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     

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     

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.     

Effects of irradiance and temperature on photosynthesis in C3, C4 and C3/C4 Panicum species

Species in the Laxa and Grandia groups of the genus Panicum are adapted to low, wet areas of tropical and subtropical America. Panicum milioides is a species with C₃ photosynthesis and low apparent photorespiration and has been classified as a C₃/C₄ intermediate. Other species in the Laxa group are C₃ with normal photorespiration. Panicum prionitis is a C₄ species in the Grandia group. Since P. milioides has some leaf characteristics intermediate to C₃ and C₄ species, its photosynthetic response to irradiance and temperature was compared to the closely related C₃ species, P. laxum and P. boliviense and to P. prionitis. The response of apparent photosynthesis to irradiance and temperature was similar to that of P. laxum and P. boliviense, with saturation at a photosynthetic photo flux density of about 1 mmol m^-2 s^-1 at 30°C and temperature optimum near 30°C. In contrast, P. prionitis showed no light saturation up to 2 mmol m^-2 s^-1 and an optimum temperature near 40°C. P. milioides exhibited low CO₂ loss into CO₂-free air in the light and this loss was nearly insensitive to temperature. Loss of CO₂ in the light in the C₃ species, P. laxum and P. boliviense, was several-fold higher than in P. milioides and increased 2- to 5-fold with increases in temperature from 10 to 40°C. The level of dark respiration and its response to temperature were similar in all four Panicum species examined. It is concluded that the low apparent photorespiration in P. milioides does not influence its response of apparent photosynthesis to irradiance and temperature in comparison to closely related C₃ Panicum species.Abbreviations AP apparent photosynthesis - I CO₂ compensation point - gl leaf conductance; gm, mesophyll conductance - PPFD photosynthetic photon flux density - PR apparent photorespiration rate - RuBPC sibulose bisphosphate carboxylase     

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C₄ photosynthesis was investigated in five C₃-C₄ intermediate species, one C₃ species, and one C₄ species in the genus Flaveria, using ¹⁴CO₂ pulse-¹²CO₂ chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating ¹⁴CO₂ into the C₄ acids malate and aspartate, following an 8-s pulse. The proportion of ¹⁴C label in these C₄ products ranged from 50–55% to 20–26% in the C₃-C₄ intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, ¹⁴CO₂ into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C₄ photosynthesis among the intermediate species based on the apparent rate of conversion of ¹⁴C label from the C₄ cycle to the C₃ cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C₄-cycle products and an increase in percentage label in C₃-cycle products during chase periods with ¹²CO₂, although the rate of change was slower than in the C₄ species, F. palmeri. In these C₃-C₄ intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C₃-C₄ intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C₄-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C₄ and C₃ cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C₃ species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C₄-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O₂, the absorbed quantum yields for CO₂ uptake (in mol CO₂·[mol quanta]^-1) averaged 0.053 in F. cronquistii (C₃), 0.051 in F. trinervia (Spreng.) Mohr (C₄), 0.052 in F. ramosissima (C₃-C₄), 0.051 in F. anomala (C₃-C₄), 0.050 in F. linearis (C₃-C₄), 0.046 in F. floridana (C₃-C₄), and 0.044 in F. pubescens (C₃-C₄). In 2% O₂ an enhancement of the quantum yield was observed in all of the C₃-C₄ intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O₂ were intermediate in value to the C₃ and C₄ species, indicating a co-function of the C₃ and C₄ cycles in CO₂ assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O₂ presumably reflect an ineffcient transfer of carbon from the C₄ to the C₃ cycle. The response of the quantum yield to four increasing O₂ concentrations (2–35%) showed lower levels of O₂ inhibition in the C₃-C₄ intermediate F. ramosissima, relative to the C₃ species. This indicates that the co-function of the C₃ and C₄ cycles in this intermediate species leads to an increased CO₂ concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O₂.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate     

A comparative immunocytochemical localization study of phosphoenolpyruvate carboxylase in leaves of higher plants

The intracellular localization of phosphoenolpyruvate (PEP) carboxylase in plants belonging to the C₄, Crassulacean acid metabolism (CAM) and C₃ types was invetigated using an immunocytochemical method with an immune serum raised against the sorghum leaf enzyme. The plants studied were sorghum, maize (C₄ type), kalanchoe (CAM type), french bean, and spinach (C₃ type). In the green leaves of C₄ plants, it was shown that the carboxylase was located in the mesophyll and stomatic cells, being largely cytosolic in the mesophyll cells. Similarly, in CAM plants, the enzyme was found mainly outside the chloroplasts. In contrast, in C₃ plants, the PEP carboxylase appeared to be distributed between the cytosol and the chloroplasts of foliar parenchyma. Examination of sections from etiolated leaves showed fluorescence emission from etioplasts and cytosol for the parenchyma of french bean as well as for the bundle sheath and mesophyll of sorghum leaves. This data indicated that during the greening process photoregulation and evolution of PEP carboxylase is dependent on the tissue and on the metabolic type of the plant considered.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate     

Direct and selective small-molecule inhibition of photosynthetic PEP carboxylase: New approach to combat C4 weeds in arable crops

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of C₄ photosynthesis. Besides, non-photosynthetic isoforms of PEPC are found in bacteria and all types of plants, although not in animals or fungi. A single residue in the allosteric feedback inhibitor site of PEPC was shown to adjust the affinity of the photosynthetic and non-photosynthetic isoforms for feedback inhibition by metabolites of the C₄ pathway. Here, we applied computational screening and biochemical analyses to identify molecules that selectively inhibit C₄ PEPC, but have no effect on the activity of non-photosynthetic PEPCs. We found two types of selective inhibitors, catechins and quinoxalines. Binding constants in the lower μM range and a strong preference for C₄ PEPC qualify the quinoxaline compounds as potential selective herbicides to combat C₄ weeds.     

On the cellular localization of phosphoenolpyruvate carboxylase in Sorghum leaves

The localization of phosphoenol pyruvate carboxylase (EC 4.1.1.3.1.) in the leaf cells of Sorghum vulgare was investigated by using three techniques: the conventional aqueous and non aqueous methods gave conflicting results; the immunocytochemical techniques clearly showed that the enzyme is predominantly located in the cytoplasm of mesophyll cells.Abbreviations PEP phosphoenol pyruvate - PAG polyacrylamide gel - NADP MDH NADP malate dehydrogenase - FITC fluorescein isothiocyanate - SAB serum albumine bovine - DTT dithiothreitol - MDH malate dehydrogenase - ME malic enzyme - PBS phosphate buffer saline - PAP peroxidase anti-peroxidase     

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.     

Incorporation of 14CO2 into C4 acids by leaves of C3-C4 intermediate and C3 species of Moricandia and Panicum at the CO2 compensation concentration

Comparative ¹⁴CO₂ pulse-¹²CO₂ chase studies performed at CO₂ compensation ()-versus air-concentrations of CO₂ demonstrated a four-to eightfold increase in assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of the C₃-C₄ intermediate species Panicum milioides Nees ex Trin., P. decipiens Nees ex Trin., Moricandia arvensis (L.) DC., and M. spinosa Pomel at . Specifically, the distribution of ¹⁴C in malate and aspartate following a 10-s pulse with ¹⁴CO₂ increases from 2% to 17% (P. milioides) and 4% to 16% (M. arvensis) when leaves are illuminated at the CO₂ compensation concentration (20 l CO₂/l, 21% O₂) versus air (340 l CO₂/l, 21% O₂). Chasing recently incorporated ¹⁴C for up to 5 min with ¹²CO₂ failed to show any substantial turnover of label in the C₄ acids or in carbon-4 of malate. The C₄-acid labeling patterns of leaves of the closely related C₃ species, P. laxum Sw. and M. moricandioides (Boiss.) Heywood, were found to be relatively unresponsive to changes in pCO₂ from air to . These data demonstrate that the C₃-C₄ intermediate species of Panicum and Moricandia possess an inherently greater capacity for CO₂ assimilation via phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) at the CO₂ compensation concentration than closely related C₃ species. However, even at , CO₂ fixation by PEP carboxylase is minor compared to that via ribulosebisphosphate carboxylase (EC 4.1.1.39) and the C₃ cycle, and it is, therefore, unlikely to contribute in a major way to the mechanism(s) facilitating reduced photorespiration in the C₃-C₄ intermediate species of Panicum and Moricandia.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - PEP phosphoenolpyruvate - CO₂ compensation concentration - 3PGA 3-phosphoglycerate - SuP sugar monophosphates - SuP₂ sugar bisphosphates Published as Paper No. 8249, Journal Series, Nebraska Agricultural Research Division     

Effects of an inhibitor of phosphoenolpyruvate carboxylase on photosynthesis of the terrestrial forms of amphibious Eleocharis species

The leafless amphibious sedge Eleocharis vivipara develops culms with C₄ traits and Kranz anatomy under terrestrial conditions, but develops culms with C₃ traits and non-Kranz anatomy under submerged conditions. The culms of the terrestrial form have high C₄ enzyme activities, while those of the submerged form have decreased C₄ enzyme activities. The culms accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the mesophyll cells (MC) and the bundle sheath cells. The Rubisco in the MC may be responsible for the operation of the C₃ pathway in the submerged form. To verify the presence of the C₃ cycle in the MC, we examined the effects of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl) -propenoate (DCDP), an inhibitor of phosphoenolpyruvate carboxylase (PEPC), on photosynthesis in culms of the terrestrial forms of E. vivipara and related amphibious species, E. baldwinii and E. retroflexa ssp. chaetaria. When 1 mM DCDP was fed via the transpiration stream to excised leaves, photosynthesis was inhibited completely in Fimbristylis dichotoma (C₄ control), but by only 20% in potato (C₃ control). In the terrestrial Eleocharis plants, the degree of inhibition of photosynthesis by DCDP was intermediate between those of the C₄ and C₃ plants, at 58–81%. These results suggest that photosynthesis under DCDP treatment in the terrestrial Eleocharis plants is due mainly to fixation of atmospheric CO₂ by Rubisco and probably the C₃ cycle in the MC. These features are reminiscent of those in C₄-like plants. Differential effects of DCDP on photosynthesis of the 3 Eleocharis species are discussed in relation to differences in the degree of Rubisco accumulation and C₃ activity in the MC. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evolution of C4 phosphoenolpyruvate carboxylase

C4 plants are known to be of polyphyletic origin and to have evolved independently several times during the evolution of angiosperms. This implies that the C4 isoform of phosphoenolpyruvate carboxylase (PEPC) originated from a nonphotosynthetic PEPC gene that was already present in the C3 ancestral species. To meet the special requirements of the C4 photosynthetic pathway the expression program of the C4 PEPC gene had to be changed to achieve a strong and selective expression in leaf mesophyll cells. In addition, the altered metabolite concentrations around C4 PEPC in the mesophyll cytoplasm necessitated changes in the enzyme's kinetic and regulatory properties. To obtain insight into the evolutionary steps involved in these altered enzyme characteristics, and even the order of these steps, the dicot genus Flaveria (Asteraceae) appears to be the experimental system of choice. Flaveria contains closely related C3, C3-C4, and C4 species that can be ordered by their gradual increase in C4 photosynthetic traits. The C4 PEPC of F. trinervia, which is encoded by the ppcA gene class, possesses typical kinetic and regulatory features of a C4-type PEPC. Its nearest neighbor is the orthologous ppcA gene of the C3 species F. pringlei. This latter gene encodes a typical nonphotosynthetic C3-type PEPC which is believed to be similar to the C3 ancestral PEPC. This pair of orthologous PEPCs has been used to map C4-specific molecular determinants for the kinetic and regulatory characteristics of C4 PEPCs. The most notable finding from these investigations was the identification of a C4 PEPC invariant site-specific mutation from alanine (C3) to serine (C4) at position 774 that was a necessary and late step in the evolution of C3 to C4 PEPC. The C3-C4 intermediate ppcA PEPCs are used to identify the sequence of events leading from a C3- to a C4-type PEPC.     

An efficient method for the determination of K m values for HCO 3 - of phosphoenolpyruvate carboxylase

One of the most serious problems in obtaining estimates of the K _m values for HCO ₃ ^- of phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) by measurement of initial rates at varying HCO ₃ ^- is the impossibility of completely excluding any contaminating HCO ₃ ^- . A method is proposed which has no need for the careful control of HCO ₃ ^- /CO₂ contamination. The kinetic data are obtained by the evaluation of progress curves of HCO ₃ ^- consumption. The method is discussed and the K _m values for HCO ₃ ^- of PEPCase from several C₄-species are presented.Abbreviations C₃, C₄ assimilated CO₂ initially found in 3-phosphoglycerate (C₃) or malate and aspartate (C₄) - OAA oxaloacetic acid - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase     

Extremely high activities of phosphoenolpyruvate carboxylase in thermogenic tissues of Araceae

Phosphoenolpyruvate carboxylase activity in extracts of a wide range of thermogenic tissues of the Araceae was shown to be in the range 10–100 mol g^-1 fresh weight min^-1 (0.5–3.7 mol mg^-1 protein min^-1). Such high activities were not found in non-thermogenic tissues of the Araceae or in thermogenic tissues of Aristolochia brasiliensis Mart. and Zucc., Victoria amazonica Schomb. and Encephalartos barteri Carruth. During development and thermogenesis in the club of Arum maculatum L. the high activities of the carboxylase did not lead to any marked accumulation of citrate, isocitrate, 2-oxoglutarate, fumarate, malate and oxaloacetate. Clubs of Arum maculatum and of Arum italicum Miller readily fixed ¹⁴CO₂ in the dark, mostly into aspartate, malate, alanine and glutamate. Pulse and chase experiments showed that most of the fixed carbon was very rapidly metabolized to CO₂. The detailed distribution suggest that this occurred largely by decarboxylation of C-4 acids. It is suggested that thermogenic tissues of the Araceae are characterized by very high activities of phosphoenolpyruvate carboxylase, and that in vivo this leads to synthesis of C-4 acids which are promptly decarboxylated.     

Sequence analysis of cDNA encoding phosphoenolpyruvate carboxylase from cultured tobacco cells

The complete nucleotide sequence of cDNA encoding phosphoenolpyruvate carboxylase (PEPCase) from cultured tobacco (a C₃ plant) cells was determined and the deduced amino acid sequence was compared with those of PEPCases from other higher plants.