Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Mitochondria isolated from leaves of Mesembryanthemum crystallinumoxidized malate by both NAD malic enzyme and NAD malate dehydrogenase.Rates of malate oxidation were higher in mitochondria from plantsgrown at 400 mil NaCl in the rooting medium and performing Crassulaceanacid metabolism (CAM) than in mitochondria from plants grownat 20 mM NaCl and exhibiting C₃-photosynthetic CO₂ fixation.The mitochondria isolated from plants both in the CAM and C₃modes were tightly coupled and gave high respiratory control.At optimum pH for malate oxidation (pH 7.0), pyruvate was themajor product in mitochondria from CAM-M. crystallinum, whereasmitochondria from C₃-M. crystallinum produced predominantlyoxaloacetate. Both the extracted NAD malic enzyme in the presenceof CoA and the oxidation of malate to pyruvate by the mitochondriafrom plants in the CAM mode had a pH optimum around 7.0 withactivity declining markedly above this pH. The activity of NAD-malicenzyme, expressed on a cytochrome c oxidase activity basis,was much higher in mitochondria from the CAM mode than the C₃mode. The results indicate that mitochondria of this speciesare adapted to decarboxylate malate at high rates during CAM. ¹Current address: Lehrstuhl für Botanik II, UniversitätWurzburg, Mittlerer Dallenbergweg 64, 8700 Würzburg, WestGermany. ²Current address: KD 120, Chemical Research Division, OntarioHydro, 800 Kipling Avenue, Toronto, Ontario M8Z5S4, Canada. ³Current address: Department of Botany, Washington State University,Pullman, Washington 99164-4230, U.S.A. (Received March 13, 1986; Accepted September 18, 1986)     

Properties of Leaf NAD-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Species Mesembryanthemum crystallinum

NAD-malic enzyme (NAD-ME) functions to decarboxylate malatein the light in leaves of certain species displaying Crassulaceanacid metabolism (CAM). The properties of NAD-ME in desaltedextracts from the inducible CAM species, Mesembryanthemum crystallinumwere examined. The shapes of the malate saturation curve andthe activity versus pH curve at 10 mM malate were dependenton the presence of the activator CoA. The malate saturationcurve was sigmoidal in the absence of an activator and hyperbolicin the presence of CoA. The pH optimum with 10mM malate andMn²⁺ as cofactor was as low as 6.5 without an activator, andincreased to 7.2 in the presence of CoA. Fumarate activationwas synergistic with CoA above pH 7.2. The enzyme displayedhysteretic behavior under suboptimal assay conditions. Rapid extraction and desalting of the enzyme (<1.5 mim) followedimmediately by assay did not reveal any difference in the propertiesof the enzyme on a day/night basis. It is proposed that diurnalregulation of the enzyme in vivo is mediated by pH and malatelevel without a change in the oligomeric form of the enzyme.The molecular weight of the enzyme was approximately 350,000at pH 6.5 or 7.8. The enzyme obtained from M. crystallinum inthe C₃ mode was very similar to the CAM enzyme except that itdisplayed a lower V_max. ³ Current address: MSU-DOE Plant Research Lab, Michigan StateUniversity, E. Lansing, Michigan, U.S.A. 48824. (Received October 2, 1984; Accepted December 20, 1984)     

Changes in Crassulacean Acid Metabolism of Kalanchoe blossfeldiana by Different Nitrogen Sources

Kalanchoe blossfeldiana Poelln. cv. Hikan (a Crassulacean acidmetabolism (CAM) plant) was grown in pots containing soil for6 months and then cultured in nutrient solution containing 10mM nitrate or ammonium as a sole nitrogen source for 2 or 3months, under a long-day (16 h) condition. Plant growth was better in the nitrate medium. Leaves of thenitrate-grown plants showed greater diurnal fluctuations intitratable acidity and malate content than those of the ammonium-grownplants. The diurnal patterns in CO₂ exchange of nitrate-grownplants were basically similar for both groups, but the amountof net CO₂ uptake at night was twice as large in the nitrate-grownplants. The leaves of the nitrate-grown plants had 1.3 to 2.5times higher activities of phosphoenolpyruvate carboxylase (PEPC),phosphofructokinase (PFK) and NAD glycelaldehyde-3-phosphatedehydrogenase (G3PDH). These results indicate that K. blossfeldianagrown in nitrate medium showed more CAM activity than thosein ammonium medium. (Received August 13, 1987; Accepted February 22, 1988)     

Two Types of Channels Involved in the Malate Ion Transport across the Tonoplast of a Crassulacean Acid Metabolism Plant

Ion channels in tonoplast of leaf cells of a Crassulacean acid metabolism plant, Graptopetalum paraguayense, using the patch clamp technique were investigated. Results showed the existence of two types of channels involved in the malate ion transport across the tonoplast. One type corresponded to the slow-activating vacuolar-type (R Hedrich, E Neher [1987] Nature 329: 833-836), probably taking part in the malate efflux from vacuoles. Another showed the membrane potential-dependent channel current of malate flux over a wide range of cytoplasmic free Ca²⁺ concentration (10⁻⁸-10⁻⁵ molar), a property favoring the malate uptake. This type seems to be different from the fast-activating vacuolar-type.     

Ecophysiology of Crassulacean Acid Metabolism (CAM)

BACKGROUND AND SCOPE: Crassulacean Acid Metabolism (CAM) as an ecophysiological modification of photosynthetic carbon acquisition has been reviewed extensively before. Cell biology, enzymology and the flow of carbon along various pathways and through various cellular compartments have been well documented and discussed. The present attempt at reviewing CAM once again tries to use a different approach, considering a wide range of inputs, receivers and outputs. INPUT: Input is given by a network of environmental parameters. Six major ones, CO(2), H(2)O, light, temperature, nutrients and salinity, are considered in detail, which allows discussion of the effects of these factors, and combinations thereof, at the individual plant level ('physiological aut-ecology'). RECEIVERS: Receivers of the environmental cues are the plant types genotypes and phenotypes, the latter including morphotypes and physiotypes. CAM genotypes largely remain 'black boxes', and research endeavours of genomics, producing mutants and following molecular phylogeny, are just beginning. There is no special development of CAM morphotypes except for a strong tendency for leaf or stem succulence with large cells with big vacuoles and often, but not always, special water storage tissues. Various CAM physiotypes with differing degrees of CAM expression are well characterized. OUTPUT: Output is the shaping of habitats, ecosystems and communities by CAM. A number of systems are briefly surveyed, namely aquatic systems, deserts, salinas, savannas, restingas, various types of forests, inselbergs and paramós. CONCLUSIONS: While quantitative census data for CAM diversity and biomass are largely missing, intuition suggests that the larger CAM domains are those systems which are governed by a network of interacting stress factors requiring versatile responses and not systems where a single stress factor strongly prevails. CAM is noted to be a strategy for variable, flexible and plastic niche occupation rather than lush productivity. 'Physiological syn-ecology' reveals that phenotypic plasticity constitutes the ecophysiological advantage of CAM.     

Crassulacean Acid Metabolism and Crassulacean Acid Metabolism Modifications in Peperomia camptotricha

Peperomia camptotricha, a tropical epiphyte from Mexico, shows variable forms of Crassulacean acid metabolism (CAM). Young leaves exhibit CAM-cycling, while mature leaves show an intermediate type of metabolism, between CAM and CAM-cycling, having approximately the same amount of nighttime gas exchange as daytime. Metabolism of young leaves appears independent of daylength, but mature leaves have a tendency toward more CAM-like metabolism under short days (8 hours). Large differences in the physical appearance of plants were found between those grown under short daylengths and those grown under long daylengths (14 hours). Some anatomical differences were also detected in the leaves. Water stress caused a switch to CAM in young and mature leaves, and as water stress increased, they shifted to CAM-idling.     

Photosynthesis in Isolated Chloroplasts of the Crassulacean Acid Metabolism Plant Sedum praealtum

Intact chloroplasts were isolated from protoplasts of the Crassulacean acid metabolism plant Sedum praealtum D.C. Typical rates of CO₂ fixation or CO₂-dependent O₂ evolution ranged from 20 to 30 micromoles per milligram chlorophyll per hour and could be stimulated 30 to 50% by several Calvin cycle intermediates. The pH optimum for CO₂ fixation was 7.0 to 7.6 with considerable activity as low as pH 6.4. Low concentrations of orthophosphate (Pi) (optimum 0.4 millimolar) stimulated photosynthesis while high concentrations (5 millimolar) caused some inhibition. Both CO₂ fixation and CO₂-dependent O₂ evolution exhibited a relatively long lag phase (4 to 6 minutes) which remained constant between 0.4 to 5 millimolar Pi. The lag phase could be decreased by addition of dihydroxyacetone-phosphate or ribose 5-phosphate. Further results are presented which suggest these chloroplasts have a functional phosphate translocator.     

Day/Night Changes in the Sensitivity of Phosphoenolpyruvate Carboxylase to Malate during Crassulacean Acid Metabolism

Phosphoenolpyruvate carboxylase (PEPC) was extracted from Mesembryanthemum crystallinum L. performing Crassulacean acid metabolism, at frequent intervals during a 12-hour light/12-hour dark cycle. Inhibition of PEPC by malate was followed at pH 8.0 and 7.5, 1 minute after homogenization of leaves. PEPC was more sensitive to malate during the light than during the dark periods and inhibition by malate was more pronounced at pH 7.5 than 8.0. For example, PEPC was not or only slightly inhibited by 0.5 millimolar malate during the dark period at both pH values and the rates per milligram chlorophyll were about the same. During the light period, 0.5 millimolar malate resulted in a 20 to 30% reduction of PEPC activity at pH 8.0 and a 80 to 90% reduction at pH 7.5. These and other experiments, in which plants were kept in prolonged dark periods, indicate that the increase in sensitivity of PEPC to malate is correlated with the change from acidification to deacidification in the tissue. These interactions account for apparent changes in pH response of PEPC in crude extracts assayed at different times of the day/night cycle.     

Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

Diurnal changes in the regulatory properties of PEP-carboxylase in Crassulacean Acid Metabolism (CAM)

Abstract. The CAM plants Kalanchoe tubiflora and K. blossfeldiana were grown under photoperiodically controlled conditions (short days). In these plants, phos-phoenolpyruvate carboxylase capacity and the sensitivity of the enzyme to the effectors L-malate (inhibitor) and glucose-6-phosphate (activator) were measured throughout the diurnal CAM cycle. In K. tubiflora , enzyme capacity was higher if measured at pH 7.0 than at pH 8.0 and displayed a rhythmical behavior with highest values at the end of the light period. As reported earlier, in K. blossfeldiana PEP-C capacity was higher during the night. It was more pronounced when plants were kept in CO₂-free air during the dark period. In both plants, the sensitivity of the enzyme to the effectors showed very clear diurnal changes: inhibition by malate and activation by glucose-6-phosphate were strikingly higher during the day than during the night; the effect depended on PEP concentration. The changing activation of the enzyme by glucose-6-phos-phate reflects diurnal changes of the Km for PEP which was found to be higher during the day than during the night. Manipulations of malate accumulation by nocturnal application of CO₂-free air did not influence these effects. The results are discussed in context with the metabolic control of CAM.     

Occurrence of Inducible Crassulacean Acid Metabolism in Leaves of Talimum triangulare (Portulacaceae)

In this paper we report for the first time the occurrence ofan inducible weak CAM in leaves of Talinwn triangulare (Jacq.)Willd. This plant is a terrestrial perennial deciduous herbwith woody stems and succulent leaves which grows under fullexposure and in the shade in northern Venezuela. Plants grownin a greenhouse (‘sun’ plants) and a growth cabinet(‘shade’ plants) with daily irrigation showed CO₂uptake only during the daytime (maximum rate, 4?0 µmolm^–2 s^–1) and a small acid accumulation during thenight (6?0 µmol H⁺g^–1 FW). Twenty-four hours aftercessation of irrigation, no CO₂ exchange was observed duringpart of the night. Dark fixation reached a maximum (1?0 µmolCO₂ m^–2 s^–1, 100 µmol H⁺ g^–1 FW) onday 9 of drought. By day 30 almost no gas exchange was observed,while acid accumulation was still 10 µmol H⁺ g^–1FW. Rewatering reverted the pattern of CO₂ exchange to thatof a C₃ plant within 24 h. Daytime and night-time phosphoenolpyruvatecarboxylase activity increased up to 100% (shade) and 62% (sun)of control values after 10 and 15 d of drought, respectively.Light compensation point and saturating irradiance were similarin well-watered sun and shade plants, values being characteristicof sun plants. CAM seems to be important for the tolerance ofplants of this species to moderately prolonged (up to 2 months)periods of drought in conditions of full exposure as well asshade, and also for regaining high photosynthetic rates shortlyafter irrigation. Key words: Talinum triwigulare, inducible CAM, PEP-C activity, recycling     

Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on Thioredoxin-Linked Enzymes of KalanchoE daigremontiana

Fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) were identified and purified from the Crassulacean acid metabolism (CAM) plant, Kalanchoë daigremontiana. FBPase and SBPase showed respective molecular weights of 180,000 and 76,000, and exhibited immunological cross-reactivity with their counterparts from chloroplasts of C₃ (spinach) and C₄ (corn) plants. Based on Western blot analysis, FBPase was composed of four identical 45,000-dalton subunits and SBPase of two identical 38,000-dalton subunits. Immunological evidence, together with physical properties, indicated that both enzymes were of chloroplast origin.

Kalanchoë FBPase and SBPase could be activated by thioredoxin f reduced chemically by dithiothreitol or photochemically by a reconstituted Kalanchoë ferredoxin/thioredoxin system. Both enzymes were activated synergistically by reduced thioredoxin f and thier respective substrates.

Kalanchoë FBPase could be partially activated by Mg²⁺ at concentrations greater than 10 millimolar; however, such activation was considerably less than that observed in the presence of reduced thioredoxin and Ca²⁺, especially in the pH range between 7.8 and 8.3. In contrast to FBPase, Kalanchoë SBPase exhibited an absolute requirement for a dithiol such as reduced thioredoxin irrespective of Mg²⁺ concentration. However, like FBPase, increased Mg²⁺ concentrations enhanced the thioredoxin-linked activation of this enzyme.

In conjunction with these studies, an NADP-linked malate dehydrogenase (NADP-MDH) was identified in cell-free preparations of Kalanchoë leaves which required reduced thioredoxin m for activity.

These results indicate that Kalanchoë FBPase, SBPase, and NADP-MDH share physical and regulatory properties with their equivalents in C₃ and C₄ plants. In contrast to previous evidence, all three enzymes appear to have the capacity to be photoregulated in chloroplasts of CAM plants, thereby providing a means for the functional segregation of glucan synthesis and degradation.

     

Crassulacean Acid Metabolism in Three Species of Commelinaceae

Diurnal patterns of CO₂ exchange and fluctuations of tissuemalic acid concentrations were investigated in three speciesof Commelinaceae: Callisia fragrans and Tripogandra multiflorafrom Jamaica, and Tradescantia brevifolia from southern Texas.Very low levels of CAM gas exchange were induced by droughtstress in C. fragrans and T. multiflora. In addition, past indicationsof CAM-cycling in the two Jamaican species were confirmed indrought-stressed plants; however, only C. fragrans exhibitedCAM-cycling under well-watered conditions. CAM-cycling underdrought stress was also found in T. brevifolia. This constitutesthe first report of CAM (sensu lato) in the genus Tradescantia.The importance of low-level CAM in these three species is discussedas a potential adaptation to drought.Copyright 1994, 1999 AcademicPress Callisia fragrans, Tradescantia brevifolia, Tripogandra multiflora, Commelinaceae, CO₂ exchange, Crassulacean acid metabolism, CAM-cycling, CAM-idling, drought stress, malic acid fluctuations     

Gas Exchange Characteristics of the Submerged Aquatic Crassulacean Acid Metabolism Plant, Isoetes howellii

The submerged aquatic plant Isoetes howellii Engelmann possesses Crassulacean acid metabolism (CAM) comparable to that known from terrestrial CAM plants. Infrared gas analysis of submerged leaves showed Isoetes was capable of net CO₂ uptake in both light and dark. CO₂ uptake rates were a function of CO₂ levels in the medium. At 2,500 microliters CO₂ per liter (gas phase, equivalent to 1.79 milligrams per liter aqueous phase), Isoetes leaves showed continuous uptake in both the light and dark. At this CO₂ level, photosynthetic rates were light saturated at about 10% full sunlight and were about 3-fold greater than dark CO₂ uptake rates. In the dark, CO₂ uptake rates were also a function of length of time in the night period. Measurements of dark CO₂ uptake showed that, at both 2,500 and 500 microliters CO₂ per liter, rates declined during the night period. At the higher CO₂ level, dark CO₂ uptake rates at 0600 h were 75% less than at 1800 h. At 500 microliters CO₂ per liter, net CO₂ uptake in the dark at 1800 h was replaced by net CO₂ evolution in the dark at 0600 h. At both CO₂ levels, the overnight decline in net CO₂ uptake was marked by periodic bursts of accelerated CO₂ uptake. CO₂ uptake in the light was similar at 1% and 21% O₂, and this held for leaves intact as well as leaves split longitudinally. Estimating the contribution of light versus dark CO₂ uptake to the total carbon gain is complicated by the diurnal flux in CO₂ availability under field conditions.     

Changes in Properties of Phosphoenolpyruvate Carboxylase with Induction of Crassulacean Acid Metabolism (CAM) in the C4 Plant Portulaca Oleracea

Aiming at understanding the odd case of CAM expression by a C₄ plant, some properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, orthophosphate: oxaloacetate carboxylyase, phosphorylating) were comparatively studied in leaves of CAM-expressing and non-expressing Portulaca oleracea L. plants. CAM expression was induced by growing plants under an 8-h photoperiod and under water-stress. CAM induction in leaves of these plants (designated as CAM) is indicated by the nocturnal acidification and by the clear diurnal oscillation pattern and amplitude of acidity, malic acid, and PEPC activity characteristic of CAM plants. Treatment of the other plant group (designated as C₄) by growth under a 16-h photoperiod and well-watered conditions did not induce expression of the tested criteria of CAM in plants. In these C₄ plants, the mentioned CAM criteria were undetectable. PEPC from CAM and C₄ Portulaca responded differently to any of the studied assay conditions or effectors. For example, extent and timing of sensitivity of PEPC to pH change, inhibition by malate, activation by glucose-6-phosphate or inorganic phosphate, and the enzyme affinity to the substrate PEP were reversed with induction of CAM from the C₄-P. oleracea. These contrasting responses indicate distinct kinetic and regulatory properties of PEPC of the two modes. Thus by shifting to CAM in the C₄ Portulaca a new PEPC isoform may be synthesised to meet CAM requirements. Simultaneous occurrence of both C₄ and CAM is suggested in P. oleracea when challenged with growth under stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on the Ferredoxin/Thioredoxin System of KalanchoE daigremontiana

Cell-free preparations of the Crassulacean acid metabolism (CAM) plant, Kalanchoë daigremontiana, were analyzed for thioredoxins and ferredoxin-thioredoxin reductase. Three distinct forms of thioredoxin were identified in Kalanchoë leaves, two of which specifically activated fructose 1,6-bisphosphatase (designated f₁ and f₂) and a third which activated NADP-malate dehydrogenase (thioredoxin m). The apparent molecular weight of both forms of thioredoxin f was 11,000 and that of thioredoxin m was 10,000. In parallel studies, ferredoxin and ferredoxin-thioredoxin reductase were purified from Kalanchoë leaf preparations. Kalanchoë ferredoxin-thioredoxin reductase was similar to that of C₃ and C₄ plants in molecular weight (31,000) and immunological cross-reactivity. Kalanchoë ferredoxin-thioredoxin reductase exhibited an affinity for ferredoxin as demonstrated by its binding to an immobilized ferredoxin affinity column. The purified components of the Kalanchoë ferredoxin-thioredoxin system could be recombined to function in the photoregulation of chloroplast enzymes. The data suggest that the ferredoxin/thioredoxin system plays a role in enzyme regulation of all higher plants irrespective of whether they show C₃, C₄, or CAM photosynthesis.