Ecophysiological Significance of CO(2)-Recycling via Crassulacean Acid Metabolism in Talinum calycinum Engelm. (Portulacaceae)

High levels of variability in gas exchange characteristics and degree of CAM-cycling were found in the same and different individuals of Talinum calycinum Engelm. collected from rock outcrops in Missouri. Differences in CO₂ assimilation were mostly correlated with differences in shoot conductance to CO₂ not shoot internal CO₂ concentration. As found previously, CAM acid fluctuations were evident in well-watered plants exhibiting C₃ gas exchange patterns (CAM-cycling) and also in drought-stressed plants with stomata closed, or nearly so, day and night (CAM-idling). Drought stress also resulted in rapid stomatal closure, conserving water during droughts. Maximal CO₂ uptake rates occurred below 35°C; higher temperatures induced decreases in CO₂ assimilation and conductance while shoot internal CO₂ concentrations remained similar. Plant water-use-efficiency was severely curtailed at temperatures above 30°C. Tissue acid fluctuations were the result of changes in malic acid concentrations. Calculations of the amount of water potentially conserved by CAM-cycling yielded values of approximately 5 to 44% of daytime water loss. Thus, CAM-cycling may be an important adaptation minimizing water loss by perennial succulents growing in shallow soil on rock outcrops.     

Characteristics of Crassulacean Acid Metabolism in the Succulent C(4) Dicot, Portulaca oleracea L

Crassulacean acid metabolism (CAM) was investigated in leaves and stems of the succulent C₄ dicot Portulaca oleracea L. Diurnal acid fluctuations, CO₂ gas exchange, and leaf resistance were monitored under various photoperiod and watering regimes. No CAM activity was seen in well watered plants grown under 16-hour days. Under 8-hour days, however, well watered plants showed a CAM-like pattern of acid fluctuation with amplitudes of 102 and 90 microequivalents per gram fresh weight for leaves and stems, respectively. Similar patterns were also observed in detached leaves and defoliated stems. Leaf resistance values indicated that stomata were open during part of the dark period, but night acidification most likely resulted from refixation of respiratory CO₂. In water-stressed plants maximum acid accumulations were reduced under both long and short photoperiods. At night, these plants showed short periods of net CO₂ uptake and stomatal opening which continued all night long during preliminary studies under natural environmental conditions. Greatest acid fluctuations, in P. oleracea, with amplitudes of 128 microequivalents per gram fresh weight, were observed in water-stressed plants which had been rewatered, especially when grown under short days. No net CO₂ uptake took place, but stomata remained open throughout the night under these conditions. These results indicate that under certain conditions, such as water stress or short photoperiods, P. oleracea is capable of developing an acid metabolism with many similarities to 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     

Crassulacean Acid Metabolism in the Succulent C(4) Dicot, Portulaca oleracea L Under Natural Environmental Conditions

Crassulacean acid metabolism (CAM) was examined under natural environmental conditions in the succulent C₄ dicot Portulaca oleracea L. Two groups of plants were monitored; one was watered daily (well watered), while the other received water once every 3 to 4 weeks to produce a ψ of −8 bars (drought stressed). Gas exchange, transpiration rate, and titratable acidity were measured for 24-hour periods during the growing season. CAM activity was greatest in drought-stressed plants during late August which had 13 hour days and day/night temperatures of 35/15°C. Under these conditions net CO₂ uptake occurred slowly throughout the night. Diurnal fluctuations of titratable acidity took place in both leaves and stems with amplitudes of 17 and 47 microequivalents per gram fresh weight, respectively. Transpiration data indicated greater opening of stomata during the night than the day. CAM was less pronounced in drought-stressed P. oleracea plants in July and September; neither dark CO₂ uptake nor positive carbon balance occurred during the July measurements. In contrast, well-watered plants appeared to rely on C₄ photosynthesis throughout the season, although some acid fluctuations occurred in stems of these plants during September.     

Salt Requirement for Crassulacean Acid Metabolism in the Annual Succulent, Mesembryanthemum crystallinum

In experiments with the facultative Crassulacean acid metabolism (CAM) species, Mesembryanthemum crystallinum, only plants which received high levels of inorganic salts fixed substantial amounts of CO₂ by the CAM pathway. Equivalent osmolarities of polyethylene glycol 6000 did not yield any CAM fixation. Plant water potential and turgor pressure had no detectable influence on the amount of CAM fixation. These observations rule out the possibility that the inorganic ions were acting as osmotic agents.     

Resistance Analysis of Nocturnal Carbon Dioxide Uptake by a Crassulacean Acid Metabolism Succulent, Agave deserti

Nocturnal CO₂ uptake by a Crassulacean acid metabolism succulent, Agave deserti Engelm. (Agavaceae), was measured so that the resistance properties of the mesophyll chlorenchyma cells and their CO₂ concentrations could be determined. Two equivalents of acidity were produced at night per mole of CO₂ taken up. The nocturnal CO₂ uptake became light-saturated at 3.5 mEinsteins cm⁻² of photosynthetically active radiation (400-700 nm) incident during the preceding day; at least 46 Einsteins were required per mole of CO₂ fixed. Variations in the daytime leaf temperature between 20 and 37 C had little effect on nocturnal CO₂ uptake. After the first few hours in the dark, the leaf liquid phase CO₂ resistance (r^liq_CO2) and the CO₂ concentration in the chlorenchyma cells (cⁱ_CO2) both increased, the latter usually reaching the ambient external CO₂ level at the end of the dark period. Increasing the leaf surface temperature above 15 C at night markedly increased the stomatal resistance, r^liq_CO2, and cⁱ_CO2.

The minimum r^liq_CO2 at night was about 1.6 seconds cm⁻¹. Based on the ratio of chlorenchyma surface area to total leaf surface area of 82, this r^liq_CO2 corresponded to a minimum cellular resistance of approximately 130 seconds cm⁻¹, comparable to values for mesophyll cells of C₃ plants. The contribution of the carboxylation reaction and/or other biochemical steps to r^liq_CO2 may increase appreciably as the nighttime temperature shifts a few degrees from the optimum or after a few hours in the dark, both of which caused large increases in r^liq_CO2. This necessitates a large internal leaf area for CO₂ diffusion into the chlorenchyma to support moderate nocturnal CO₂ uptake rates by these succulent leaves.

     

Isoforms of NADP-Malic Enzyme from Mesembryanthemum crystallinum L. That are Involved in C3 Photosynthesis and Crassulacean Acid Metabolism

Exposure of the facultative halophyte Mesembryanthemum crystallinumL. to salt stress induces a shift from C₃ photosynthesis toCrassulacean acid metabolism (CAM). During induction of CAM,the activity of NADP-malic enzyme (EC 1.1.1.40 [EC] ) increased asmuch as 12-fold in leaves, while the enzymatic activity in rootsfell to half of the original level. These changes in the activityof the enzyme corresponded to changes in levels of the enzymeprotein. NADP-malic enzymes extracted from leaves in the C₃and CAM modes could be distinguished by differences in electrophoreticmobility during electrophoresis on a non-denaturing polyacrylamidegel. NADP-malic enzyme extracted from roots in the C₃-mode andin the CAM mode migrated as fast as the enzyme extracted fromleaves in the CAM mode on the same gel. Although the patternof peptide fragments from NADP-malic enzyme from CAM-mode leaveswas similar to that from C₃-mode leaves, as indicated by peptidemapping, both immunoprecipitation and an enzyme-linked immunosorbentassay revealed some antigenic differences between the enzymesextracted from leaves in the C₃ and the CAM modes. These resultssuggest the existence of at least two isoforms of NADPmalicenzyme that differ in their levels of expression during inductionof CAM. (Received April 21, 1994; Accepted September 5, 1994)     

The Influence of Leaf "Windows" on Crassulacean Acid Metabolism in the South African Succulent Senecio Rowleyanus (Asteraceae)

Net CO₂ exchange rate (P_N) of shoots and diel fluctuations in titratable acidity of leaves of Senecio rowleyanus were measured to determine whether penetration of radiant energy through leaf “windows” (narrow, translucent strips on the leaf epidermis) resulted in increased CAM. Nocturnal P_N and nighttime increases in acidity were compared among plants with windows covered with reflective adhesive tape, transparent adhesive tape (to control for potential effects of the adhesive), and no tape. The windows did not significantly enhance the degree of CAM in S. rowleyanus. This revised version was published online in September 2006 with corrections to the Cover Date.     

The Relation between Photosynthesis, Respiration, and Crassulacean Acid Metabolism in Leaf Slices of Aloe arborescens Mill

Leaves and leaf slices from Aloe arborescens Mill. were used to study the interrelations between Crassulacean acid metabolism, photosynthesis, and respiration. Oxygen exchange of leaf slices was measured polarographically. It was found that the photosynthetic utilization of stored malic acid resulted in a net evolution of oxygen. This oxygen production, and the decrease in acid content of the leaf tissue, were completely inhibited by amytal, although the rate of respiratory oxygen uptake was hardly affected by the presence of this inhibitor of mitochondrial electron transport. Other poisons of respiration (cyanide) and of the tricarboxylic acid cycle (trifluoroacetate, 2-diethyl malonate) also were effective in preventing acid-dependent oxygen evolution. It is concluded that the mobilization of stored acids during light-dependent deacidification of the leaves depends on the operation of the tricarboxylic acid cycle and of the electron transport of the mitochondria.     

Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C(3) Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism

Mesembryanthemum crystallinum, a halophilic, inducible Crassulacean acid metabolism (CAM) species, was grown at NaCl concentrations of 20 and 400 millimolar in the rooting medium. Plants from the low salinity treatment showed exclusively C₃-photosynthetic net CO₂ fixation, whereas plants exposed to the high salinity level exhibited net CO₂ dark fixation involving CAM. Mesophyll protoplasts, isolated from both tissues, were gently ruptured, and the intracellular localization of enzymes was studied following differential centrifugation and Percoll density gradient centrifugation of protoplast extracts. Both centrifugation techniques resulted in the separation of intact chloroplasts, with up to 90% yield, from other organelles and the nonparticulate fraction of cells. Enzymes were identified by determination of activity and by sodium dodecyl sulfate gel electrophoresis of enzyme protein.     

Correlation between CAM-Cycling and Photosynthetic Gas Exchange in Five Species of Talinum (Portulacaceae)

Photosynthetic gas exchange and malic acid fluctuations were monitored in 69 well-watered plants from five morphologically similar species of Talinum in an investigation of the ecophysiological significance of the Crassulacean acid metabolism (CAM)-cycling mode of photosynthesis. Unlike CAM, atmospheric CO₂ uptake in CAM-cycling occurs exclusively during the day; at night, the stomata are closed and respiratory CO₂ is recaptured to form malic acid. All species showed similar patterns of day-night gas exchange and overnight malic acid accumulation, confirming the presence of CAM-cycling. Species averages for gas exchange parameters and malic acid fluctuation were significantly different such that the species with the highest daytime gas exchange had the lowest malic acid accumulation and vice versa. Also, daytime CO₂ exchange and transpiration were negatively correlated with overnight malic acid fluctuation for all individuals examined together, as well as within one species. This suggests that malic acid may effect reductions in both atmospheric CO₂ uptake and transpiration during the day. No significant correlation between malic acid fluctuation and water-use efficiency was found, although a nonsignificant trend of increasing water-use efficiency with increasing malic acid fluctuation was observed among species averages. This study provides evidence that CO₂ recycling via malic acid is negatively correlated with daytime transpirational water losses in well-watered plants. Thus, CAM-cycling could be important for survival in the thin, frequently desiccated soils of rock outcrops on which these plants occur.     

Effects of Irradiance on Crassulacean Acid Metabolism in the Epiphyte Tillandsia usneoides L. (Bromeliaceae)

Spanish moss (Tillandsia usneoides L.) was collected in South Carolina, maintained in a greenhouse, then exposed to five levels of photosynthetic photon flux density (PPFD) for 3 weeks. Following this treatment, plants were sampled for chlorophyll concentrations, nocturnal acid accumulations, and photosynthetic responses to subsequent exposure at a range of PPFD. No acclimation to PPFD was observed; all plants exhibited similar patterns of nocturnal CO₂ uptake and acid accumulation regardless of initial PPFD treatment. These patterns revealed that at a PPFD level of approximately 200 micromoles per square meter per second (daytime integrated PPFD of 10 moles per square meter per day), CAM saturated or, in low-PPFD plants, was optimal. The results of this study indicate that adaptation to high PPFD is not necessarily a requirement of CAM.     

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.     

Short-Term and Long-Term Responses of Crassulacean Acid Metabolism Plants to Elevated CO(2)

For the leaf succulent Agave deserti and the stem succulent Ferocactus acanthodes, increasing the ambient CO₂ level from 350 microliters per liter to 650 microliters per liter immediately increased daytime net CO₂ uptake about 30% while leaving nighttime net CO₂ uptake of these Crassulacean acid metabolism (CAM) plants approximately unchanged. A similar enhancement of about 30% was found in dry weight gain over 1 year when the plants were grown at 650 microliters CO₂ per liter compared with 350 microliters per liter. Based on these results plus those at 500 microliters per liter, net CO₂ uptake over 24-hour periods and dry weight productivity of these two CAM succulents is predicted to increase an average of about 1% for each 10 microliters per liter rise in ambient CO₂ level up to 650 microliters per liter.     

A Diel Flux Balance Model Captures Interactions between Light and Dark Metabolism during Day-Night Cycles in C3 and Crassulacean Acid Metabolism Leaves

Although leaves have to accommodate markedly different metabolic flux patterns in the light and the dark, models of leaf metabolism based on flux-balance analysis (FBA) have so far been confined to consideration of the network under continuous light. An FBA framework is presented that solves the two phases of the diel cycle as a single optimization problem and, thus, provides a more representative model of leaf metabolism. The requirement to support continued export of sugar and amino acids from the leaf during the night and to meet overnight cellular maintenance costs forces the model to set aside stores of both carbon and nitrogen during the day. With only minimal constraints, the model successfully captures many of the known features of C₃ leaf metabolism, including the recently discovered role of citrate synthesis and accumulation in the night as a precursor for the provision of carbon skeletons for amino acid synthesis during the day. The diel FBA model can be applied to other temporal separations, such as that which occurs in Crassulacean acid metabolism (CAM) photosynthesis, allowing a system-level analysis of the energetics of CAM. The diel model predicts that there is no overall energetic advantage to CAM, despite the potential for suppression of photorespiration through CO₂ concentration. Moreover, any savings in enzyme machinery costs through suppression of photorespiration are likely to be offset by the higher flux demand of the CAM cycle. It is concluded that energetic or nitrogen use considerations are unlikely to be evolutionary drivers for CAM photosynthesis.Photosynthetic metabolism continues to be studied intensively because of its importance for crop performance and the global carbon cycle in relation to climate change. The metabolic pathways and enzymes involved in carbon fixation and related metabolic processes, such as the synthesis of Suc and starch, have been well-characterized. However, it is apparent that full appreciation of leaf metabolism requires these metabolic processes to be placed in the context of the wider metabolic network (Szecowka et al., 2013). This is particularly important for predicting how strategies for engineering improved photosynthesis (Maurino and Weber, 2013) may affect network properties, such as redox and energy balancing (Kramer and Evans, 2011).Flux balance analysis (FBA) has emerged as the method of choice for predicting fluxes in large metabolic network models (Sweetlove and Ratcliffe, 2011), and several flux balance models have explicitly considered photosynthetic metabolism in a variety of plants species and microorganisms, including cyanobacteria (Synechocystis sp. PCC 6803; Knoop et al., 2010, 2013; Montagud et al., 2010; Nogales et al., 2012; Saha et al., 2012), Chlamydomonas reinhardtii (Boyle and Morgan, 2009; de Oliveira Dal''Molin et al., 2011), Arabidopsis (Arabidopsis thaliana; de Oliveira Dal’Molin et al., 2010a), rapeseed (Brassica napus) embryos (Hay and Schwender, 2011), rice (Oryza sativa; Poolman et al., 2013), maize (Zea mays; Saha et al., 2011), and several C₄ plants (de Oliveira Dal’Molin et al., 2010b). These models successfully predicted the metabolic routes involved in the fixation of CO₂ into different biomass components in the light. However, one major feature of metabolism of photosynthetic organisms, namely the interaction between light and dark metabolism, is neglected in most of these studies. Effectively, most models assume that the organism grows in constant light, which is rarely true in natural conditions.Apart from the obvious switch from photoautotrophic to heterotrophic metabolism between day and night, interactions between the two phases can occur through the temporal separation of storage compound synthesis and subsequent mobilization. For example, it has been shown that the carbon skeletons used for nitrogen assimilation during the day are largely provided by carboxylic acids that were synthesized and stored during the previous night (Gauthier et al., 2010). Such temporal shifts of carbon and nitrogen metabolism have substantial implications for fluxes in the central metabolic network of leaves in the light (Tcherkez et al., 2009). Interactions between temporally separated metabolic events are also a critical feature of Crassulacean acid metabolism (CAM) photosynthesis, in which CO₂ is initially fixed at night by phosphoenolpyruvate carboxylase (PEPC), leading to night storage of carboxylic acids (mainly malic acid) that are decarboxylated during the day to provide CO₂ for the conventional photosynthetic carbon assimilation cycle. Although this is principally an adaptation to arid environments, there are unresolved questions as to whether CAM photosynthesis is energetically more efficient than C₃ photosynthesis (Winter and Smith, 1996). Such questions are becoming more important in the light of the proposed use of CAM plants as a source of biofuel (Yan et al., 2011).One recent study used FBA to consider both light and dark metabolism in Synechocystis sp. PCC 6803 over a complete diel cycle divided into 192 time steps (Knoop et al., 2013). Time courses of metabolic flux predictions over a diel cycle were simulated by altering the constraints on metabolic outputs (biomass composition) depending on the time point and based on empirical rules. This simulation led to a highly constrained model and did not allow the range of potential interactions between the day and night phases to be fully explored. We have developed an alternative modeling framework for integrated day-night FBA, in which the metabolic fluxes in the light and dark phases were simulated simultaneously in a single optimization problem. A predefined list of storage compounds that can accumulate freely over the diurnal cycle was made available to the model. The model was then free to choose among these storage compounds, the choice being dictated by the need to satisfy the objective function within the applied constraints. This diurnal modeling framework was used to explore the interactions between light and dark metabolism and to predict the metabolic fluxes in the light in both C₃ and CAM photosynthesis. We show that accounting for day-night interactions leads to an altered pattern of fluxes during the day that provides a better match with experimental observations. We were also able to simulate network flux distributions in CAM metabolism. The model successfully predicts the classic CAM cycle in the different CAM subtypes and allows a comparison of the energetic efficiency and metabolic costs between CAM and C₃ photosynthetic metabolism.     

Short-Term Regulation of Crassulacean Acid Metabolism Activity in a Tropical Hemiepiphyte,Clusia uvitana

Diel courses of net CO2 exchange of leaves were studied in Clusia uvitana (Clusiaceae), a tropical Crassulacean acid metabolism (CAM) hemiepiphyte, growing in the crown of a 47-m tall kapok tree on Barro Colorado Island, Panama. Measurements on days without precipitation showed that net uptake of atmospheric CO2 occurred at night, a feature of CAM, as well as in the early morning and late afternoon. During 36 h of almost continuous rainfall, nocturnal net CO2 uptake was abolished and the diel pattern of net CO2 exchange became similar to that of a C3 plant. Exposing well-watered, potted plants of Clusia in the laboratory to temperatures and photosynthetic photon flux densities similar to those during the tropical rainstorm also abolished nocturnal net CO2 uptake. In contrast, Kalanchoe pinnata (Crassulaceae), an obligate CAM plant, still showed net CO2 dark fixation following the same low-light and moderate-temperature conditions, albeit at decreased rates. During these 12-h photoperiods, titratable acidity in Clusia increased slightly above its high level measured at the end of the previous dark period, whereas in Kalanchoe, the acid content decreased by about 40%. A survey among outer canopy leaves of Clusia on Barro Colorado Island showed that leaves that exhibited little or no nocturnal acidification maintained high levels of H+ at dawn and dusk. Progressively lower levels of H+ at dusk were accompanied by progressively higher nocturnal increases in H+. The data suggest that in C. uvitana the rapid switching between CAM- and C3-type carbon fixation that may occur within 24 h in response to environmental changes is controlled by the acidity status of the leaves in the light. Nocturnal CO2 fixation is enhanced by conditions that decrease the organic acid content during the light period.     

Relationships between Photosynthetically Active Radiation, Nocturnal Acid Accumulation, and CO(2) Uptake for a Crassulacean Acid Metabolism Plant, Opuntia ficus-indica

The influences of photosynthetically active radiation (PAR) and water status on nocturnal Crassulacean acid metabolism (CAM) were quantitatively examined for a widely cultivated cactus, Opuntia ficus-indica (L.) Miller. When the total daily PAR was maintained at 10 moles photons per square meter per day but the instantaneous PAR level varied, the rate of nocturnal H⁺ accumulation (tissue acidification) became 90% saturated near 700 micromoles per square meter per second, a PAR level typical for similar light saturation of C₃ photosynthesis. The total nocturnal H⁺ accumulation and CO₂ uptake reached 90% of maximum for a total daily PAR of about 22 moles per square meter per day. Light compensation occurred near 0 moles per square meter per day for nocturnal H⁺ accumulation and 4 moles per square meter per day for CO₂ uptake. Above a total daily PAR of 36 moles per square meter per day or for an instantaneous PAR of 1150 micromoles per square meter per second for more than 6 hours, the nocturnal H⁺ accumulation actually decreased. This inhibition, which occurred at PAR levels just above those occurring in the field, was accompanied by a substantial decrease in chlorophyll content over a 1-week period.

A minimum ratio of H⁺ accumulated to CO₂ taken up of 2.5 averaged over the night occurred for a total daily PAR of 31 moles per square meter per day under wet conditions. About 2 to 6 hours into the night under such conditions, a minimum H⁺-to-CO₂ ratio of 2.0 was observed. Under progressively drier conditions, both nocturnal H⁺ accumulation and CO₂ uptake decreased, but the H⁺-to-CO₂ ratio increased. A ratio of two H⁺ per CO₂ is consistent with the H⁺ production accompanying the conversion of starch to malic acid, and it apparently occurs for O. ficus-indica when CAM CO₂ uptake is strongly favored over respiratory activity.

     

Purification by Immunoadsorption and Immunochemical Properties of NADP-Dependent Malic Enzymes from Leaves of C(3), C(4), and Crassulacean Acid Metabolism Plants

NADP:malic enzyme from corn (Zea mays L.) leaves was purified by conventional techniques to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies raised against this protein in rabbits were purified, coupled covalently to protein A-Sepharose CL-4B, and used as an immunoaffinity resin to purify the NADP:malic enzymes of the C₃ plants spinach (Spinacia oleracea L.) and wheat (Triticum aestivum L.), of the Crassulacean acid metabolism (CAM) plant Bryophyllum daigremontianum R. Hamed et Perr. de la Bathie and the C₄ plants corn, sugarcane (Saccharum officinarum L.), and Portulaca grandiflora L. Such procedures yielded homogeneous protein preparations with a single protein band, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, except for P. grandiflora L. with two bands. The specific activities of the purified proteins ranged between 56 and 91 units (milligrams per protein). NADP:malic enzyme represented up to 1% of the total soluble protein in C₄ plants, 0.5% in the CAM plant, and less than 0.01% in C₃ plants. In immunotitration tests involving immunoprecipitation and immunoinhibition of activity by an antiserum against the corn leaf enzyme, the NADP:malic enzymes of corn and sugarcane showed virtually full identity of epitopes, while the NADP:malic enzymes of the C₃ and CAM plants exhibited a cross-reaction of one-twentieth and one-fourth by these tests, respectively. The NADP:malic enzyme of P. grandiflora exhibited characteristics more closely related to the enzymes of C₃ and CAM plants than to those of C₄ plants.