Dramatic difference in the responses of phosphoenolpyruvate carboxylase to temperature in leaves of C3 and C4 plants

Temperature caused phenomenal modulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in leaf discs of Amaranthus hypochondriacus (NAD-ME type C(4) species), compared to the pattern in Pisum sativum (a C(3) plant). The optimal incubation temperature for PEPC in A. hypochondriacus (C(4)) was 45 degrees C compared to 30 degrees C in P. sativum (C(3)). A. hypochondriacus (C(4)) lost nearly 70% of PEPC activity on exposure to a low temperature of 15 degrees C, compared to only about a 35% loss in the case of P. sativum (C(3)). Thus, the C(4) enzyme was less sensitive to supra-optimal temperature and more sensitive to sub-optimal temperature than that of the C(3) species. As the temperature was raised from 15 degrees C to 50 degrees C, there was a sharp decrease in malate sensitivity of PEPC. The extent of such a decrease in C(4) plants (45%) was more than that in C(3) species (30%). The maintenance of high enzyme activity at warm temperatures, together with a sharp decrease in the malate sensitivity of PEPC was also noticed in other C(4) plants. The temperature-induced changes in PEPC of both A. hypochondriacus (C(4)) and P. sativum (C(3)) were reversible to a large extent. There was no difference in the extent of phosphorylation of PEPC in leaves of A. hypochondriacus on exposure to varying temperatures, unlike the marked increase in the phosphorylation of enzyme on illumination of the leaves. These results demonstrate that (i) there are marked differences in the temperature sensitivity of PEPC in C(3) and C(4) plants, (ii) the temperature induced changes are reversible, and (iii) these changes are not related to the phosphorylation state of the enzyme. The inclusion of PEG-6000, during the assay, dampened the modulation by temperature of malate sensitivity of PEPC in A. hypochondriacus. It is suggested that the variation in temperature may cause significant conformational changes in C(4)-PEPC.     

Heat inactivation of leaf phosphoenolpyruvate carboxylase: Protection by aspartate and malate in C4 plants

The activity of phosphoenolpyruvate (PEP) carboxylase EC 4.1.1.31 in leaf extracts of Eleusine indica L. Gaertn., a C₄ plant, exhibited a temperature optimum of 35–37° C with a complete loss of activity at 50° C. However, the enzyme was protected effectively from heat inactivation up to 55° C by L-aspartate. Activation energies (Ea) for the enzyme in the presence of aspartate were 2.5 times lower than that of the control enzyme. Arrhenius plots of PEP carboxylase activity (±aspartate) showed a break in the slope around 17–20° C with a 3-fold increase in the Ea below the break. The discontinuity in the slopes was abolished by treating the enzyme extracts with Triton X-100, suggesting that PEP carboxylase in C₄ plants is associated with lipid and may be a membrane bound enzyme. Depending upon the species, the major C₄ acid formed during photosynthesis (malate or aspartate) was found to be more protective than the minor C₄ acid against the heat inactivation of their PEP carboxylase. Oxaloacetate, the reaction product, was less effective compared to malate or aspartate. Several allosteric inhibitors of PEP carboxylase were found to be moderately to highly effective in protecting the C₄ enzyme while its activators showed no significant effect. PEP carboxylase from C₃ species was not protected from thermal inactivation by the C₄ acids. The physiological significance of these results is discussed in relation to the high temperature tolerance of C₄ plants.Abbreviations CAM crassulaccan acid metabolism - Chl chlorophyll - Ea activation energy - PEP phosphoenolypyruvate Journal Series Paper, New Jersey Agricultural Experiment Station     

Light/dark modulation of phosphoenolpyruvate carboxylase in C3 and C4 species

In this report, the effects of light on the activity and allosteric properties of phosphoenolpyruvate (PEP) carboxylase were examined in newly matured leaves of several C₃ and C₄ species. Illumination of previously darkened leaves increased the enzyme activity 1.1 to 1.3 fold in C₃ species and 1.4 to 2.3 fold in C₄ species, when assayed under suboptimal conditions (pH 7) without allosteric effectors. The sensitivities of PEP carboxylase to the allosteric effectors malate and glucose-6-phosphate were markedly different between C₃ and C₄ species. In the presence of 5 mM malate, the activity of the enzyme extracted from illuminated leaves was 3 to 10 fold higher than that from darkened leaves in C₄ species due to reduced malate inhibition of the enzyme from illuminated leaves, whereas it increased only slightly in C₃ species. The Ki(malate) for the enzyme increased about 3 fold by illumination in C₄ species, but increased only slightly in C₃ species. Also, the addition of the positive effector glucose-6-phosphate provided much greater protection against malate inhibition of the enzyme from C₄ species than C₃ species. Feeding nitrate to excised leaves of nitrogen deficient plants enhanced the degree of light activation of PEP carboxylase in the C₄ species maize, but had little or no effect in the C₃ species wheat. These results suggest that post-translational modification by light affects the activity and allosteric properties of PEP carboxylase to a much greater extend in C₄ than in C₃ species.     

Patterns of phosphoenolpyruvate carboxylase activity and cytosolic pH during light activation and dark deactivation in C3 and C4 plants

The rate and extent of light activation of PEPC may be used as another criterion to distinguish C₃ and C₄ plants. Light stimulated phosphoenolypyruvate carboxylase (PEPC) in leaf discs of C₄ plants, the activity being three times greater than that in the dark but stimulation of PEPC was limited about 30% over the dark-control in C₃ species. The light activation of PEPC in leaves of C₃ plants was complete within 10 min, while maximum activation in C₄ plants required illumination for more than 20 min, indicating that the relative pace of PEPC activation was slower in C₄ plants than in C₃ plants. Similarly, the dark-deactivation of the enzyme was also slower in leaves of C₄ than in C₃ species. The extent of PEPC stimulation in the alkaline pH range indicated that the dark-adapted form of the C₄ enzyme is very sensitive to changes in pH. The pH of cytosol-enriched cell sap extracted from illuminated leaves of C₄ plants was more alkaline than that of dark-adapted leaves. The extent of such light-dependent alkalization of cell sap was three times higher in C₄ leaves than in C₃ plants. The course of light-induced alkalization and dark-acidification of cytosol-enriched cell sap was markedly similar to the pattern of light activation and dark-deactivation of PEPC in Alternanthera pungens, a C₄ plant. Our report provides preliminary evidence that the photoactivation of PEPC in C₄ plants may be mediated at least partially by the modulation of cytosolic pH.Abbreviations CAM Crassulacean acid metabolism - G-6-P glucose-6-phosphate - PMSF phenylmethylsulfonyl fluoride - PEPC phosphoenolpyruvate carboxylase - PEPC-PK phosphoenolpyruvate ca carboxylase-protein kinase     

Illumination increases the affinity of phosphoenolpyruvate carboxylase to bicarbonate in leaves of a C4 plant, Amaranthus hypochondriacus

Illumination increased markedly the affinity to bicarbonate of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) in leaves of Amaranthus hypochondriacus L., a C4 plant. When leaves were illuminated, the apparent Km for (HCO3-) of PEPC decreased by about 50% concurrent with a 2- to 5-fold increase in Vmax and 3- to 4-fold increase in Ki for malate. The inclusion of ethoxyzolamide, an inhibitor of carbonic anhydrase, during the assay had no effect on kinetic and regulatory properties of PEPC indicating that carbonic anhydrase was not involved during light-induced sensitization of PEPC to HCO3-. Pretreatment of leaf discs with cycloheximide (CHX), a cytosolic protein synthesis inhibitor, suppressed significantly the light-enhanced decrease in apparent Km (HCO3-). Further, in vitro phosphorylation of purified dark-form PEPC by protein kinase A (PKA) decreased the apparent Km (HCO3-) of the enzyme, in addition increasing Ki (malate) as expected. Such changes, due to in vitro phosphorylation of purified PEPC by PKA, occurred only with wild-type PEPC, but not in the mutant form of maize (S15D) which is already a mimic of the phosphorylated enzyme. These results suggest that phosphorylation of the enzyme is important during the sensitization of PEPC to HCO3- by illumination in C4 leaves. Since illumination is expected to increase the cytosolic pH and the availability of dissolved HCO3- in mesophyll cells, the sensitization by light of PEPC to HCO3- could be physiologically quite significant.     

Regulation of phosphoenolpyruvate carboxylase in C4 plants: inhibition by pyrophosphate of the enzyme from Amaranthus viridis

Phosphoenolpyruvate carboxylase from the leaves of Amaranthus viridis was inhibited by pyrophosphate. The inhibition was competitive with respect to phosphoenolpyruvate (K_i 0.85 mm) and noncompetitive with respect to bicarbonate.     

Activity regulation and physiological impacts of maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants

Phosphoenolpyruvate carboxylase (PEPC) was overproduced in the leaves of rice plants by introducing the intact maize C₄-specific PEPC gene. Maize PEPC in transgenic rice leaves underwent activity regulation through protein phosphorylation in a manner similar to endogenous rice PEPC but contrary to that occurring in maize leaves, being downregulated in the light and upregulated in the dark. Compared with untransformed rice, the level of the substrate for PEPC (phosphoenolpyruvate) was slightly lower and the product (oxaloacetate) was slightly higher in transgenic rice, suggesting that maize PEPC was functioning even though it remained dephosphorylated and less active in the light. ¹⁴CO₂ labeling experiments indicated that maize PEPC did not contribute significantly to the photosynthetic CO₂ fixation of transgenic rice plants. Rather, it slightly lowered the CO₂ assimilation rate. This effect was ascribable to the stimulation of respiration in the light, which was more marked at lower O₂ concentrations. It was concluded that overproduction of PEPC does not directly affect photosynthesis significantly but it suppresses photosynthesis indirectly by stimulating respiration in the light. We also found that while the steady-state stomatal aperture remained unaffected over a wide range of humidity, the stomatal opening under non-steady-state conditions was destabilized in transgenic rice. This revised version was published online in August 2006 with corrections to the Cover Date.     

Thermal regulation of phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase in c(3) and c(4) plants native to hot and temperate climates

Exposure of leaf sections from 2-week-old seedlings of sorghum (Sorghum bicolor L.) (C₄ plant), corn (Zea mays L.) (C₄), peanut (Arachis hypogaea L.) (C₃ plant), and soybean (Glycine max L.) (C₃) to 40 or 45°C for up to 4 hours resulted in significant increases in the levels of 102 kilodalton (C₄), 52 kilodalton (C₃ and C₄), and 15 kilodalton (C₃ and C₄) polypeptides. These proteins comigrated, respectively, with authentic phosphoenolpyruvate carboxylase (PEPC) and the large (RLSU) and small (RSSU) subunits of ribulose-1,5-bisphosphate carboxylase (Rubisco) during both one- and two-dimensional SDS-PAGE and reacted with antisera raised against these enzymes. After 4 hours at 50°C, levels of the polypeptides either remained relatively stable (PEPC, RLSU) or increased (RSSU) in sorghum and peanut (plants native to hot climates). In corn and soybean (plants native to temperate climates), levels of the proteins either fell sharply (corn) or showed strong evidence of incomplete processing and/or aggregation (soybean). In addition to changes in levels of the proteins, the activities of PEPC and Rubisco in extracts of leaves exposed to 50°C fell by 84% and 11% of their respective control values in sorghum and by 54% each in peanut. In corn and soybean, the activities of both enzymes were depressed at 40°C, with measured values at 50°C not exceeding 5% of those from the nonstressed controls.     

Protein degradation in C3 and C4 plants with particular reference to ribulose bisphosphate carboxylase and glycolate oxidase

Determining the degradation characteristics of proteins is difficult due to the lack of appropriate methodologies, particularly in the case of leaf proteins. Previous studies suggest that ribulose bisphosphate carboxylase (RuBP carboxylase; EC 4.1.1.39) proteolysis may be fundamentally different in C3 and C4 plants. To test this hypothesis, the relative degradation rates of the total soluble protein, RuBP carboxylase and glycolate oxidase (EC 1.1.3.1) in the second leaves of intact C3 (Triticum aestivum L.) and C4 (Zea mays L) and Sorghum bicolor L.)plants was measured. The methodology utilized involved an efficient procedure to label the leaf proteins, the use of a double-labelling method to measure protein degradation and a single-step purification of the labelled proteins under study. RuBP carboxylase is subjected to continuous degradation in all plants investigated. Its rate of degradation is higher for Z. mays, intermediate for T. aestivum and lower for S. bicolor. When the rate of RuBP carboxylase degradation was compared with that of the total soluble protein a differential pattern was obtained for the plant species examined: whereas maize presents a faster rate of RuBP carboxylase degradation than of the total soluble protein, wheat and sorghum show similar rates. However, the rate of RuBP carboxylase proteolysis in the three plant species studied is much lower than the rate of glycolate oxidase degradation. The results obtained indicate that, under the conditions of study, the degradation characteristics of plant RuBP carboxylase, as those of glycolate oxidase, are species specific, in a way suggesting that they do not depend on the type of photosynthetic metabolism of the species considered (C3 or C4).     

Evolution of C4 phosphoenolpyruvate carboxylase in the genus Alternanthera: gene families and the enzymatic characteristics of the C4 isozyme and its orthologues in C3 and C3/C4 Alternantheras

Phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.3) is a key enzyme of C₄ photosynthesis. It has evolved from ancestral non-photosynthetic (C₃) isoforms and thereby changed its kinetic and regulatory properties. We are interested in understanding the molecular changes, as the C₄ PEPCases were adapted to their new function in C₄ photosynthesis and have therefore analysed the PEPCase genes of various Alternanthera species. We isolated PEPCase cDNAs from the C₄ plant Alternanthera pungens H.B.K., the C₃/C₄ intermediate plant A. tenella Colla, and the C₃ plant A. sessilis (L.) R.Br. and investigated the kinetic properties of the corresponding recombinant PEPCase proteins and their phylogenetic relationships. The three PEPCases are most likely derived from orthologous gene classes named ppcA. The affinity constant for the substrate phosphoenolpyruvate (K _0.5 PEP) and the degree of activation by glucose-6-phosphate classified the enzyme from A. pungens (C₄) as a C₄ PEPCase isoform. In contrast, both the PEPCases from A. sessilis (C₃) and A. tenella (C₃/C₄) were found to be typical C₃ PEPCase isozymes. The C₄ characteristics of the PEPCase of A. pungens were accompanied by the presence of the C₄-invariant serine residue at position 775 reinforcing that a serine at this position is essential for being a C₄ PEPCase (Svensson et al. 2003). Genomic Southern blot experiments and sequence analysis of the 3′ untranslated regions of these genes indicated the existence of PEPCase multigene family in all three plants which can be grouped into three classes named ppcA, ppcB and ppcC.     

Evolution of C(4) phosphoenolpyruvate carboxylase in Flaveria: determinants for high tolerance towards the inhibitor L-malate

During the evolution of angiosperms, C4 phosphoenolpyruvate carboxylases have evolved several times independently from ancestral non-photosynthetic isoforms. They show distinct kinetic and regulatory properties when compared with the C3 isozymes. To identify the evolutionary alterations which are responsible for C4-specific properties, particularly the increased tolerance towards the allosteric inhibitor L-malate, the photosynthetic phosphoenolpyruvate carboxylase of Flaveria trinervia Mohr C4 and its ortholog from the closely related C3 plant Flaveria pringlei Gand. were examined using reciprocal enzyme chimeras. The main determinants for a high tolerance towards L-malate were located in the C-terminal region of the C4 enzyme. The effect of interchanging the region between amino acids 296 and 437 was strongly dependent upon the activation of the enzyme by glucose-6-phosphate. This confirms earlier observations that this region is important for the regulation of the enzyme by glucose-6-phosphate and that it harbours determinants for the different response of the C3 and the C4 enzyme towards this allosteric activator. In addition, it was possible to demonstrate that the only C4-specific amino acid, a serine in the C-terminal part of the enzyme, is not involved in conferring an increased L-malate tolerance to the C4 enzyme.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: activity and role of mitochondria in bundle sheath cells

Mitochondria from bundle sheath cells of the phosphoenolpyruvate carboxykinase-type C4 species Urochloa panicoides were shown to have metabolic properties consistent with a role in C4 photosynthesis predicted from earlier studies. The rate of O2 uptake in response to added malate plus ADP was at least five times the activity observed with NADH, glycine, or succinate. With malate plus ADP the O2 uptake rate averaged about 150 nmol O2 min-1 mg-1 protein, equivalent to about 0.6 mumol min-1 mg-1 of extracted chlorophyll. About half of this activity was apparently phosphorylation-linked with ADP/O2 ratios of about 4. Studies with electron transport inhibitors suggested that about 65% of this malate oxidation is cytochrome oxidase-terminated with a minor component mediated via the alternative oxidase. These mitochondria supported rapid rates of pyruvate production from malate and this activity was also stimulated by ADP but blocked by inhibitors of electron transport. Adding oxaloacetate increased pyruvate production but inhibited O2 uptake. The results were consistent with the notion that in this subgroup of C4 species mitochondrial-located NAD malic enzyme contributes substantially to total C4 acid decarboxylation. This enzyme is apparently also the primary source of NADH necessary to generate the ATP required for phosphoenolpyruvate carboxykinase-mediated oxaloacetate decarboxylation.     

Tree stem phosphoenolpyruvate carboxylase (PEPC): lack of biochemical and localization evidence for a C4-like photosynthesis system

Here, the kinetic properties and immunolocalization of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in young stems of Fagus sylvatica were investigated. The aim of the study was to test the hypothesis that there is a C4-like photosynthesis system in the stems of this C3 tree species. The activity, optimal pH and L-malate sensitivity of PEPC, and the Michaelis-Menten constant (Km) for phosphoenolpyruvate (PEP), were measured in protein extracts from current-year stems and leaves. A gel blot experiment and immunolocalization studies were performed to examine the isozyme complexity of PEPC and the tissue distribution of PEPC and Rubisco in stems. Leaf and stem PEPCs exhibited similar, classical values characteristic of C3 PEPCs, with an optimal pH of c. 7.8, a Km for PEP of c. 0.3 mM and a IC50 for L-malate (the L-malate concentration that inhibits 50% of PEPC activity at the Km for PEP) of c. 0.1 mM. Western blot analysis showed the presence of two PEPC subunits (molecular mass c. 110 kDa) both in leaves and in stems. Immunogold labelling did not reveal any differential localization of PEPC and Rubisco, neither between nor inside cells. This study suggests that C4-type photosynthesis does not occur in stems of F. sylvatica and underlines the importance of PEPC in nonphotosynthetic carbon fixation by most stem tissues (fixation of respired CO2 and fixation via the anaplerotic pathway).