Effects of temperature on the activity of phosphoenolpyruvate carboxylase and on the control of CO2 fixation in Bryophyllum fedtschenkoi

The phosphorylation state and the malate sensitivity of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in Bryophyllum fedtschenkoi Hamet et Perrier are altered by changes in the ambient temperature. These effects, in turn alter the in-vivo activity of the enzyme. Low temperature (3 °C or less), stabilizes the phosphorylated form of the enzyme, while high temperature (30 °C) promotes its dephosphorylation. The catalytic activity of the phosphorylated and dephosphorylated forms of PEPCase increases with temperature, but the apparent K _i values for malate of both forms of the enzyme decrease. Results of experiments with detached leaves maintained in darkness in normal air indicate that the changes in malate sensitivity and phosphorylation state of PEPCase with temperature are of physiological significance. When the phosphorylated form of PEPCase is stabilized by reducing the temperature of leaves 9 h after transfer to constant darkness at 15 °C, a prolonged period of CO₂ fixation follows. When leaves are maintained in constant darkness at 15 °C until CO₂ output reaches a low steady-state level and the PEPCase is dephosphorylated, reducing the temperature to 3 °C results in a further period of CO₂ fixation even though the phosphorylation state of PEPCase does not change.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase We thank the Agricultural and Food Research Council for financial support for this work.     

Regulation of C4-phosphoenolpyruvate carboxylase activity by ambient CO2

Light activation of phosphoenolpyruvate carboxylase from the leaves of the C₄ plant Setaria verticillata (L.) is more pronounced at low CO₂ levels. The 2-fold activation observed at physiological ambient CO₂ becomes 3.64-fold at 5 L/L and completely abolished above 700 L/L. When the stomata close under the influence of abscisic acid at 330 L/L CO₂, the extent of light activation is high (3.59-fold), probably because the increased diffusive resistance keeps the internal CO₂ at much lower levels. Under darkness. CO₂ and absicisic acid do not affect the extractable phosphoenolpyruvate carboxylase activity. Internal CO₂ levels may determine phosphoenolpyruvate concentratio in the cytoplasm through the control of its utilization by phosphoenolpyruvate carboxylase. We have recently proposed (Samaras et al. 1988) that photosynthetically produced phosphoenolpyruvate could be an activator of the enzyme. It is therefore suggested that CO₂ indirectly affects the activation state of phosphoenolpyruvate carboxylase by controlling the levels of phosphoenolpyruvate which may act as an activator.Abbreviations PEPCase phosphoenolpyruvate carboxylase - PEP phosphoenolpyruvate - PAR photosynthetically active radiation - G6P glucose-6-phosphate - ABA abscisic acid - MDH malate dehydrogenase - PPDK pyruvate, Pi, dikinase - CAM Crassulacean Acid Metabolism     

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     

Gas-exchange of ears of cereals in response to carbon dioxide and light

Data for the maximum carboxylation velocity of ribulose-1,5-biosphosphate carboxylase, V_m, and the maximum rate of whole-chain electron transport, J_m, were calculated according to a photosynthesis model from the CO₂ response and the light response of CO₂ uptake measured on ears of wheat (Triticum aestivum L. cv. Arkas), oat (Avena sativa L. cv. Lorenz), and barley (Hordeum vulgare L. cv. Aramir). The ratio J_m/V_m is lower in glumes of oat and awns of barley than it is in the bracts of wheat and in the lemmas and paleae of oat and barley. Light-microscopy studies revealed, in glumes and lemmas of wheat and in the lemmas of oat and barley, a second type of photosynthesizing cell which, in analogy to the Kranz anatomy of C₄ plants, can be designated as a bundle-sheath cell. In wheat ears, the CO₂-compensation point (in the absence of dissimilative respiration) is between those that are typical for C₃ and C₄ plants.A model of the CO₂ uptake in C₃–C₄ intermediate plants proposed by Peisker (1986, Plant Cell Environ. 9, 627–635) is applied to recalculate the initial slopes of the A(p^c) curves (net photosynthesis rate versus intercellular partial pressure of CO₂) under the assumptions that the J_m/V_m ratio for all organs investigated equals the value found in glumes of oat and awns of barley, and that ribulose-1,5-bisphosphate carboxylase is redistributed from mesophyll to bundle-sheath cells. The results closely match the measured values. As a consequence, all bracts of wheat ears and the inner bracts of oat and barley ears are likely to represent a C₃–C₄ intermediate type, while glumes of oat and awns of barley represent the C₃ type.Abbreviations A net photosynthesis rate (mol·m^-2·s^-1) - J_m maximum rate of whole-chain electron transport (mol·e^-·m^-2·s^-1) - p^c (bar) intercellular partial pressure of CO₂ - PEP phosphoenolpyruvate - PPFD photosynthetic photon flux density (mol quanta·m^-2·s^-1) - RuBPCase ribulose bisphosphate carboxylase/oxygenase - RuBP ribulose bisphosphate - V_m maximum carboxylation velocity of RuBPCase (mol·m^-2·s^-1) - T^* CO₂ compensation point in the absence of dissimilative respiration (bar)     

Activities of carboxylating enzymes in the CAM species Opuntia ficus-indica grown under current and elevated CO2 concentrations

Responses of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPCase) to an elevated atmospheric CO₂ concentration were determined along with net CO₂ uptake rates for the Crassulacean acid metabolism species Opuntia ficus-indica growing in open-top chambers. During the spring 13 months after planting, total daily net CO₂ uptake of basal and first-order daughter cladodes was 28% higher at 720 than at 360 l CO₂ l^-1. The enhancement, caused mainly by higher CO₂ assimilation during the early part of the night, was also observed during late summer (5 months after planting) and the following winter. The activities of Rubisco and PEPCase measured in vitro were both lower at the elevated CO₂ concentration, particularly under the more favorable growth conditions in the spring and late summer. Enzyme activity in second-order daughter cladodes increased with cladode age, becoming maximal at 6 to 10 days. The effect ofelevated CO₂ on Rubisco and PEPCase activity declined with decreasing irradiance, especially for Rubisco. Throughout the 13-month observation period, O. ficus-indica thus showed increased CO₂ uptake when the atmospheric CO₂ concentration was doubled despite lower activities of both carboxylating enzymes.     

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.     

Molecular properties of phosphoenolpyruvate carboxylase from C3, C3−C4 intermediate,and C4 Flaveria species

Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) from Flaveria trinervia Mohr (C₄), F. floridana Johnston (C₃–C₄), and F. cronquistii Powell (C₃) leaves were compared by electrotransfer blotting/enzyme-linked immunoassay (Western-blot analysis), mobility of the native enzyme in polyacrylamide gels and in isoelectric focusing (IEF) gels, peptide mapping, and in-vitro translation of RNA isolated from each plant. The PEPCases from the C₃ and C₃–C₄ plants were very similar to each other in terms of electrophoretic mobilities on gels and isoenzyme patterns on IEF gels, and identical in peptide mapping. Quantitative differences were noted, however, in that the C₃–C₄ intermediate plant contained more PEPCase overall and that the relative activity of individual isoenzymes shifted between the C₃ and C₃–C₄ intermediate PEPCases. The PEPCase from the C₄ plant had a different isoenzyme pattern, a different peptide map, and was far more abundant than the other two enzymes. Western blot analysis demonstrated the cross-reactivity of PEPCases from all three Flaveria species with antibody raised against maize PEPCase. The results provide evidence, at the molecular level, that supports the view of C₃–C₄ intermediate species as C₃-like plants with some C₄-like photosynthetic characteristics, but there are differences from the C₃ plant in the quantity and properties of the PEPCase from the C₃–C₄ intermediate plant.Abbreviations IEF isoelectric focusing - kDa kilodalton - PEPCase phosphoenolpyruvate carboxylase - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase     

Effects of temperature and photosynthetic inhibitors on light activation of C4-phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase from leaves of the C₄ plant Setaria verticillata (L.) Beauv. is activated by light; day levels of activity are reached after 30 minutes of illumination. Photoactivation is prevented by inhibitors of photosynthetic electron flow or of photophosphorylation and by D,L-glyceraldehyde, which inhibits the reductive pentose phosphate pathway.Although the extractable activity in the dark is not affected by temperature the photoactivation is prevented when both illumination and extraction are done under low temperature (5 C). High temperature (30 C) during either illumination or extraction is needed for activation. Once the enzyme is photoactivated at 30 C, a transfer of the leaves to 5 C does not abolish the extra activity.The results suggest that both unimpaired electron flow and photophosphorylation are prerequisites for the activation of phosphoenolpyruvate carboxylase. Low temperature apparently suppresses either the transport to the cytoplasm of a photosynthetic intermediate or the activating reaction itself. The inclusion of phosphoenolpyruvate in the extraction medium increases the night activity.On the basis of the available information, it is suggested that phosphoenolpyruvate could be the activator in vivo. In that case, the activation of phosphoenolpyruvate carboxylase would depend on internal CO₂ level and prior photoactivation of both pyruvate, orthophosphate, dikinase and NADP malate dehydrogenase.Abbreviations PEPCase phosphoenolpyruvate carboxylase - PEP phosphoenolpyruvate - PAR photosynthetically active radiation - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea - DSPD disalicylidenpropanediamine - MV methylviologen - ME malic enzyme - MDH malate dehydrogenase - PPDK pyruvate, Pi dikinase - CAM Crassulacean Acid Metabolism     

Immunocytochemical localization of phosphoenolpyruvate carboxylase and photosynthetic gas-exchange characteristics in ears of Triticum durum Desf.

The presence and distribution of phosphoenolpyruvate carboxylase (PEPCase) in the glumes and immature grains of durum wheat (Triticum durum Desf.) were studied by electron-microscopical immunolabeling of PEPCase with polyclonal antibodies followed by protein A-gold. Plants were grown under mediterranean field conditions and samples were obtained two weeks after anthesis. In the kernels, high gold label was associated with the unstained areas of the protein bodies of aleurone cells, whereas labeling in the cytoplasm and chloroplasts of the pericarp was slight, although significantly above the background. In the glumes, high gold label was only located in cytoplasmic granules (vesicles) of the mesophyll cells, although labeling in the cytoplasm and chloroplasts was also significantly above the background. These observations in immature kernels and glumes are in accordance with the anaplerotic role of this enzyme, as evidenced in C₃ plants. Measurements of apparent photosynthesis and its O₂ dependence and CO₂ compensation concentration were made on ears and flag leaves of durum wheat. In addition, an analog of phosphoenolpyruvate, 3,3-dichloro-2-dihydroxy-phosphinoylmethyl-2-propenoate, was used to inhibit PEPCase and, thereby, to assess the contribution of the PEPCase to photosynthesis in detached ears. There was no effect of the inhibitor on the apparent photosynthesis of ears. Whereas inhibition of apparent photosynthesis by 210 mL · L^–1 O₂ in flag leaves was typical of C₃ species, inhibition in ears was even greater. The CO₂ compensation concentrations in different ear parts were similar to or higher than in flag leaves and the O₂ dependence was also comparable (about 70%). Therefore, gas-exchange data give further support to the assumption that a C₄ cycle is absent or limited to very low rates in ears of durum wheat.Abbreviations and Symbol AP apparent photosynthesis - BSA bovine serum albumin - DCDP 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate - PBS phosphate-buffered saline - PEP phosphoenolpyruvate - PEPCase PEP carboxylase - CO₂ compensation concentration We thank Dr. Sam Sun (University of Hawaii) for kindly providing the PEPCase antibodies and Drs. C.L.D. Jenkins and M.D. Hatch (Division of Plant Industry, CSIRO, Canberra, Australia) for supplying DCDP for these experiments. We are also grateful to Dr. Robert Bargalló and Mrs. Ana Ribero (Servei de Microscopia Electronica, Universitat de Barcelona) for technical assistance in electron-microscopy studies. Participation of Drs. J.L. Araus and J. Bort in this work was supported by the Research Project of CICYT AGF92-0301, Spain.     

Photoautotrophic growth of soybean cells in suspension culture III. Characterization of carbon fixation products under high and low CO2 levels

A photoautotrophic soybean suspension culture (SB-P) was used to study CO₂ assimilation while exposed to elevated or ambient CO₂ levels. These studies showed that under elevated CO₂ (5% v/v) malate is the dominant fixation product, strongly suggesting that phosphoenolpyruvate carboxylase (PEPCase) is the primary enzyme involved in carbon fixation in these cells under their normal growth conditions. Citrate and [aspartate + glutamate] were also significant fixation products during fifteen minutes of exposure to ¹⁴CO₂. During the ten minute unlabeled CO₂ chase however, ¹⁴C-malate continued to increase while citrate and [aspartate + glutamate] declined. Fixation of ¹⁴CO₂ under ambient CO₂ levels (0.037%) showed a very different product pattern as 3-phosphoglycerate was very high in the first one to two minutes followed by increases in [serine + glycine] and [aspartate + glutamate]. Hexose phosphates were also quite high initially but then declined relatively rapidly. Thus, the carbon fixation pattern at ambient CO₂ levels resembles somewhat that seen in C3 leaf cells while that seen at elevated CO₂ levels more closely resembles that of a C₄ plant. The initial fixation product of C₃ plants, 3-PGA, was never detectable under high CO₂ conditions. These data suggest that an in vitro photoautotrophic system would be suitable for studying carbon fixation physiology during photosynthetic and non-photosynthetic growth.Abbreviations SB-P photoautotrophic soybean cells - PEPCase phosphoenol-pyruvate carboxylase - RuBPCase ribulose bisphosphate carboxylase/oxygenase - 3-PGA 3-phosphoglycerate     

Some relationships between contents of photosynthetic intermediates and the rate of photosynthetic carbon assimilation in leaves of Zea mays L.

The relationship between the gas-exchange characteristics of attached leaves of Zea mays L. and the contents of photosynthetic intermediates was examined at different intercellular partial pressure of CO₂ and at different irradiances at a constant intercellular partial pressure of CO₂. (i) The behaviour of the pools of the C₄-cycle intermediates, phosphoenolpyruvate and pyruvate, provides evidence for light regulation of their consumption. However, light regulation of phosphoenolpyruvate carboxylase does not influence the assimilation rate at limiting intercellular partial pressures of CO₂. (ii) A close correlation between the pools of phosphoenolpyruvate and glycerate-3-phosphate exists under many different flux conditions, consistent with the notion that the pools of C₄ and C₃ cycles are connected via the interconversion of glycerate-3-phosphate and phosphoenolpyruvate. (iii) The ratio of triose-phosphate to glycerate-3-phosphate is used as an indicator of the availability of ATP and NADPH. Changes of this ratio with CO₂ and with irradiance are compared with results obtained in C₃ leaves and indicate that the mechanism of regulation of carbon assimilation by light in leaves of C₄ plants may differ from that in C₃ plants. (iv) The behaviour of the ribulose-1,5-bisphosphate pool with CO₂ and irradiance is contrasted with the behaviour of these pools measured in leaves of C₃ plants.Abbreviations P _i intercellular CO₂ pressure - RuBP ribulose-1,5-bisphosphate - PEP phosphoenolpyruvate - triose-P triose phosphates - PGA glycerate-3-phosphate     

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     

Kinetic variation in grass phosphoenolpyruvate carboxylases provides opportunity to enhance C4 photosynthetic efficiency

The high rates of photosynthesis and the carbon-concentrating mechanism (CCM) in C₄ plants are initiated by the enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC). The flow of inorganic carbon into the CCM of C₄ plants is driven by PEPC’s affinity for bicarbonate (K_HCO3), which can be rate limiting when atmospheric CO₂ availability is restricted due to low stomatal conductance. We hypothesize that natural variation in K_HCO3 across C₄ plants is driven by specific amino acid substitutions to impact rates of C₄ photosynthesis under environments such as drought that restrict stomatal conductance. To test this hypothesis, we measured K_HCO3 from 20 C₄ grasses to compare kinetic properties with specific amino acid substitutions. There was nearly a twofold range in K_HCO3 across these C₄ grasses (24.3 ± 1.5 to 46.3 ± 2.4 μm ), which significantly impacts modeled rates of C₄ photosynthesis. Additionally, molecular engineering of a low-HCO₃⁻ affinity PEPC identified key domains that confer variation in K_HCO3. This study advances our understanding of PEPC kinetics and builds the foundation for engineering increased-HCO₃⁻ affinity and C₄ photosynthetic efficiency in important C₄ crops.     

Growth characteristics of hormone and vitamin independent photoautotrophic cell suspension cultures fromChenopodium rubrum

Summary This communication reports the photoautotrophic growth of hormone and vitamin independent cell suspension cultures ofChenopodium rubrum. The transfer of cells from stationary growth into fresh culture medium results in a high protein formation, followed by an exponential phase of cell division, whereas the onset of rapid chlorophyll formation is delayed for 4 days. At the stage of most rapid cell division there is no net synthesis of starch and sugar. When the cells enter stationary growth, there is a progressive accumulation of chlorophyll, sugar, and starch.Photoautotrophic cell cultures assimilate about 80–90 mol CO₂/mg chlorophyll X hour. Dark CO₂ fixation is about 3.7% to 2.2% of the light values during exponential and stationary growth, respectively. As shown by short-term¹⁴CO₂ fixation, CO₂ is predominantly assimilated through ribulosebisphosphate carboxylase via the Calvin pathway. There is a significant increase in the¹⁴C label of C₄ carboxylic acids in exponentially dividing cells as compared to cells from stationary growth. Thein vitro activity of phosphoenolpyruvate carboxylase and ribulosebisphosphate carboxylase is almost equal during exponential cell division. A decrease in cell division activity is accompanied by a significant change in the specific activities of both carboxylation enzymes. In non dividing cells from stationary growth the activity of ribulosebisphosphate carboxylase is greately enhanced and that of phosphoenolpyruvate carboxylase is reduced, documenting the development of carboxylation capacities typical for C₃-plants.The experimental results provide evidence that phosphoenolpyruvate carboxylase activity might be regulated by ammonia and could be involved in anaplerotic CO₂ fixation which supplies carbon skeletons of the citric acid cycle.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylene-diamine-tetraacetic acid - FDP fructose bisphosphate - F-6-P fructose-6-phosphate - G-6-P glucose-6-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PGA 3-phosphoglyceric acid - PEP phosphoenolpyruvate - RuDP ribulosebisphosphate     

Carbon-isotope discrimination by leaves of Flaveria species exhibiting different amounts of C3-and C4-cycle co-function

Carbon-isotope ratios were examined as ¹³C values in several C₃, C₄, and C₃–C₄ Flaveria species, and compared to predicted ¹³C, values generated from theoretical models. The measured ¹³C values were within 4 of those predicted from the models. The models were used to identify factors that contribute to C₃-like ¹³C values in C₃–C₄ species that exhibit considerable C₄-cycle activity. Two of the factors contributing to C₃-like ¹³C values are high CO₂ leakiness from the C₄ pathway and pi/pa values that were higher than C₄ congeners. A marked break occurred in the relationship between the percentage of atmospheric CO₂ assimilated through the C₄ cycle and the ¹³C value. Below 50% C₄-cycle assimialtion there was no significant relationship between the variables, but above 50% the ¹³C values became less negative. These results demonstrate that the level of C₄-cycle expression can increase from, 0 to 50% with little integration of carbon transfer from the C₄ to the C₃ cycle. As expression increaces above 50%, however, increased integration of C₃- and C₄-cycle co-function occurs.Abbreviations and symbols RuBP carboxylase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - PEP carboxylase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - pa atmospheric CO₂ partial pressure - pi intercellular CO₂ partial pressure - isotope ratio - quantum yield for CO₂ uptake     

Variable HCO 3 - affinity of Elodea canadensis Michaux in response to different HCO 3 - and CO2 concentrations during growth

Summary Elodea canadensis grows over a wide range of inorganic carbon, nutrient, and light conditions in lakes and streams. Affinity for HCO ₃ ^- use during photosynthesis ranged from strong to weak in Elodea collected from seven localities with different HCO ₃ ^- and CO₂ concentrations. The response to HCO ₃ ^- was also very plastic in plants grown in the laboratory at high HCO ₃ ^- concentrations and CO₂ concentrations varying from 14.8 to 2,200 M. Bicarbonate affinity was markedly reduced with increasing CO₂ concentrations in the growth medium so that ultimately HCO ₃ ^- use was not detectable. High CO₂ concentrations also decreased CO₂ affinity and induced high CO₂ compensation points (360M CO₂) and tenfold higher half-saturation values (800 M CO₂).The variable HCO ₃ ^- affinity is probably environmentally based. Elodea is a recently introduced species in Denmark, where it reproduces only vegetatively, leaving little opportunity for genetic variation. More important, local populations in the same water system had different HCO ₃ ^- affinities, and a similar variation was created by exposing one plant collection to different laboratory conditions.Bicarbonate use enabled Elodea to photosynthesize rapidly in waters of high alkalinity and enhanced the carbon-extracting capacity by maintaining photosynthesis above pH 10. On the other hand, use of HCO ₃ ^- represents an investment in transport apparatus and energy which is probably not profitable when CO₂ is high and HCO ₃ ^- is low. This explanation is supported by the findings that HCO ₃ ^- affinity was low in field populations where HCO ₃ ^- was low (0.5 and 0.9 m M) or CO₂ was locally high, and that HCO ₃ ^- affinity was suppressed in the laboratory by high CO₂ concentrations.Abbreviations DIC dissolved inorganic carbon (CO₂+ HCO ₃ ^- +CO ₃ ^- ) - CO₂ compensation point - K _1/2 apparent halfsaturation constant - P_{HCO 3} ^– interpolated photosynthesis in pure HCO ₃ ^- and zero CO₂ - P_max photosynthetic rate under carbon and light saturation     

Mass-spectrometric evidence for the double-carboxylation pathway of malate synthesis by Crassulacean acid metabolism plants in light

Phyllodia of the Crassulacean acid metabolism (CAM) plant Kalanchoë tubiflora were allowed to fix ¹³CO₂ in light and darkness during phase IV of the diurnal CAM cycle, and during prolongation of the regular light period. After ¹³CO₂ fixation in darkness, only singly labelled [¹³C]malate molecules were found. Fixation of ¹³CO₂ under illumination, however, produced singly labelled malate as well as malate molecules which carried label in two, three or four carbon atoms. When the irradiance during ¹³CO₂ fixation was increased, the proportion of singly labelled malate decreased in favour of plurally labelled malate. The irradiance, however, did not change either the ratio of labelled to unlabelled malate molecules found in the tissue after the ¹³CO₂ application, or the magnitude of malate accumulation during the treatment with label. The ability of the tissue to store malate and the labelling pattern changed throughout the duration of the prolonged light period. The results indicate that malate synthesis by CAM plants in light can proceed via a pathway containing two carboxylation steps, namely ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) which operate in series and share common intermediates. It can be concluded that, in light, phosphoenolpyruvate carboxylase can also synthesize malate independently of the proceeding carboxylation step by ribulose-1,5-bisphosphate carboxylase/oxygenase.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - TMS trimethylsilyl     

Responses of carboxylating enzymes,sucrose metabolizing enzymes and plant hormones in a tropical epiphytic CAM orchid to CO2 enrichment

After exposure to a doubled CO₂ concentration of 750 µL L^?1 for 2 months, average relative growth rate (RGR) of Mokara Yellow increased 25%. The two carboxylating enzymes, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPCase), responded differently to CO₂ enrichment. There was a significant daytime down‐regulation in Rubisco activity in the leaves of CO₂‐enriched plants. However, PEPCase activity in CO₂‐enriched plants was much higher in the dark period, although it was slightly lower during the daytime than that at ambient CO₂. Leaf sucrose–phosphate synthase (SPS) and sucrose synthase (SS) activities in CO₂‐enriched plants increased markedly, along with a night‐time increase in total titratable acidity and malate accumulation. There was a remarkable increase in the levels of indole‐3‐acetic acid (IAA), gibberellins A₁ and A₃ (GA₁₊₃), isopentenyladenosine (iPA) and zeatin riboside (ZR) in the expanding leaves of plants grown at elevated CO₂. It is suggested that (1) the down‐regulation of Rubisco and up‐regulation of SPS and SS are two important acclimation processes that are beneficial because it enhanced both photosynthetic capacity at high CO₂ and reduced resource investment in excessive Rubisco capacity; (2) the increased levels of plant hormones in CO₂‐enriched M. Yellow might play an important role in controlling its growth and development.     

Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)^-1·min^-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. K_m values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (K_a) for Mg²⁺ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW molecular weight - PEP (Case) phosphoenolpyruvate (carboxylase) - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Carbon assimilation by different developmental stages of Laminaria saccharina

Various stages of the life cycle of the marine brown alga Laminaria saccharina (L.) Lamour. (Laminariales, Phaeophyta) including male and female gametophytes, female gametes, zygotes and young sporophytes of different age were investigated for their potentials of carbon dioxide (¹⁴CO₂) fixation. Rates of photosynthesis attain the same order of magnitude in all stages. Photosynthetic ¹⁴CO₂-fixation is accompanied by a substantial light independent carbon assimilation. This is confirmed by rate determinations of the equivalent carboxylating enzymes present in the plants, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxokinase (EC 4.1.1.32) as well as by chromatographic analyses of the appropriate [¹⁴C]-assimilate patterns.Abbreviations RuBP-C ribulose-1,5-bisphosphate carboxylase - PEP-CK phosphoenolpyruvate carboxykinase - PEP phosphoenolpyruvate - PS photosynthesis - DF dark fixation