Properties of phosphoenolpyruvate carboxylase in rapidly prepared,desalted leaf extracts of the Crassulacean acid metabolism plant Mesembryanthemum crystallinum L.

Properties of phosphoenolpyruvate (PEP) carboxylase, obtained from leaves of Mesembryanthemum crystallinum L. performing Crassulacean acid metabolism (CAM), were determined at frequent time points during a 12-h light/12-h dark cycle. Leaf extracts were rapidly desalted and PEP carboxylase activity as a function of PEP concentration, malate concentration, and pH was measured within 2 min after homogenization of the tissue. Maximum velocity of PEP carboxylase was similar in the light and dark at pH 7.5 and pH 8.0. However, PEP carboxylase had as much as a 12-fold lower K _m for PEP and as much as a 20-fold higher K _i for malate during the dark than during the light periods, the magnitude of these differences being dependent on the assay pH. Assuming that enzyme properties immediately after isolation reflect the approximate state of the enzyme in vivo, these differences in enzyme properties reduce the potential for CO₂ fixation via PEP carboxylase in the light. A small decrease in cytoplasmic pH in the light would greatly magnify the above differences in day/night properties of PEP carboxylase, because the sensitivity of PEP carboxylase to inhibition by malate increased with decreasing pH. Properties of PEP carboxylase were also studied in plants exposed to short-term perturbations of the normal 12-h light/12-h dark cycle (e.g., prolonged light period, prolonged dark period). Under all light/dark regimes, there was a close correlation between change in properties of PEP carboxylase and changes of the tissue from acidification to deacidification, and vice versa. Changes in properties of PEP carboxylase were not merely light/dark phenomena because they were also observed in plants exposed to continuous light or dark. the data indicate that, during CAM, PEP carboxylase exists in two stages which differ in their capacity for net malate synthesis. The physiologically-active state is distinguished by a low K _m for PEP and a high K _i for malate and favors malate synthesis. The physiologically-inactive state has a high K _m for PEP and a low K _i for malate and exists during periods of deacidification and other periods lacking synthesis of malic acid.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPC PEP carboxylase - RuBP ribulose 1,5-bisphosphate - RH relative humidity     

Temperature effects on malic-acid efflux from the vacuoles and on the carboxylation pathways in crassulacean-acid-metabolism plants

The studies described in the paper were conducted with tissue slices of Crassulacean acid metabolism (CAM) plants floating in isotonic buffer. In a first series of experiments, temperature effects on the efflux of [¹⁴C]malate and¹⁴CO₂ were studied. An increase of temperature increased the efflux from the tissue in a non-linear manner. The efflux was markedly influenced also by the temperatures applied during the pretreatment. The rates of label export in response to the temperature and the relative contributions of¹⁴CO₂ and [¹⁴C]malate to the label export were different in the two studied CAM plants (Kalanchoë daigremontiana, Sempervivum montanum). In further experiments, temperature response of the labelling patterns produced by¹⁴CO₂ fixation and light and darkness were studied. In tissue which had accumulated malate (acidified state) an increase of temperature decreased the rates of dark CO₂ fixation whilst the rates of CO₂ fixation in light remained largely unaffected. An increase of temperature shifted the labelling patterns from a C₄-type (malate being the mainly labelled compound) into a C₃-type (label in carbohydrates). No such shift in the labelling patterns could be observed in the tissue which had depleted the previously stored malate (deacidified state). The results indicate that in the acidified tissue the increase of temperature increases the efflux of malate from the vacuole by changing the properties of the tonoplast. It is assumed that the increased export of malic acid lowers the in-vivo activity of phosphoenol pyruvate carboxylase by feedback inhibition.Abbreviations CAM Crassulacean acid metabolism - FW fresh weight - PEPCase phosphoenolpyruvate carboxylase Dedicated to Professor O.L. Lange, Würzburg, on the occasion of his 60th birthday     

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     

Characterization of carbon metabolism in Opuntia ficus-indica Mill. exhibiting the idling mode of Crassulacean acid metabolism

Upon transfer from well-watered conditions to total drought, long-day-grown cladodes of Opuntia ficus-indica Mill. shift from full Crassulacean acid metabolism (CAM) to CAM-idling. Experiments using ¹⁴C-tracers were conducted in order to characterize the carbon-flow pattern in cladodes under both physiological situations. Tracer was applied by ¹⁴CO₂ fumigations and NaH¹⁴CO₃ injections during the day-night cycle. The results showed that behind the closed stomata, mesophyll cells of CAM-idling plants retained their full capacity to metabolize CO₂ in light and in darkness. Upon the induction of CAM-idling the level of the capacity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) was maintained. By contrast, malate pools decreased, displaying finally only a small or no day-night oscillation. The capacity of NADP-malic enzyme (EC 1.1.1.40) decreased in parallel with the reduction in malate pools. Differences in the labelling patterns, as influenced by the mode of tracer application, are discussed.Abbreviations CAM Crassulacean acid metabolism - PEP-Case phosphoenolpyruvate carboxylase     

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 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     

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     

CO2 and malate metabolism in starch-containing and starch-lacking guard-cell protoplasts

Isolated, purified mesophyll and guard-cell protoplasts of Vicia faba L. and Allium cepa L. were exposed to ¹⁴CO₂ in the light and in the dark. The guard-cell protoplasts of Vicia and Allium did not show any labeling in phosphorylated products of the Calvin cycle, thus appearing to lack the ability to reduce CO₂ photosynthetically. In Vicia, high amounts of radioactivity (35%) appeared in starch after 60-s pulses of ¹⁴CO₂ both in the light and in the dark. Presumably, the ¹⁴CO₂ is fixed into the malate via PEP carboxylase and then metabolized into starch as the final product of gluconeogenesis. This is supported by the fact that guard-cell protoplasts exposed to malic acid uniformly labeled with ¹⁴CO₂ showed high amounts of labeled starch after the incubation, whereas cells labeled with [4-¹⁴C]malate had minimal amounts of labeled starch (1/120).In contrast, the starch-deficient Allium, guard-cell protoplasts did not show any significant ¹⁴CO₂ fixation. However, adding PEP to an homogenate stimulated ¹⁴CO₂ uptake, thus supporting the interpretation that the presence of starch as a source of PEP is necessary for incorporating CO₂ and delivering malate. With starch-containing Vicia guard-cell protoplasts, the correlation between changes in volume and the interconversion of malate and starch was demonstrated. It was shown that the rapid gluconeogenic conversion of malate into starch prevents an increase of the volume of the protoplasts, whereas the degradation of starch to malate is accompanied by a swelling of the protoplasts.Abbreviations GCPs guard-cell protoplasts - MCPs mesophyll cell protoplasts - PEP phosphoenolpyruvate - DTT dithiothreitol - 3-PGA 3-phosphoglyceric acid - RiBP ribulose 1,5 bisphosphate - MDH malate dehydrogenase - MES 2-(N-morpholino)ethane sulfonic acid - CAM crassulacean acid metabolism     

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     

Carbon dioxide assimilation in some aerial plant organs and tissues

Summary CO₂ fixation characteristics of a number of mature (but not senescing) tissues and organs (the outer layers of green pod and the seed testa of Vicia faba L.; the outer layers of green pod and seeds of Trigonella foenum-graecum L.; the outer layers of the green fruit of Lycopersicon esculentum Mill.) were studied and compared with their respective C₃ leaf characteristics. On a chlorophyll basis phosphoenolpyruvate carboxylase, malic enzyme (NADP) and malate dehydrogenase (NAD and NADP) acitivites were much higher in the non-leaf tissues (except for V. faba seed testa) than the leaf tissues. Generally, on a protein basis the differences were less significant. All tissues possessed ribulose-1.5-diphosphate carboxylase activity though there was great variation in activities both on a protein and chlorophyll basis. Protein: chlorophyll ratios varied greatly from tissue to tissue being lowest in the leaf tissue (11.5–14.0) and highest in V. faba seed testa (805.5). Chlorophyll a:b ratios were all between 2 and 3. ¹⁴CO₂ uptake in the dark by L. esculentum fruit slices was about 1/3 that in the light and the major, initially labelled product was malate both in the light and dark. Neither typical C₄-photosynthesis or crassulacean acid metabolism were exhibited by the non-leaf tissues and it was considered that the increased levels of certain enzyme activities were present to refix and recycle respired CO₂.Abbreviations PEP phosphoenolpyruvate - RuDP ribulose -1,5-, diphosphate - MDH malate dehydrogenase - CAM Crassulacean acid metabolism - OAA oxaloacetic acid     

Studies on carbon flow in Crassulacean acid metabolism during the initial light period

In the Crassulacean acid metabolism (CAM) plants Kalanchoë tubiflora and Sedum morganianum a shift in the pathways occurs by which external CO₂ enters the metabolism during the initial light period (phase II of the diurnal CAM cycle). At the beginning of phase II, CO₂ is fixed mainly by the C₄ pathway; during late phase II, however, it is fixed mainly via the C₃ pathway. The C₃ pathway contributes to the phosphoenolpyruvate-carboxylase-mediated CO₂ fixation by the provision of three-carbon skeletons. Since the shift in the carbon-flow pathway is delayed after a CO₂-free night when malic-acid accumulation in the vacuoles is prevented, it is very likely that the amount of malic acid in the vacuole is integrated in the mechanism which controls CAM during the initial light period. A light-on signal at the beginning of phase II is not required to bring about the shifts in the carbon-flow pathways, as is shown by the reaction of plants to a prolonged dark period. A model of carbon flow during phase II is proposed.Abbreviations CAM Crassulacean acid metabolism - PEP-Case phosphoenolpyruvate carboxylase     

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.     

Enzymes of nitrogen assimilation in maize roots

The intracellular distribution of enzymes involved in the Crassulacean acid metabolism (CAM) has been studied in Bryophyllum calycinum Salisb. and Crassula lycopodioides Lam. After separation of cell organelles by isopycnic centrifugation, enzymes of the Crassulacean acid metabolism were found in the following cell fractions: Phosphoenolpyruvate carboxylase in the chloroplasts; NAD-dependent malate dehydrogenase in the mitochondria and in the supernatant; NADP-dependent malate dehydrogenase and phosphoenolpyruvate carboxykinase in the chloroplasts; NADP-dependent malic enzyme in the supernatant and to a minor extent in the chloroplasts; NAD-dependent malic enzyme in the supernatant and to some degree in the mitochondria; and pyruvate; orthophosphate dikinase in the chloroplasts. The activity of the NAD-dependent malate dehydrogenase was due to three isoenzymes separated by (NH₄)₂SO₄ gradient solubilization. These isoenzymes represented 17, 78, and 5% of the activity recovered, respectively, in the order of elution. The isoenzyme eluting first was associated with the mitochondria and the second isoenzyme was of cytosolic origin, while the intracellular location of the third isoenzyme was probably the peroxisome. Based on these findings, the metabolic path of Crassulacean acid metabolism within cells of CAM plants is discussed. New address: Institut für Pflanzenphysiologie und Zellbiologie, Freie Universität Berlin, Königin-Luise-Straße 12-16a. D-1000 Berlin 33     

Activity and quantity of ribulose bisphosphate carboxylase-and phosphoenolpyruvate carboxylase-protein in two Crassulacean acid metabolism plants in relation to leaf age,nitrogen nutrition,and point in time during a day/night cycle

Activity of ribulose 1,5-bisphosphate (RuBP) carboxylase in leaf extracts of the constitutive Crassulacean acid metabolism (CAM) plant Kalanchoe pinnata (Lam.) Pers. decreased with increasing leaf age, whereas the activity of phosphoenolpyruvate (PEP) carboxylase increased. Changes in enzyme activities were associated with changes in the amount of enzyme proteins as determined by immunochemical analysis, sucrose density gradient centrifugation, and SDS gel electrophoresis of leaf extracts. Young developing leaves of plants which received high amounts of NO ₃ ^- during growth contained about 30% of the total soluble protein in the form of RuBP carboxylase; this value declined to about 17% in mature leaves. The level of PEP carboxylase in young leaves of plants at high NO ₃ ^- was an estimated 1% of the total soluble protein and increased to approximately 10% in mature leaves, which showed maximum capacity for dark CO₂ fixation. The growth of plants at low levels of NO ₃ ^- decreased the content of soluble protein per unit leaf area as well as the extractable activity and the percentage contribution of both RUBP carboxylase and PEP carboxylase to total soluble leaf protein. There was no definite change in the ratio of RuBP carboxylase to PEP carboxylase activity with a varying supply of NO ₃ ^- during growth. It has been suggested (e.g., Planta 144, 143–151, 1978) that a rhythmic pattern of synthesis and degradation of PEP carboxylase protein is involved in the regulation of -carboxylation during a day/night cycle in CAM. No such changes in the quantity of PEP carboxylase protein were observed in the leaves of Kalanchoe pinnata (Lam.) Pers. or in the leaves of the inducible CAM plant Mesembryanthemum crystallinum L.Abbreviations CAM Crassulacean acid metabolism - RuBP ribulose 1,5-bisphosphate - PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate     

Photosynthetic carbon metabolism during leaf ontogeny in Zea mays L.: Enzyme studies

The activities of several enzymes, including ribulose-1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured as a function of leaf age in Z. mays. Mature leaf tissue had a RuDP-carboxylase activity of 296.7 mol CO₂ g^-1 fresh weight h^-1 and a PEP-carboxylase activity of 660.6 mol CO₂ g^-1 fresh weight h^-1. In young corn leaves the activity of the two enzymes was 11 and 29%, respectively, of the mature leaves. In senescent leaf tissue, RuDP carboxylase activity declined more rapidly than that of any of the other enzymes assayed. On a relative basis the activities of NADP malic enzyme (EC 1.1.1.40), aspartate (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2), and NAD malate dehydrogenase (EC 1.1.1.37) exceeded those of both PEP and RuDP carboxylase in young and senescent leaf tissue. Pulse-chase labeling experiments with mature and senescent leaf tissue show that the predominant C₄ acid differs between the two leaf ages. Labeling of alanine in senescent tissue never exceeded 4% of the total ¹⁴C remaining during the chase period, while in mature leaf tissue alanine accounted for 20% of the total after 60 s in ¹²CO₂. The activity of RuDP carboxylase during leaf ontogeny in Z. mays parallels the development of the activity of this enzyme in C₃ plants.Abbreviations RuDP ribulose-1,5-diphosphate - PEP phosphoenol pyruvate - PGA 3-phosphoglycerate     

Structural and metabolic properties of green tissue cultures from a CAM plant, Kalanchoë blossfeldiana hybr. Montezuma

Abstract Crassulacean acid metabolism (CAM) was studied in mixotrophic callus tissue cultures of Kalanchoë blossfeldiana hybr. Montezuma and compared with plants propagated from the calli. The ultrastructural properties of the green callus cells are similar to mesophyll cells of CAM plants except that occasionally abnormal mitochondria were observed. There was permanent net CO₂ output by the calli in light and darkness, which was lower in darkness than in light. The calli exhibited a diurnal rhythm of malic acid, with accumulation during the night and depletion during the day. ¹⁴C previously incorporated by dark CO₂ fixation into malate was transferred upon subsequent illumination into end products of photosynthesis. All these data indicate that CAM operates in the calli tissue. The results revealed that the capacity for CAM is obviously lower in the calli compared with plantlets developing from the calli, or with ‘adult’ plants. The data suggest also that CAM in the calli was not limited by the activities of CAM enzymes.     

Perturbations of malate accumulation and the endogenous rhythms of gas exchange in the Crassulacean acid metabolism plant<Emphasis Type="Italic"> Kalanchoë daigremontiana</Emphasis>: testing the tonoplast-as-oscillator model

In continuous light, leaves of the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier exhibit a circadian rhythm of CO₂ uptake, stomatal conductance and leaf-internal CO₂ pressure. According to a current quantitative model of CAM, the pacemaking mechanism involves periodic turgor-related tension and relaxation of the tonoplast, which determines the direction of the net flux of malate between the vacuole and the cytoplasm. Cytoplasmic malate, in turn, through its inhibitory effect on phosphoenolpyruvate carboxylase, controls the rate of CO₂ uptake. According to this mechanism, when the accumulation of malate is disrupted by removing CO₂ from the ambient air, the induction of a phase delay with respect to an unperturbed control plant is expected. First, using the mathematical model, such phase delays were observed in numerical simulations of three scenarios of CO₂ removal: (i) starting at a trough of CO₂ uptake, lasting for about half a cycle (ca. 12 h in vivo); (ii) with the identical starting phase, but lasting for 1.5 cycles (ca. 36 h); and (iii) starting while CO₂ increases, lasting for half a cycle again. Applying the same protocols to leaves of K. daigremontiana in vivo did not induce the predicted phase shifts, i.e. after the end of the CO₂ removal the perturbed rhythm adopted nearly the same phase as that of the control plant. Second, when leaves were exposed to a nitrogen atmosphere for three nights prior to onset of continuous light to prevent malate accumulation, a small, 4-h phase advance was observed instead of a delay, again contrary to the model-based expectations. Hence, vacuolar malic acid accumulation is ruled out as the central pacemaking process. This observation is in line with our earlier suggestion [T.P. Wyka, U. Lüttge (2003) J Exp Bot 54:1471–1479] that in extended continuous light, CO₂ uptake switches gradually from a CAM-like to a C3-like mechanism, with oscillations of the two CO₂ uptake systems being tightly coordinated. It appears that the circadian rhythm of gas exchange in this CAM plant emerges from one or several devices that are capable of generating temporal information in a robust manner, i.e. they are protected from even severe metabolic perturbations.Abbreviations CAM Crassulacean acid metabolism - c_ia Ratio of mesophyll CO₂ concentration to external CO₂ concentration - J_C Rate of carbon dioxide uptake - J_W Transpiration rate - g_W Stomatal conductance - LL Continuous light conditions - PEPC Phosphoenolpyruvate carboxylase - Rubisco d-Ribulose-1,5-bisphosphatecarboxylase/oxygenase - Effective quantum yield of photosystem II     

Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism

Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C₃-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO₂ fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP⁺-or a mitochondrial NAD⁺-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C₃-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD⁺-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme     

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     

Oxygen and carbon dioxide exchanges in crassulacean-acid-metabolism-plants

The 24 h O₂ uptake and release together with the CO₂ balance have been measured in two CAM plants, one a non-succulent Sempervivum grandifolium, the other a succulent Prenia sladeniana. The O₂ uptake was estimated by the use of ¹⁸O₂. It was found that the mean hourly O₂ uptake in the light was 7 times that in the dark for Sempervivum and 5 times that for Prenia, after correction for the lightdark temperature difference. It was estimated that oxygen uptake in the light was 2.4 times greater than oxygen release (=net photosynthesis) in Sempervivum and 1.4 times greater in Prenia. In both plants there was a positive carbon balance over the 24 h period under the experimental conditions. It was estimated that malate formed during the night could, if completely oxidized to CO₂ and water, account for 74% of the light phase O₂ uptake in Sempervivum. In Prenia the O₂ uptake was more than sufficient to account for a full oxidation of malate.Abbreviations CAM Crassulacean acid metabolism - PAR photosynthetically active radiation - PEP phosphoenolpyruvate - RrBP ribulose-1,5-bisphosphate - TCA tricarboxylic acid cycle