligomeric state of the enzyme in response to pH or temperature, isolation of multiple cDNAs encoding different forms of PEPC and cloning and expression of maize/sorghum PEPC in transgenic tobacco or transformed E. coli cells. Further experiments using advanced techniques of biochemistry and molecular biology would help in understanding the molecular mechanism of reaction, regulation of enzyme activity, gene expression and evolutionary pattern of C₄ PEPC.     

The Light-Dependent Transduction Pathway Controlling the Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase in Protoplasts from Digitaria sanguinalis

Phosphoenolpyruvate carboxylase (PEPC) was characterized in extracts from C4 mesophyll protoplasts isolated from Digitaria sanguinalis leaves and shown to display the structural, functional, and regulatory properties typical of a C4 PEPC. In situ increases in the apparent phosphorylation state of the enzyme and the activity of its Ca2+-independent protein-serine kinase were induced by light plus NH4Cl or methylamine. The photosynthesis-related metabolite 3-phosphoglycerate (3-PGA) was used as a substitute for the weak base in these experiments. The early effects of light plus the weak base or 3-PGA treatment were alkalinization of protoplast cytosolic pH, shown by fluorescence cytometry, and calcium mobilization from vacuoles, as suggested by the use of the calcium channel blockers TMB-8 and verapamil. The increases in PEPC kinase activity and the apparent phosphorylation state of PEPC also were blocked in situ by the electron transport and ATP synthesis inhibitors DCMU and gramicidin, respectively, the calcium/calmodulin antagonists W7, W5, and compound 48/80, and the cytosolic protein synthesis inhibitor cycloheximide. These results suggest that the production of ATP and/or NADPH by the illuminated mesophyll chloroplast is required for the activation of the transduction pathway, which presumably includes an upstream Ca2+-dependent protein kinase and a cytosolic protein synthesis event. The collective data support the view that the C4 PEPC light transduction pathway is contained entirely within the mesophyll cell and imply cross-talk between the mesophyll and bundle sheath cells in the form of the photosynthetic metabolite 3-PGA.     

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.     

Temperature Effects on Phosphoenolpyruvate Carboxylase from a CAM and a C(4) Plant : A Comparative Study

The effect of temperature in the range from 10 to 35°C on various characteristics of phosphoenolpyruvate carboxylase from the leaves of a CAM plant, Crassula argentea and a C₄ plant Zea mays shows a number of different effects related to the environment in which these distinct types of metabolic specialization normally operate. The Arrhenius plot of V_max for the two enzyme forms shows that the CAM enzyme has a linear increase with temperature while the C₄ enzyme has an inflection at 27°C implying a conformational or aggregational change in the enzyme or a shift in reaction mechanism to one requiring a lower activation energy. The Arrhenius plot of K_m for the two enzymes reveals the startling fact that at temperatures above 20°C an increasing temperature causes an increase in K_mPEP for the CAM enzyme while the C₄ enzyme displays a decreased K_m as the temperature increases. The inhibitory effect of 5 millimolar malate also shows opposite trends for the two enzymes. For the CAM enzyme the percent inhibition by malate increases from essentially none at 15°C to 70% at 35°C. For the C₄ enzyme the percent inhibition drops from about 60% at 20°C to 2% at 30°C. Similar opposite behavior of the two enzymes is found with the K_i for malate. Pretreatment at high temperatures for periods up to 2 hours was found to result in differences similar to those described above if the treated enzyme were subsequently assayed at 25°C.     

Modulation by Bicarbonate of Catalytic and Regulatory Properties of C4Phosphoenolpyruvate Carboxylase from Amaranthus hypochondriacus: Desensitization to Malate and Glucose 6-Phosphate and Sensitization to Mg2+

The catalytic and regulatory properties of phosphoenolpyruvate(PEP) carboxylase (PEPC) are modulated remarkably by the increasein the level of bicarbonate in the assay medium. The activityof PEPC increased by two-fold as the concentration of bicarbonatewas raised from 0.05 to 10 mM. During this state, there wasonly marginal effect on K_m for PEP, while the affinity of PEPCto Mg²⁺ increased by >2 fold. In contrast, the sensitivityof PEPC to malate decreased with increasing concentration ofHCO₃^–. Similarly, the stimulation by glucose 6-phosphate(G-6-P) at optimal concentration (10 mM) of HCO₃^– wasmuch less than that at suboptimal concentration (0.05 mM). K₁for malate increased by about 3 fold and K_a for G-6-P risedby fourfold as bicarbonate concentration was rised from 0.05to 10 mM. These results suggest that HCO₃^– desensitizesPEPC to both malate and G-6-P. Further, these changes were manifestedin both dark- as well as light-forms of the enzyme. Similarresults were obtained with PEPC in leaf extracts or in purifiedform. We therefore propose that bicarbonate-induced changesare independent of phospho-rylation and possibly through a significantchange in the conformation of the enzyme. This is the firstdetailed report indicating marked modulation of regulatory andcatalytic properties of PEPC by bicarbonate, one of its substrate. (Received April 14, 1998; Accepted September 22, 1998)     

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.     

Detecting Photoactivation of Phosphoenolpyruvate Carboxylase in C(4) Plants : An Effect of pH

Photoactivation of phosphoenolpyruvate carboxylase in C₄ plants is detected more efficiently when activity is assayed at suboptimum pH (e.g. 7.2); the magnitude of the light effect is often larger at low phosphoenolpyruvate concentration.

Darkness and low assay pH induce an allosteric behavior (positive cooperativity with phosphoenolpyruvate) which is relieved in light or by higher pH; thus, normal Michaelis-Menten kinetics are exhibited only when the enzyme is extracted during the day and assayed at pH 8.2.

Light activation, pH, and substrate level appear to be components of a regulatory device suppressing the activity in darkness and enhancing it under light.

     

Effects of the Phosphoenolpyruvate Carboxylase Inhibitor 3,3-Dichloro-2-(Dihydroxyphosphinoylmethyl)propenoate on Photosynthesis: C(4) Selectivity and Studies on C(4) Photosynthesis

The effect of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl)-propenoate (DCDP), an analog of phosphoenolpyruvate (PEP), on PEP carboxylase activity in crude leaf extracts and on photosynthesis of excised leaves was examined. DCDP is an effective inhibitor of PEP carboxylase from Zea mays or Panicum miliaceum; 50% inhibition was obtained at 70 or 350 micromolar, respectively, in the presence of 1 millimolar PEP and 1 millimolar HCO₃⁻. When fed to leaf sections via the transpiration stream, DCDP at 1 millimolar strongly inhibited photosynthesis in C₄ species (79-98% inhibition for a range of seven C₄ species), but only moderately in C₃ species (12-46% for four C₃ species), suggesting different mechanisms of inhibition for each photosynthetic type. The response of P. miliaceum (C₄) net photosynthesis to intercellular pCO₂ showed that carboxylation efficiency, as well as the CO₂ saturated rate, are lowered in the presence of DCDP and supported the view that carboxylation efficiency in C₄ species is directly related to PEP carboxylase activity. A fivefold increase in intercellular pCO₂ over that occurring in P. miliaceum under normal photosynthesis conditions only increased net photosynthesis rate in the presence of 1 millimolar DCDP from zero to about 5% of the maximal uninhibited rate. Therefore, it seems unlikely that direct fixation of atmospheric CO₂ by the bundle sheath cells makes any significant contribution to photosynthetic CO₂ assimilation in C₄ species. The results support the concept that C₄-selective herbicides may be developed based on inhibitors of C₄ pathway reactions.     

The Promoter of the Gene Encoding the C4 Form of Phosphoenolpyruvate Carboxylase Directs Mesophyll-Specific Expression in Transgenic C4 Flaveria spp

The function of the C4 mechanism of photosynthesis depends on the strict compartmentation of the enzymes involved. Here, we investigate the regulatory mechanisms that ensure the mesophyll-specific expression of the C4 isoform of phosphoenolpyruvate carboxylase. We show that 2 kb of the 5[prime] flanking region of the Flaveria trinervia C4 PpcA1 gene is sufficient to direct mesophyll-specific expression of the [beta]-glucuronidase reporter gene in transgenic F. bidentis (C4) plants. In young leaves of seedlings, the activity of this promoter is dependent on the developmental stage of the mesophyll cells. It is induced in a basipetal fashion (leaf tip to base) during leaf development. The promoter region of the orthologous nonphotosynthetic Ppc gene of F. pringlei (C3) induces reporter gene expression mainly in the vascular tissue of leaves and stems as well as in mesophyll cells of transgenic F. bidentis plants. Our experiments demonstrate that during the evolution of the C4 Flaveria species, cis-acting elements of the C4 Ppc gene must have been altered to achieve mesophyll-specific expression.     

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)     

Significance of the Conserved Tyr352 and Asp380 Residues in the Catalytic Activity of Bacillus stearothermophilus Aminopeptidase II as Evaluated by Site-directed Mutagenesis

The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.5-fold. Detailed analysis of the kinetic consequences in the mutant proteins revealed that the K (m) values were increased 1.9- to 2.6-fold with respect to wild-type enzyme. Catalytic efficiencies (k (cat)/K (m)) of mutant proteins were between 3.5- and 31-fold lower than the corresponding value of the wild-type enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results indicate that residues Tyr352 and Asp380 are essential for the proper function of AP-II.     

Uptake of Lead and Cadmium by Maize Seedlings and the Effect of Heavy Metals on the Activity of Phosphoenolpyruvate Carboxylase Isolated from Maize

Maize seeds and five-day-old maize seedlings were incubated in media containing Pb²⁺ at concentrations of 50, 100, and 200 mg 1^-1 and Cd²⁺ at concentrations of 1, 5, 10 and 50 mg 1^-1. After five days of incubation, both heavy metals were determined by means of AAS following wet mineralisation of roots and shoots. The results obtained indicate that Pb²⁺ were transported to shoots from roots at a lower rate than Cd²⁺. Phosphoenolpyruvate carboxylase (PEPC) isolated from germinating maize seeds was inhibited to a comparable degree by solutions containing 0.001 mmol 1^-1 Pb²⁺, 0.01 mmol 1^-1 Cd²⁺, and 0.005 mmol 1^-1 Cu²⁺. The enzyme was protected against this inhibition by the addition of mercaptoethanol, the substrate (PEP), or the cofactor (Mg²⁺). The inhibition increased during a 20 min incubation of the enzyme with salts of the metals. Mn²⁺, Ni²⁺, and Co²⁺ ions could partially substitute for the metal cofactor Mg²⁺. K_m values for these metal ions were as follows: for Mg²⁺ 0.07 mmol 1^-1 in the range from 0 to 0.30 mmol 1^-1 Mg²⁺; 0.71 mmol 1^-1 for 0.30 to 2.50 mmol 1^-1 Mg²⁺; for Mn²⁺ 0.36 mmol 1^-1; for Ni²⁺ 0.34 mmol 1{-1}; and for Co²⁺ 0.20 mmol 1^-1. The activity of the enzyme reached with Mn²⁺ 85 %, with Ni²⁺ 65 %, and with Co²⁺ 55 % of the activity recorded with Mg²⁺.     

Photosynthetic Characteristics of C3-C4 Intermediate Flaveria Species II. Kinetic Properties of Phosphoenolpyruvate Carboxylase from C3, C4 and C3-C4 Intermediate Species

The kinetic properties of phosphoenolpyruvate (PEP) carboxylasehave been studied among several Flaveria species: the C₃ speciesF. cronquistii, the C₃–C₄ species F. pubescens and F.linearis, and the C₄ species F. trinervia. At either pH 7 or8, the maximum activities (in µmol.mg Chl^–1.h^–1)for F. pubescens and linearis (187–513) were intermediateto those of the C₃ species (12–19) and the C₄ species(2,182–2,627). The response curves of velocity versusPEP concentration were hyperbolic for the C₃ and C₃–C₄species at either pH 7 or 8 while they were sigmoidal for theC₄ species at pH 7 and hyperbolic at pH 8. The K_m values forPEP determined from reciprocal plots were lowest in the C₃ species,and of intermediate value in the C₃–C₄ species comparedto the K' values of the C₄ species determined from Hill plotsat either pH 7 or 8. Glucose-6-phosphate (G6P) decreased theK_m values for PEP at both pH 7 and 8 in the C₃ and C₃–C₄species. In the C₄ species, G6P decreased the K' values at pH8 but increased the K' values at pH 7. In all cases, G6P hadits effect by influencing the activity at limiting PEP concentrationswith little or no effect on the maximum activity. At pH 8 andlimiting concentrations of PEP the degree of stimulation ofthe activity by G6P was greatest in the C₄ species, intermediatein F. linearis, a C₃–C₄ species, and lowest in the C₃species. In several respects, the PEP carboxylases of the C₃–C₄Flaveria species have properties intermediate to those of theC₃ and C₄ species. (Received April 30, 1983; Accepted August 22, 1983)