Leaf inner structure and immunogold localization of some key enzymes involved in carbon metabolism in CAM plants

There are relatively few reports on the leaf structure and in situ immunolocalization of carbon metabolism enzymes in crassulacean acid metabolism (CAM) plants, compared with reports on C4 plants. The leaf inner structure and the subcellular location of some key CAM enzymes for a phosphoenolpyruvate carboxykinase (PCK) CAM species, Ananas comosus, and three malic enzyme (ME) CAM species, Mesembryanthemum crystallinum, Kalanchoe daigremontiana, and K. pinnata, was investigated by immunogold labelling and electron microscopy in this study. The leaves of these species had few intercellular air spaces in the mesophyll. A large vacuole occupied the mesophyll cells, and many vesicles of various sizes occurred in the cytosol. Immunocytochemical study revealed that labelling was present for phosphoenolpyruvate carboxylase in the cytosol and for ribulose-1,5-bisphosphate carboxylase/oxygenase in the chloroplasts of the mesophyll cells in all species. No specific labelling for pyruvate orthophosphate dikinase (PPDK) was observed in the PCK-CAM species. In the ME-CAM species, the patterns of labelling for PPDK differed. In M. crystallinum labelling for PPDK was present only in the chloroplasts, whereas in the two Kalanchoe species it occurred in the cytosol as well as in the chloroplasts. These results suggest that the subcellular localization of PPDK varies with ME-CAM species, in contrast to the conventional belief that it is localized in the chloroplasts.Key words: Crassulacean acid metabolism, immunolocalization, leaf inner structure, phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase.      

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     

Effects of glycerol on the in vitro stability and regulatory activation/inactivation of pyruvate,orthophosphate dikinase of Zea mays L.

Glycerol stabilizes the activity of pyruvate, orthophosphate dikinase extracted from darkened or illuminated maize leaves. It serves as a better protectant of activity than dithiothreitol for the active day-form and the glycerol concentration needed for full protection is inversely related to the level of protein. The night-form of the enzyme is also protected by glycerol not only against inactivation, but also against partial reactivation in storage. Glycerol does not prevent the P_i-dependent activation nor the ADP-dependent inactivation of pyruvate, orthophosphate dikinase, but the rates of both processes are substantially decreased. The ability of the inactive night-form for P_i-dependent activation is also sustained by glycerol for at least 2 h at 20°C, apparently through stabilization of the labile regulatory protein.Abbreviations BSA bovine serum albumin - G-6-P glucose-6-phosphate - MDH malate dehydrogenase - PCMB p-chloromercuribenzoate - PEP phosphoenolpyruvate - PEPCase phosphoenol-pyruvate carboxylase - PPDK pyruvate, orthophosphate dikinase - PVP polyvinylpyrrolidone     

Cold Inactivation of Phosphoenolpyruvate Carboxylase and Pyruvate Orthophosphate Dikinase from the C4 Perennial Plant Atriplex halimus

Phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) cold inactivation was studied in leaf extracts from Atriplex halimus L. Both enzyme activities gradually reduced as the temperature and the total soluble protein decreased. Mg²⁺ at a concentration of 10 mM stabilized PEPC and PPDK activities against cold inactivation. At low Mg²⁺ concentration (4 mM), PEPC was strongly protected by phosphoenolpyruvate, glucose-6-phosphate, and, partially, byL-malate, while PPDK was protected by PEP, but not by its substrate, pyruvate. High concentrations of compatible solutes (glycerol, betaine, proline, sorbitol and trehalose) proved to be good protectants for both enzyme activities against cold inactivation. When illuminated leaves were exposed to low temperature, PPDK was partially inactivated, while the activity of PEPC was not altered.     

Assaying for pyruvate,orthophosphate dikinase activity: Necessary precautions with phosphoenolpyruvate carboxylase as coupling enzyme

Phosphoenolpyruvate carboxylase (EC 4.1.1.31), used as a coupling enzyme in the assay of the pyruvate, orthophosphate dikinase (EC 2.7.9.1) forward reaction, is a serious limiting factor for the overall rate when added at a level of 0.2–0.3 unit/ml of assay medium. Nonlimiting assay conditions are obtained by either increasing the level of the coupling enzyme to 3 units/ml or adding 6mM glucose-6-phosphate as an activator/stabilizer of phosphoenolpyruvate carboxylase.Abbreviations G-6-P glucose-6-phosphate - LDH lactate dehydrogenase - MDH malate dehydrogenase - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PVP polyvinylpyrrolidone - PPDK pyruvate, orthophosphate dikinase - U unit of enzyme activity (mol/min)     

The compartmentation of carboxylating and decarboxylating enzymes in guard cell protoplasts

Guard cell and mesophyll cell protoplasts of Vicia faba L. were purified and separated into cytoplasmic and plastid fractions by a selective silicone-oil filtration. Before fractionation, the protoplasts were ruptured by a low speed centrifugation through a narrow-aperture nylon net placed in a plastic vial. This protoplast homogenation and subsequently the silicone-oil fractionation offer the possibility of investigating the comparatmentation of the enzymatic carboxylating (ribulose bisphosphate carboxylase EC 4.1.1.39, phosphoenolpyruvate carboxylase EC 4.1.1.31, NAD⁺ and NADP⁺ linked malate dehydrogenase EC 1.1.1.37) and decarboxylating pathways of malic (malic enzyme EC 1.1.1.40, phosphoenolpyruvate carboxykinase EC 4.1.1.32, pyruvate orthophosphate dikinase EC 2.7.9.1) which occur during the swelling and shrinking of the guard cell protoplasts. A model is proposed which describes the transport processes of malic acid during the starch-malate balance as correlated to the volume changes of the protoplasts. As the enzymes and their compartmentation in the guard cell protoplasts seem to be consistent with those of crassulacean acid metabolism (CAM) plants, the metabolism of stomata and of CAM cells is compared.Abbreviations AQ anthraquinone-2-sulfonic acid - CAM Crassulacean acid metabolism - DCPIP_red 2,6-dichlorophenol-indophenol - DTT dithiothreitol - EDTA ethylendiamine tetraacetic acid - GAPDH glyceraldehyde-3-phosphate dehydrogenase - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane sulphonic acid - MDH malante dehydrogenase - MES 2(N-morpholino) ethane sulphonic acid - OAA oxaloacetic acid - PEP phosphoenolpyruvate - PSI photosystem I - KuP₂ ribulose bisphosphate     

Oligomeric enzymes in the C4 pathway of photosynthesis

This review deals with the factors controlling the aggregation-state of several enzymes involved in C₄ photosynthesis, namely phosphoenolpyruvate carboxylase, NAD-and NADP-malic enzyme, NADP-malic dehydrogenase and pyruvate, phosphate dikinase and its regulatory protein. All of these enzymes are oligomeric and have been shown to undergo changes in their quaternary structure in vitro under different conditions. The activity changes linked to variations in aggregation-state are discussed in terms of their putative physiological role in the regulation of C₄ metabolism.Abbreviations P-enolpyruvate phosphoenolpyruvate - NAD-ME NAD-dependent malic enzyme - NADP-ME NADP-dependent malic enzyme - NADP-MDH NADP-dependent malic dehydrogenase - PPDK pyruvate, phosphate dikinase - PPDK-RP pyruvate, phosphate dikinase regulatory protein - V_max maximal velocity - K_m Michaelis constant - CAM Crassulacean acid metabolism     

Metabolic networks to generate pyruvate,PEP and ATP from glycerol in Pseudomonas fluorescens

Glycerol is a major by-product of the biodiesel industry. In this study we report on the metabolic networks involved in its transformation into pyruvate, phosphoenolpyruvate (PEP) and ATP. When the nutritionally-versatile Pseudomonas fluorescens was exposed to hydrogen peroxide (H₂O₂) in a mineral medium with glycerol as the sole carbon source, the microbe reconfigured its metabolism to generate adenosine triphosphate (ATP) primarily via substrate-level phosphorylation (SLP). This alternative ATP-producing stratagem resulted in the synthesis of copious amounts of PEP and pyruvate. The production of these metabolites was mediated via the enhanced activities of such enzymes as pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC). The high energy PEP was subsequently converted into ATP with the aid of pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) with the concomitant formation of pyruvate. The participation of the phospho-transfer enzymes like adenylate kinase (AK) and acetate kinase (ACK) ensured the efficiency of this O₂-independent energy-generating machinery. The increased activity of glycerol dehydrogenase (GDH) in the stressed bacteria provided the necessary precursors to fuel this process. This H₂O₂-induced anaerobic life-style fortuitously evokes metabolic networks to an effective pathway that can be harnessed into the synthesis of ATP, PEP and pyruvate. The bioconversion of glycerol to pyruvate will offer interesting economic benefit.     

Elevated pyruvate,orthophosphate dikinase (PPDK) activity alters carbon metabolism in C3 transgenic potatoes with a C4 maize PPDK gene

Changes in carbon metabolism and δ¹³C value of transgenic potato plants with a maize pyruvate,orthophosphate dikinase (PPDK; EC 2.7.9.1) gene are reported. PPDK catalyzes the formation of phospho enol pyruvate (PEP), the initial acceptor of CO₂ in the C₄ photosynthetic pathway. PPDK activities in the leases of transgenic potatoes were up to 5.4‐fold higher than those of control potato plants (wild‐type and treated control plants). In the transgenic potato plants, PPDK activity in leaves was negatively correlated with pyruvate content (r²= 0.81), and was positively correlated with malate content (r²= 0.88). A significant increase in the δ¹³C value was observed in the transgenic potato plants, suggesting a certain contribution of PEP carboxylase as the initial acceptor of atmospheric CO₂. These data suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C₄‐type carbon metabolism. However, since parameters associated with CO₂ gas exchange were not affected, the altered carbon metabolism had only a small effect on the total photosynthetic characteristics of the transgenic plants.     

Immunogold localization of photosynthetic enzymes in leaves of various C4 plants, with particular reference to pyruvate orthophosphate dikinase

Cellular localization of photosynthetic enzymes was investigated by immunogold electron microscopy for leaves of nine C4 grasses (three NADP-malic enzyme (NADP-ME)subtype species, three NAD-malic enzyme (NAD-ME) subtype species, and three phosphoenolpyruvate carboxykinase (PCK) subtype species), two C4 sedges (NADP-ME subtype species) and two C4 dicots (an NADP-ME and an NADP/NAD-ME subtype species). In leaves of all species, immunogold labelling was present for phosphoenolpyruvate carboxylase in the cytosol of the mesophyll cells (MC) and for ribulose-1,5-bisphosphate carboxylase/oxygenase in the chloroplasts of the bundle sheath cells (BSC). However, considerable specific variation was found in the intercellular patterns of labelling for pyruvate orthophosphate dikinase (PPDK). In the NADP-ME grasses, two NAD-ME grasses, and the dicots, significant labelling for PPDK was present in the both the BSC and the MC chloroplasts. In the other NAD-ME grass, the PCK grasses, and the sedges, labelling for PPDK was present almost exclusively in the chloroplasts of the MC. These patterns were observed in the leaves of both young seedlings and mature plants. These results indicate that the accumulation of PPDK in leaves of C4 plants is not necessarily restricted to the MC, although the chloroplasts of the MC accumulate more than those of the BSC.Key words: C4 plants, immunolocalization, phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, ribulose-1,5-bisphosphate carboxylase/oxygenase.      

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     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Variation in amounts of pyruvate, orthophosphate dikinase, and some other enzymes of the c(4) pathway in some wheat species

First leaves and flag leaves of the wheat species Triticum aestivum cv Anza (6×), T. boeoticum Boiss (2×) L. were examined for content of pyruvate, orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and ribulose 1,5-bisphosphate carboxylase (RuBPC) by protein blot analyses using antibodies to maize leaf enzymes and by activity assays. In agreement with previous reports, the amount of RuBPC per mesophyll cell was about 3 times more in the hexaploid species, T. aestivum, than in the diploid species, T. boeoticum, both in first leaves and in flag leaves. In contrast, the level of PPDK polypeptide was nearly 3-fold higher per unit leaf area in the first leaf and 63% higher in the flag leaf of this diploid species compared to this hexaploid species. There was no significant difference in the levels of polypeptide and enzyme activity of PEPC between diploid and hexaploid wheat. Despite this significantly greater level of PPDK in the diploid species, the actual amount of PPDK could still supply only a limited amount of the enzyme activity necessary to provide phosphoenolpyruvate (PEP) for any putative intracellular C₄ carbon shuttle providing carbon to RuBPC. Thus, this difference in enzyme amount could not by itself account for the reported high rates of net photosynthesis at high light intensity in T. boeoticum. Together with reported anatomical differences between the diploid and hexaploid species, however, this biochemical difference may be of physiological importance.     

The role of inorganic phosphate in the regulation of C4 photosynthesis

Inorganic phosphate participates in many fundamental processes within the plant cell. Its broad influence on plant metabolism is related to such key operations as metabolite transport, enzyme regulation and carbohydrate metabolism in general. This review discusses these topics with special emphasis on the role assigned to this ubiquitous anion within the C₄ pathway of photosynthesis.Abbreviations DHAP dihydroxyacetone phosphate - Ga3P glyceraldehyde-3-phosphate - NAD(P)-ME-NAD(P) dependent malic enzyme - PEP phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - PFK and PFP-ATP- and PPi dependent fructose-6-phosphate 1-phosphotransferase - PPDK pyruvate:orthophosphate dikinase - RPPC reductive pentose-phosphate cycle - RuBisCO ribulose bisphosphate carboxylase-oxygenase - SPS sucrose-6-phosphate synthase     

Regulation of levels of nuclear transcripts for C4 photosynthesis in bundle sheath and mesophyll cells of maize leaves

In vitro translation of polyA⁺ mRNAs isolated from purified maize bundle sheath and mesophyll cells results in the production of distinctive, cell-specific polypeptides. Immunoprecipitation experiments show that translatable polyA⁺ mRNAs for phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK) and NADP-malate dehydrogenase (MDH) are prominent in mesophyll but not bundle sheath cells. On the contrary, those for sedoheptulose-1,7-bisphosphatase (SBP), fructose-1,6-bisphosphatase (FBP), NADP-malic enzyme (ME) and the small subunit of ribulose-1,5-bisphosphate carboxylase (RuBPC SS) are present only in bundle sheath cells. Moreover, polyA⁺ mRNAs encoding the 33 kD, 23 kD and 16 kD polypeptides of the oxygen-evolving complex (OE33, OE23 and OE16) and the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCP II) are much more abundant in mesophyll than in bundle sheath cells. Northern blot analyses with cDNA clones of PEPC, PPDK, ME, RuBPC SS, OE33, OE23, OE16 and LHCP II are consistent with the conclusion that the cell-specific expression of these genes is regulated at the RNA level. The RNA level differences are especially dramatic in dark-grown maize seedlings after illumination for 24 h.     

Enzymic activities of carbohydrate,purine, and pyrimidine metabolism in the Anaeroplasmataceae (class Mollicutes)

Cell-free extracts of two strictly anaerobic mollicutes, Anaeroplasma intermedium 5LA and Asteroleplasma anaerobium 161^T, were tested for enzymic activities of intracellular carbohydrate metabolism. Asteroleplasma anaerobium was also tested for enzymes of purine and pyrimidine metabolism. Both organisms had enzymic activities associated with the nonoxidative portion of the pentose phosphate pathway, and with the Embden-Meyerhoff-Parnas pathway. The 6-phosphofructokinase (PFK) of Asteroleplasma anaerobium was ATP-dependent, whereas the PFK of Anaeroplasma intermedium was PP_i-dependent. The two anaerobic mollicutes also differed with respect to the enzymes that converted phosphoenolpyruvate (PEP) to pyruvate; Anaeroplasma intermedium had pyruvate kinase activity, but Asteroleplasma anaerobium had pyruvate, orthophosphate dikinase activity (PP_i-dependent). Both organisms had lactate dehydrogenase activity which was activated by fructose 1,6-bisphosphate (Fru-1,6-P ₂). Anaeroplasma intermedium had activity for PEP carboxykinase (activated by Fru-1,6-P ₂), but Asteroleplasma anaerobium did not. PEP carboxytransphosphorylase activity was not detected in either organism. Anaeroplasma intermedium had malate dehydrogenase and isocitrate dehydrogenase activities, but it had no activities for the three other tricarboxylic acid cycle enzymes examined; Asteroleplasma anaerobium had malate dehydrogenase activity only. Asteroleplasma anaerobium had enzymic activities for the interconversion of purine nucleobases, (deoxy)ribonucleosides, and (deoxy)ribomononucleotides, including PP_i-dependent nucleoside kinase, reported heretofore only in some other mollicutes. Asteroleplasma anaerobium could synthesize dTDP by the thymine salvage pathway if deoxyribose 1-phosphate was provided, and it had dUTPase, ATPase, and dCMP kinase activities. It lacked (deoxy)cytidine deaminase, dCMP deaminase, and deoxycytidine kinase activities.Abbreviations EMP Embden-Meyerhof-Parnas - ICDH isocitrate dehydrogenase - LDH lactate dehydrogenase - PEP phosphoenolpyruvate - PFK phosphofructokinase - PPDK pyruvate, orthophosphate dikinase - TCA cycle tricarboxylic acid cycle Note: Other abbreviations used are as per the instruction to authors, or the reference cited therein (Eur J Biochem 1:259), or Biochem J 120:449 (which supercedes a portion of the first reference)