Photosynthesis in enzymatically isolated leaf cells from the CAM plant Sedum telephium L.

A technique has been developed for the enzymatic isolation of leaf cells from the Crassulacean acid-metabolism plant Sedum telephium. The cells exhibited high activity in both ¹⁴CO₂ incorporation (30–70 mol CO₂ mg^-1 chlorophyll h^-1) and O₂ evolution in the presence of bicarbonate (60–110 mol O₂ mg^-1 chlorophyll h^-1). Half-maximum saturation of ¹⁴CO₂ incorporation occurred at a bicarbonate concentration of ca. 2 mM (20 M CO₂) at pH 8.4 and 30°C. Two types of light-dependent O₂ evolution are reported: O₂ evolution in the absence of exogenously supplied bicarbonate (endogenous O₂ evolution), and bicarbonate-stimulated O₂ evolution. Oxygen evolution in the presence of approximately ambient concentrations of CO₂ appeared to be a combination of the endogenous O₂ evolution and O₂ evolution from fixation of the exogenously supplied CO₂.Abbreviations CAM Crassulacean acid metabolism - cirlo chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - RuDP ribulose-1,5-diphosphate     

Anti-inflammatory and antioxidant activity of a medicinal tincture from Pedilanthus tithymaloides

Pedilanthus tithymaloides (L.) Poit. (Euphorbiaceae) is a low tropical American shrub with a reported wide range of healing properties such as emetic, anti-inflammatory, antibiotic, antiseptic, antihemorrhagic, antiviral, antitumoral, and abortive. In the present study, a tincture from P. tithymaloides collected in Cuba was evaluated for its in vivo anti-inflammatory activity, using the rat paw oedema assay, and for its in vitro scavenging effects on reactive oxygen species (ROS) (HO*, O2*-, HOCl, ROO* and H2O2), reactive nitrogen species (RNS) (ONOO- and *NO), and DPPH* radical. The protein, free amino acid, and phenolic contents of the tincture were also determined. Pertaining to the anti-inflammatory activity, the intraperitoneal administration of the tincture inhibited carrageenan-induced rat paw oedema, whereas in the scavenging assays the tincture showed to be effective against all the assayed ROS and RNS, specially for HO* (IC50 = 345+/-77 microg/mL), O2*- (IC50 = 143+/-7 microg/mL), HOCl (IC50 = 113+/-20 microg/mL), ONOO- (IC50 = 44+/-3 microg/mL), and *NO (IC50 = 54+/-4 microg/mL), but displayed weak activity in the DPPH* assay. The protein content of the tincture was 0.70%, and twenty free amino acids were identified and quantified. The content of total phenolics was 17.4+/-0.15 mg of gallic acid equivalents (GAE)/g dry material. These results provide scientific support for the empirical use of P. tithymaloides tincture as an anti-inflammatory medicine.     

Na+/H+-transporter, H+-pumps and an aquaporin in light and heavy tonoplast membranes from organic acid and NaCl accumulating vacuoles of the annual facultative CAM plant and halophyte Mesembryanthemum crystallinum L.

Crassulacean acid metabolism (CAM) was induced in Mesembryanthemum crystallinum L. by either NaCl- or high light (HL)- stress. This generated in mesophyll cells predominantly of NaCl-stressed plants two different types of vacuoles: the generic acidic vacuoles for malic acid accumulation and additionally less acidic (“neutral”) vacuoles for NaCl sequestration. To examine differences in the tonoplast properties of the two types of vacuoles, we separated microsomal membranes of HL- and NaCl-stressed M. crystallinum plants by centrifugation in sucrose density gradients. Positive immunoreactions of a set of antibodies directed against tonoplast specific proteins and tonoplast specific ATP- and PP_i-hydrolytic activity were used as markers for vacuolar membranes. With these criteria tonoplast membranes were detected in both HL- and NaCl-stressed plants in association with the characteristic low sucrose density but also at an unusual high sucrose density. In HL-stressed plants most of the ATP- and PP_i-hydrolytic activity and cross reactivity with antibodies including that directed against the Na⁺/H⁺-antiporter from Arabidopsis thaliana was detected with light sucrose density. This relationship was inverted in NaCl-stressed plants; they exhibited most pump activity and immunoreactivity in the heavy fraction. The relative abundance of the heavy membrane fraction reflects the relative occurrence of “neutral” vacuoles in either HL- or NaCl-stressed plants. This suggests that tonoplasts of the “neutral” vacuoles sediment at high sucrose densities. This is consistent with the view that this type of vacuoles serves for Na⁺ sequestration and is accordingly equipped with a high capacity of proton pumping and Na⁺ uptake via the Na⁺/H⁺-antiporter.     

Photosynthetic changes and oxidative stress caused by iron ore dust deposition in the tropical CAM tree Clusia hilariana

The effect of iron solid particulate matter (SPM_Fe) deposited onto soil and leaves on photosynthesis and oxidative stress was evaluated in Clusia hilariana, a CAM tropical tree of high occurrence in Brazilian restingas. Significant increases in iron content were found in plants exposed to SPM_Fe applied onto leaf and soil surfaces. However, only the application of SPM_Fe on leaves of C. hilariana caused significant reductions in some evaluated characteristics such as photosynthetic rate, stomatal conductance, transpiration, organic acid accumulation, potential quantum yield of PSII, and changes in daily CAM photosynthesis pattern. Increase in relative membrane permeability and reduction in catalase and superoxide dismutase activities in the leaves of plants exposed to SPM_Fe also were observed; however, lipid peroxidation did not change. These responses seem to be due to the combination of physical effects such as increase of leaf temperature, reduction in light absorption, obstruction of stomatal pores, and biochemical effects triggered by oxidative stress.     

Expression of crassulacean acid metabolism in <Emphasis Type="Italic">Clusia hilariana</Emphasis> Schlechtendal in different stages of development in the field

Expression of crassulacean acid metabolism (CAM) in the obligate CAM-tree Clusia hilariana SCHLTDL. was studied in the restinga of Jurubatiba National Park, on the Atlantic coast of Rio de Janeiro state, Brazil, comparing plants at different developmental stages. Between young and mature plants there were trends of differences in six parameters, which are all related to CAM expression. From young to mature plants there were tendencies for a decrease of (1) the degree of succulence, (2) the degree of day/night changes of malic acid levels, (3) titratable acidity with nocturnal acid accumulation, (4) the degree of day/night changes of free hexoses with nocturnal break down, (5) effective quantum use efficiency of photosystem II at high photosynthetic photon flux density, and (6) protection from photoinhibition. These tendencies form a clear pattern which suggests that CAM was somewhat more pronounced in leaves of young plants than in leaves of mature plants. A developmental regulation may be involved. However, the observations are probably best explained by stress, since in the dry soils of the restinga young plants have no access to the ground water table while adult trees develop extensive root systems.     

<Emphasis Type="Italic">δ</Emphasis><Superscript>13</Superscript>C values and crassulacean acid metabolism in <Emphasis Type="Italic">Clusia</Emphasis> species from Panama

The genus Clusia is notable in that it contains arborescent crassulacean acid metabolism (CAM) plants. As part of a study of CAM in Clusia, titratable acidities were measured in 25 species and ¹³C values were measured for 38 species from Panamá, including seven undescribed species, and 11 species from Colombia, Costa Rica and Honduras. CAM was detected in 12 species. Clusia flava, C. rosea and C. uvitana exhibited ¹³C values or diurnal fluctuations in acidity indicative of strong CAM. In C. croatii, C. cylindrica, C. fructiangusta, C. lineata, C. odorata, C. pratensis, C. quadrangula, C. valerioi and C. sp. D diurnal fluctuations in acidity were consistent with weak CAM but the ¹³C values were C₃-like. All of the species that exhibited strong or weak CAM were in the C. flava or C. minor species groups. CAM was not detected in any member of the C. multiflora species group. Strong CAM species were not collected at altitudes above 680 m a.s.l. On the basis of ¹³C values, the expression of CAM was similar in terrestrial, hemi-epiphytic and epiphytic species and did not differ between individuals of the same species that exhibited different life-forms. This study indicates that phylogenetic affiliation may be a predictor of an ability to exhibit CAM in Clusia species from the Panamanian region, and that weak CAM is probably a common photosynthetic option in many Clusia species. ¹³C value is not a particularly good indicator of a potential of Clusia species growing in the field to exhibit CAM because it appears that the contribution in most species of CAM to carbon gain is generally rather small when integrated over the life-time of leaves.     

Expression of P-Enolpyruvate Carboxylase and other Aspects of CAM during the Development of Peperomia camptotricha Leaves

During the development of Peperomia camptotricha leaves, metabolism changes from C₃-photosynthesis to Crassulacean acid metabolism (CAM). The youngest leaves showed no diurnal fluctuation of organic acids or P-enolpyruvate carboxylase (PEPc) activity. There was little evidence for PEPc protein using PEPc antibodies prepared from the CAM form of PEPc, nor was there evidence for PEPc mRNA when tested using a cDNA probe made from CAM P. scandens. As leaves matured, there was a parallel increase in titratable acidity, PEPc activity, PEPc protein, and PEPc mRNA. In leaf whorls 1 through 6, there was a significant linear correlation between the diurnal fluctuation of organic acids and PEPc activity indicating a functional relationship. The specific activity of PEPc increased as leaves matured and the K_m (PEP) decreased indicating that the enzyme was becoming more active. The ratio of PEPc protein to PEPc mRNA decreased as leaves matured. During the expression of CAM, the spongy mesophyll where most of the CAM activity occurs increased in thickness and per cent air space, whereas the palisade mesophyll where most of the C₃ activity occurs did not increase in size dramatically. The diurnal fluctuation of organic acids and the expression of PEPc activity, protein, and mRNA increased as the thickness of the spongy mesophyll increased. During the expression of CAM in Peperomia camptotricha, there appears to be coordinated expression of PEPc mRNA, protein, and activity, the commencement of diurnal organic acid fluctuation, and the development of the CAM-like spongy mesophyll. Thus the evidence suggests that CAM in this species is expressed during normal development and not in response to environmental signals.     

Photosynthetic flexibility and ecophysiological plasticity: questions and lessons from Clusia, the only CAM tree, in the neotropics

The discovery of crassulacean acid metabolism (CAM) in the trees of Clusia: arrival in the limelight of international research 8 II. Phylogeny 8 III. Photosynthetic physiotypes 10 IV. Metabolic flexibility: organic acid variations 12 V. The environmental control of photosynthetic flexibility 13 VI. Phenotypic plasticity: physiotypes and morphotypes 16 VII. Ecological amplitude and habitat impact 16 VIII. Conclusions and outlook 21 Acknowledgements 22 References 22 Summary It is the aim of this review to present a monographic survey of the neotropical genus Clusia on scaling levels from molecular phylogeny, metabolism, photosynthesis and autecological environmental responses to ecological amplitude and synecological habitat impact. Clusia is the only dicotyledonous genus with real trees performing crassulacean acid metabolism (CAM). By way of introduction, a brief historical reminiscence describes the discovery of CAM in Clusia and the consequent increase in interest in studying this particular genus of tropical shrubs and trees. The molecular phylogeny of CAM in the genus is compared with that in Kalancho? and the Bromeliaceae. At the level of metabolism and photosynthesis, the great plasticity of expression of photosynthetic physiotypes, i.e. (i) C(3) photosynthesis, (ii) CAM including CAM idling, (iii) CAM cycling and (iv) C(3)/CAM-intermediate behaviour, as well as metabolic flexibility in Clusia is illustrated. At the level of autecology, the factors water, irradiance and temperature, which control photosynthetic flexibility, are assessed. The phenotypic plasticity of physiotypes and morphotypes is described. At the level of synecology, the ecological amplitude of Clusia in the tropics and the relations to habitat are surveyed.     

Superoxide dismutase activity in callus from the C3-CAM intermediate plant Mesembryanthemum crystallinum

In light-grown callus obtained from M. crystallinum hypocotyls, three classes of superoxide dismutase (SOD): Mn-, Fe- and Cu/ZnSOD were identified. Callus cultured on a medium containing 0.4 M NaCl showed an increase in FeSOD activity on day 4 of the experiment. In contrast, Cu/ZnSOD activity was higher over 16 days of the experiment. Salinity stress induces oxidative stress mainly for the cytosolic SOD form (Cu/ZnSOD). After 16 days of callus culture on salt-containing medium, diurnal malate oscillations, and an increase in NADP-malic enzyme activity were noticed. These results strongly suggest that C₃-CAM transition can also be expressed at the cellular level. Therefore, callus tissue could be a useful model, similar to a whole plant, for investigation of mechanisms of stress responses in M. crystallinum.     

Photosynthesis but not CAM responded flexibly to changes in irradiance in Plectranthus marrubioides (Lamiaceae)

Well-watered plants of Plectranthus marrubioides Benth., a crassulacean acid metabolism (CAM) species naturally inhabiting sun exposed succulent places, were grown at photosynthetically active photon flux densities (PPFD) of either 150 (LL) or 300 (HL) μmol m-2 s-1 in a controlled environment. Photosynthesis of LL plants was saturated by irradiance of ca. 500 μmol m-2 s-1 while in HL plants saturation was not reached up to 1200 μmol m-2 s-1 and photosynthetic capacity was nearly 50 % higher than in the LL plants. However, maximum photon yield was 55 % lower and compensation irradiance was 25 % higher in LL plants. The former also had larger, more succulent leaves, i.e., they were morphologically more sun adapted. On the other hand, nocturnal accumulation of malic and citric acid, nighttime CO2 gain, and the low relative carbon recycling were independent of the prevailing PPFD. Furthermore, photosynthetic performance was flexibly and reversibly adjusted in HL plants after transfer to 600 or 150 μmol m-2 s-1 while nocturnal CO2 uptake was not influenced. Photosynthesis showed a high acclimation potential to high PPFD and patterns of gas exchange became more C3-like the higher the irradiance was, without a direct effect on CAM in P. marrubioides. This revised version was published online in June 2006 with corrections to the Cover Date.     

Antioxidative protection in the inducible CAM plant Sedum album L. following the imposition of severe water stress and recovery

The antioxidative protection during the C₃-CAM shift induced by water stress was investigated in the temperate succulent Sedum album L. The C₃-CAM shift was characterized in terms of CO₂ exchange, titratable acidity and phosphoenolpyruvate carboxylase activity. Well-watered plants displayed C₃-like patterns of gas exchange and exhibited a mild day-night acid fluctuation indicating that those plants were performing CAM-cycling metabolism. Imposed drought highly stimulated CAM cycling, decreasing the net CO₂ uptake during the day, eliminating net CO₂ efflux at night and stimulating tissue acid fluctuations. As water deficit developed, chlorophyll fluorescence measurements showed a decrease in the Fv/Fm ratio, indicating that photoinhibition could follow after severe drought. Protection might be performed by the increased activity of enzymes involved in the destruction of free radicals and oxidants, but their response depended on the water status of the plant. Ascorbate peroxidase and superoxide dismutase activities increased in plants subjected to mild stress but declined during severe water stress. Catalase activity, however, was quite stable under mild water stress and was clearly inhibited under severe water stress. At this stage, glutathione reductase and monodehydroascorbate reductase seemed to be very important in the protection against oxidants, both increasing considerably their activities under severe water stress. Even if recycling has been shown to alleviate photoinhibition, our results clearly demonstrate that antioxidative enzymes play an important role in the protection of plants from oxidants during the C₃-CAM shift induced by water stress.     

Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts,vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana

Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H⁺-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces.Abbveviations CAM Crassulacean acid metabolism - H⁺-ATPase proton-translocating ATPase     

CAM induction by photoperiodism in green callus cultures from a CAM plant

Abstract Green calli obtained from leaves of the CAM-inducible plant Kalanchoe blossfeldiana cv. Montezuma were grown either under long-day or short-day regimes in the Phytotron of Gif-sur-Yvette. Callus cells were found to be CAM inducible by the short-day treatment, according to levels and diurnal oscillations of malate pools and phosphor-enolpyruvate (PEP) carboxylase (EC.4.1.1.31) capacity. De novo synthesis of PEP carboxylase was shown to occur under the short-day regime. In spite of continuous net CO² output, CAM-like patterns of CO₂ exchange by calli were obtained under the short-day treatment. After 2 months under both photoperiodic conditions, spontaneous organogenesis in callus tissues gave rise to numerous shootlets which were determined as photoperiod dependent for CAM; the same was so for plants originating from these shootlets.     

A Comparative Study by δ13C-Analysis of Crassulacean Acid Metabolism (CAM) in Kalanchoë (Crassulaceae) Species of Africa and Madagascar

The carbon isotope ratios (δ¹³C values) of samples of Kalanchoë species collected in Africa were compared with previous data obtained with species from Madagascar. In contrast to the Malagasy species which cover the whole range of δ¹³C values from ?10 to ? 30%_o, indicating high inter- and intraspecific diversity of CAM performance, in the African species nearly all δ¹³C values were less negative than ?18%₀. Thus, in the African species the CAM behaviour is characterized by CO₂ uptake proceeding mainly during the night. The distribution of δ¹³C values among the species clearly mirrors the taxonomic groups and the three sections of the genus Kalanchoë sensu lato. The Kitchingia section comprises only groups having CAM with a high proportion of carbon acquisition by the C₃-pathway of photosynthesis. The same holds true for the first three groups of the Bryophyllum section, whereas in the following groups of the section CAM with CO₂ proceeding mainly during the night is common. The latter CAM mode is typical also for the majority of groups and species in the section Eukalanchoë. The African Kalanchoë species belong to the Eukalanchoë section, whereas in Madagascar all three sections are abundant. The data support the view that the centre of adaptive radiation of the genus is located in Madagascar. They also suggest that high CAM variability is abundant in the more primitive taxa of the genus, whereas the phylogenetically more derived taxa show a stereotype CAM with CO₂ uptake taking place only during the night.     

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     

Induction of a Crassulacean acid like metabolism in the C4 succulent plant,Portulaca oleracea L.: physiological and morphological changes are accompanied by specific modifications in phosphoenolpyruvate carboxylase

The induction of a Crassulacean acid like metabolism (CAM) was evidenced after 21–23 days of drought stress in the C₄ succulent plant Portulaca oleracea L. by changes in the CO₂ exchange pattern, in malic acid content and in titratable acidity during the day–night cycle. Light microscopy studies also revealed differences in the leaf structure after the drought treatment. Following the induction of the CAM-like metabolism, the regulatory properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the enzyme responsible for the diurnal fixation of CO₂ in C₄ plants but nocturnal in CAM plants, were studied. The enzyme from stressed plants showed different kinetic properties with respect to controls, notably its lack of cooperativity, higher sensitivity to L-malate inhibition, higher PEP affinity and lower enzyme content on a protein basis. In both conditions, PEPC's subunit mass was 110 kDa, although changes in the isoelectric point and electrophoretic mobility of the native enzyme were observed. In vivo phosphorylation and native isoelectrofocusing studies indicated variations in the phosphorylation status of the enzyme of samples collected during the night and day, which was clearly different for the control and stressed groups of plants. The results presented suggest that PEPC activity and regulation are modified upon drought stress treatment in a way that allows P. oleracea to perform a CAM-like metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.     

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