Nocturnal Malic Acid Accumulation Research Articles

The contribution of weak CAM to the photosynthetic metabolic activities of a bromeliad species under water deficit

The Crassulacean acid metabolism (CAM) can be a transitory strategy for saving water during unfavourable conditions, like a dry season. In some cases, CAM can also contribute to the maintenance of photosynthetic integrity, even if carbon gain and growth are impaired. CAM occurs in different intensities, being stronger or weaker depending on the degree of nocturnal malic acid accumulation. For example, Guzmania monostachia is an epiphytic tank bromeliad that shows an increase in its nocturnal organic acid accumulation and a variable CAM behaviour when exposed to water deficit. In this context, this study aimed at investigating whether the weak CAM displayed by this species may mitigate the harmful effects of water limitation on its photosynthetic activity. To this, bromeliads were submitted to well-watered and water deficit conditions. Guzmania monostachia plants under water deficiency conditions showed a reduction on atmospheric carbon assimilation without exhibiting changes in PSII integrity and carbohydrate production while showed an increase in nocturnal malic acid accumulation. Additionally, spots with high PSII efficiency in the leaf portion with a greater nocturnal malic acid accumulation were observed in plants exposed to water shortage conditions. These high-efficiency spots might be associated with a greater malate decarboxylation capacity. Also, the malic acid contributed to approximately 50% of the total carbon assimilated under water deficit. These results suggest that weak CAM may participate in photo-protection and it appears to meaningfully contribute to the overall carbon balance, being an important metabolic strategy to maintain plant fitness during water deficit periods.

Cladode growth dynamics in Opuntia ficus-indica under drought

Cactus pear (Opuntia ficus-indica L. Miller) is a CAM plant with an extraordinary capacity to store water in its succulent stems (cladodes). However, the daily variations of cladode thickness is unknown. Studying cladode thickness fluctuations may be useful for the early prediction of plant dehydration stress. The objective of this study was to determine if age, water availability and temperature influence diel cladode shrinkage and enlargement dynamics in cactus pear. The experiment was conducted in a greenhouse from April to July 2014, using cactus pear plants, equally split into irrigated and unirrigated treatments, and unrooted cladodes detached from mother plants. Soil moisture content (SMC), soil water depletion over 24-h, cladode relative water content (RWC), cladode thickness, stomatal conductance (gs), cladode growth rates (area increase) and nocturnal malic acid accumulation were monitored in plants of various ages. Cladode shrinkage and enlargement dynamics were assessed using stem gauges and expressed as absolute growth rate (AGRthickness, μmmin−1). In unirrigated pots, drought decreased SMC, RWC,cladode thickness, cladode growth rate and gs. Younger cladodes lost water later than older ones. Detached cladodes exhibited gs activity 3–4 months after detachment. Trends of AGRthickness showed that there was a progressive reduction of diel swelling and shrinkage fluctuations as cladodes aged. Such fluctuations were minimized under severe drought when 1-year-old cladodes reached 8mm thickness and a RWC of about 45%. A positive correlation was found between SMC and AGRthickness. Temperatures were also directly correlated with AGRthickness, although this relationship was gradually lost as SMC decreased. Overall, cactus pears were able to maintain some growth at very low hydration levels, and cladode growth was highly responsive to rehydration after long periods of drought.

Multiple isoforms of phosphoenolpyruvate carboxylase in the Orchidaceae (subtribe Oncidiinae): implications for the evolution of crassulacean acid metabolism.

Phosphoenolpyruvate carboxylase (PEPC) catalyses the initial fixation of atmospheric CO2 into oxaloacetate and subsequently malate. Nocturnal accumulation of malic acid within the vacuole of photosynthetic cells is a typical feature of plants that perform crassulacean acid metabolism (CAM). PEPC is a ubiquitous plant enzyme encoded by a small gene family, and each member encodes an isoform with specialized function. CAM-specific PEPC isoforms probably evolved from ancestral non-photosynthetic isoforms by gene duplication events and subsequent acquisition of transcriptional control elements that mediate increased leaf-specific or photosynthetic-tissue-specific mRNA expression. To understand the patterns of functional diversification related to the expression of CAM, ppc gene families and photosynthetic patterns were characterized in 11 closely related orchid species from the subtribe Oncidiinae with a range of photosynthetic pathways from C3 photosynthesis (Oncidium cheirophorum, Oncidium maduroi, Rossioglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) and strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthagenense). Phylogenetic analysis revealed the existence of two main ppc lineages in flowering plants, two main ppc lineages within the eudicots, and three ppc lineages within the Orchidaceae. Our results indicate that ppc gene family expansion within the Orchidaceae is likely to be the result of gene duplication events followed by adaptive sequence divergence. CAM-associated PEPC isoforms in the Orchidaceae probably evolved from several independent origins.

Vacuolar malate uptake is mediated by an anion-selective inward rectifier.

Electrophysiological studies using the patch-clamp technique were performed on isolated vacuoles from leaf mesophyll cells of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana to characterize the malate transport system responsible for nocturnal malic acid accumulation. In the presence of malate on both sides of the membrane, the current-voltage relations of the tonoplast were dominated by a strongly inward-rectifying anion-selective channel that was active at cytoplasmic-side negative voltages. Rectification of the macroscopic conductance was reflected in the voltage-dependent gating of a 3-pS malate-selective ion channel, which showed a half-maximal open probability at -43 mV. Also, the time-averaged unitary currents following a step to a negative voltage corresponded to the time-dependent kinetics of the macroscopic currents, suggesting that the activity of this channel underlies the anion-selective inward rectifier. The inward rectifier showed saturation kinetics with respect to malate (apparent Km of 2.5 mm malate2- activity), a selectivity sequence of fumarate2- > malate2- > Cl- > maleate2- approximately citrate3-, and greater activity at higher pH values (with an apparent pK of 7.1 and maximum activity at around pH 8.0). All these properties were in close agreement with the characteristics of malate transport observed in isolated tonoplast vesicles. Further, 100 microM niflumate reversibly blocked the activity of the 3-pS channel and inhibited both macroscopic currents and malate transport into tonoplast vesicles to the same extent. The macroscopic current densities recorded at physiological voltages and the estimated channel density of 0.2 microm-2 are sufficient to account for the observed rates of nocturnal malic acid accumulation in this CAM plant, suggesting that the 3-pS, inward-rectifying, anion-selective channel represents the principal pathway for malate influx into the vacuole.

Interactive effects of photon fluence rates and drought on CAM-cycling in Delosperma tradescantioides (Mesembryanthemaceae).

The ability of photosynthesis and CAM to acclimate to low (220 µmol m-2 s-1 ; LL) and relatively high (550 µmol m-2 s-1 ; HL) photosynthetic photon flux densities (PPFD) was investigated in the CAM-cycling species Delosperma tradescantioides by means of CO2 gas exchange and chlorophyll fluorescence analysis. Furthermore, the influence of short-term drought on malic acid accumulation and the activity of photosystem II (PSII) was studied to assess the possible interactions between drought and the prevailing PPFD in this species. HL plants showed features of sun versus shade acclimation relative to LL plants. Nocturnal malic acid accumulation (Δ-malate) and leaf water content also tended to be higher in HL plants. Irrespective of the PPFD during growth, the weak Δ-malate doubled within 3 days of drought. Despite largely restricted CO2 uptake, photosynthetic activity as estimated from fluorescence analysis declined only ca 5%. After 7 days of drought, when plants showed CAM-idling and Δ-malate had decreased again, potential carbon assimilation was still ca 84% of that in well-watered plants and remained relatively constant throughout the day. Decarboxylation of malic acid accounted for ca 23% of potential assimilation assuming total oxidation of a maximum portion of this organic acid. Drought did not affect predawn maximum photochemical efficiency (Fv /Fm ). Nonphotochemical quenching (qN) increased (24%) in response to desiccation and resulted in a more or less constant reduction state of PSII. This increase in qN resulted mainly from the change in its fast-relaxing component (qNF), while the slow component (qNS) was significant only at or above saturating PPFD in both HL and LL plants. The photon response characteristics of PSII, which differed between LL and HL plants, were unaffected by short-term drought. Photon harvesting and photon use were always adjusted to guarantee a low reduction state of PSII. Results suggest that in both LL and HL plants CAM-cycling may help to stabilize photosynthesis but to a large extent by other means than simply providing internally derived CO2 .

Physiological and Anatomical Responses to Water Deficits in the Cam Epiphyte Tillandsia ionantha (Bromeliaceae)

Although physiological responses to drought have been examined in several species of epiphytic bromeliads, few have included a comprehensive methodological approach to the study of the carbon and water relations of a single species undergoing drought stress. Thus, physiological and anatomical responses to an imposed drought treatment were examined in the atmospheric Crassulacean acid metabolism (CAM) epiphyte Tillandsia ionantha. From 0 through 20 d without water, nocturnal malic acid accumulation and CO2 uptake rates did not change despite a 17% reduction in relative water content. In addition, water potentials averaged -0.40 MPa and, unlike leaf water content, did not decline. The avoidance of further declines in leaf water content was attributed to the restriction of stomatal opening to the night (a characteristic feature of CAM), to low stomatal densities and small stomatal pores, and to a thick boundary layer resulting from a dense foliar trichome cover. The maintenance of high physiological activity during the first 20 d of the drought treatment was most likely a result of the high water potentials in the chlorenchyma, which were attributed, in part, to water movement from the water-storage parenchyma (= "hydrenchyma") to the chlorenchyma. Nocturnal malic acid accumulation and the rate of net CO2 exchange declined in a linear fashion from 30 to 60 d without water, as did leaf water potential and osmotic potential. During this time, CO2 recycling increased from ca. 20% to nearly 75%. Though declining throughout this later stage of the drought treatment, metabolic activity remained relatively high, possibly as a result of the observed osmotic adjustment as well as a potentially high cell wall elasticity.

Influence of leaf water content on the C3 -CAM transition in Mesembryanthemum crystallinum.

Changes in leaf water content, night-time accumulation of malic (δ-malate) and citric acid (δ-citrate) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activity were followed for 60 d after germination in well watered and salt-stressed plants of the facultatively halophytic ephemeral Mesembryanthemum crystallinum L. To separate the effects of development, salt stress and water deficit on crassulacean acid metabolism (CAM) induction plants were stressed initially 10 d after germination and then successively at 1-wk intervals (five sets). Related to dry mass or organic matter (i.e. dry mass corrected for the mass of inorganic ions) water content started to decrease during the late embryonal phase of the life cycle. Water content on a dry mass basis was always lower in salt-stressed than in well watered individuals. However, on an organic matter basis no difference was detectable. This indicated that salt treatment did not reduce leaf water content but falsified the basis (dry mass). Increases in leaf succulence and in pressure potential prevented long-term water deficit in well watered and in salt-stressed plants. Instead, these changes displayed enhanced vacuolisation, which is an essential prerequisite for the development of CAM. The end of that differentiation process might allow the initiation of nocturnal malic acid accumulation in a threshold response. At the onset of each salt treatment, short-term water deficits occurred due to an incomplete osmotic adaptation independent of plant age. As δ-malate only appeared when plants were c. 35 d old this water deficit was unlikely to be a decisive CAM-inducing factor. About 2 wk after germination water content began to decline during the light periods in plants of all treatments. This pattern disappeared again when CAM had been fully established. Daytime transpirational water loss is therefore unlikely to be the decisive factor because it failed to induce the metabolic shift in young plants. Environmental stress (e.g. salt or drought) can therefore only induce δ-malate when leaf and plant differentiation has reached a certain stage.

Stomatal responses to changes in air humidity are not necessarily linked to nocturnal CO2 uptake in the CAM plant Plectranthus marrubioides Benth. (Lamiaceae)

ABSTRACTPlants of the crassulacean acid metabolism (CAM) species Plectranthus marrubioides (Lamiaceae) were subjected to short‐ and long‐term changes in air humidity in controlled‐environment experiments. Stomata of well‐watered individuals of this all‐cell leaf‐succulent taxon responded directly, quickly and reversibly to variations of the water vapour gradient between leaf and air (Δw). Mean night‐time leaf conductance to water vapour decreased curvilinearly with increasing Δw but linearly with lowered relative air humidity. Stomatal response was generally independent of the prevailing temperature and was not linked to CO2 uptake rates. Therefore, net night‐time carbon gain, nocturnal malic acid accumulation and, thus, relative carbon recycling were not influenced by changes in air humidity in the temperature range tested. Mean nocturnal molar water use efficiency, however, decreased with decreasing air humidity because of the increased transpirational water loss. If watering was repeatedly withheld for several days during the experiments, employing a temperature regime of 35/30°C day and night, stomatal conductance became low enough to inhibit CO2 uptake, but only at the highest Δw. The results suggest that drought stress was necessary to increase responsiveness of plants to the point where CAM was also inhibited by decreases in air humidity.

Ecophysiological investigations on plants of the genus Plectranthus (fam. Lamiaceae) native to Yemen and southern Africa

Summary In this first survey of the ecophysiology of 8 succulent and semi-succulent species from the genus Plectranthus (Larniaceae) native to Yemen and southern Africa it has been shown that three species, Plectranthus arabicus, P. hadiensis and P. marrubioides are obligate CAM plants. Well-watered plants of these yemenitic species, which are found primarily with other succulents at altitudes up to 2 600 m, exhibit a nocturnal accumulation of malic acid, and, to a much smaller extent, citric acid as well as night-time CO 2 uptake under controlled-environment conditions. While the semi-succulent P. cyrpiculoides utilises the CAM-cycling mode, the semi-succulent species, P. barbatus, P. purpuratus, P. zatarhendi, and P. zatarhendi var. tomentosus, are typical C 3 plants. Thus, the ability to perform CAM was well correlated with succulence. Neither CAM nor succulence was, however, directly related to maximum desiccation resistance in these species. Increasing day-time temperatures from 20 to 35°C reduces net daily carbon gain in all, leading to CO 2 losses in most of the C 3 species. In the CAM species, nocturnal CO 2 uptake at least partially alleviated the effect of the high temperature conditions. On the other hand, the C 3 species P. barbatus , growing in succulent plant communities in high mountain habitats (up to 3000 m), was also relatively insensitive to high day-time temperatures. This is the first extensive description of CAM and CAM-cycling in the Lamiaceae. The presence of CAM in Plectranthus correlated with habitat aridity in situ.

Nocturnal Respiration Rates and Malic Acid Accumulation in Five Species of Talinum (Portulacaceae) during CAM-Cycling

Summary Dark respiration rates of leaf slices of five species of Talinum (Portulacaceae) did not significantly correlate with nocturnal increases in leaf malic-acid concentrations. Thus, respiration rate does not appear to be the primary limiting factor regarding the degree of nocturnal malic acid accumulation in plants exhibiting CAM-cycling. Instead, these findings support the possibility that the biochemical capacity for CAM limits the amount of malic acid synthesized at night during CAM-cycling.

Soluble Sugars as the Carbohydrate Reserve for CAM in Pineapple Leaves

Neutral ethanol-soluble sugar pools serve as carbohydrate reserves for Crassulacean acid metabolism (CAM) in pineapple (Ananas comosus (L.) Merr.) leaves. Levels of neutral soluble sugars and glucans fluctuated reciprocally with concentrations of malic acid. Hexose loss from neutral soluble-sugar pools was sufficient to account for malic acid accumulation with about 95% of the required hexose accounted for by turnover of fructose and glucose pools. Hexose loss from starch or starch plus lower molecular weight glucan pools was insufficient to account for nocturnal accumulation of malic acid. The apparent maximum catalytic capacity of pyrophosphate:6-phosphofructokinase (PPi-PFK) at 15 degrees C was about 16 times higher than the mean maximum rate of glycolysis that occurred to support malic acid accumulation in pineapple leaves at night and 12 times higher than the mean maximum rate of hexose turnover from all carbohydrate pools. The apparent maximum catalytic capacity of ATP-PFK at 15 degrees C was about 70% of the activity required to account for the mean maximal rate of hexose turnover from all carbohydrate pools if turnover were completely via glycolysis, and marginally sufficient to account for mean maximal rates of acidification. Therefore, at low night temperatures conducive to CAM and under subsaturating substrate concentrations, PPi-PFK activity, but not ATP-PFK activity, would be sufficient to support the rate of glycolytic carbohydrate processing required for acid accumulation. These data for pineapple establish that there are at least two types of CAM plants with respect to the nature of the carbohydrate reserve utilized to support nighttime CO(2) accumulation. The data further indicate that the glycolytic carbohydrate processing that supports acidification proceeds in different subcellular compartments in plants utilizing different carbohydrate reserves.

Day-night changes in the levels of adenine nucleotides, phosphoenolpyruvate and inorganic pyrophosphate in leaves of plants having Crassulacean acid metabolism

The levels of phosphorylated compounds studied during the dark period of Crassulacean acid metabolism (CAM) in Kalanchoë leaves showed increases for ATP and pyrophosphate and decreases for ADP, AMP and phosphenolpyruvate; levels of inorganic phosphate remained constant. Changes in adenylate levels and the correlated nocturnal increase in adenylate-energycharge were closely related to changes in malate levels. The increase in ATP levels was much inhibited in CO2-free air and stimulated after induction of CAM in short-day-treated plants of K. blossfeldiana cv. Tom Thumb. Changes in levels of phosphoenolpyruvate and pyrophosphate were independent of the presence of CO2. The results show the operation of complex regulatory mechanisms in the energy metabolism of CAM plants during nocturnal malic-acid accumulation.

CARBON DIOXIDE AND WATER DEMAND: CRASSULACEAN ACID METABOLISM (CAM), A VERSATILE ECOLOGICAL ADAPTATION EXEMPLIFYING THE NEED FOR INTEGRATION IN ECOPHYSIOLOGICAL WORK.

Plants having crassulacean acid metabolism (CAM) tend to occupy habitats where the prevailing environmental stress is scarcity of water. These are semi-arid or arid regions, salinas or epiphytic sites. CAM plants manage the dilemma of desiccation or starvation by nocturnal malic acid accumulation in the vacuoles. Malic acid serves as a form of CO2 storage and as an osmoticum. In this way malic acid accumulation allows, firstly, separation of uptake and assimilation of atmospheric CO2 with water-saving daytime stomatal closure and, secondly, osmotic acquisition of water. There is no very special trait which is specific for CAM. An array of biophysical and biochemical functional elements, which are also found in other plants, is integrated in CAM performance. This leads to a large diversity of behaviour which makes CAM plants highly versatile in their response to environmental variables. Besides CO2 dark fixation, transport of malic acid across the tonoplast is one of the key elements in CAM function. This is examined in detail at the level of membrane biophysics and biochemistry. The versatility of CAM is illustrated by examples from field work, with comparisons involving different species, seasons, modes of photosynthesis (CAM vs C3 ), kinds of stress and ways of stress imposition. Contents Summary 593 I. Studies of CAM: an example for the ecophysiological approach 594 II. Malic acid transport at the tonoplast 602 III. Regulation 605 IV. Desiccation or starvation 610 V. Comparative autecology 614 VI. Ecology: promise of integration 621 Acknowledgements 622 References 622.

Nocturnal water storage in plants having Crassulacean acid metabolism

Measurements of water uptake and transpiration, during the dark period of plants having Crassulacean acid metabolism (CAM) allow calculation of leaf-volume changes (ΔV). Nocturnal leaf-volume changes of CAM plants have also been reported in the literature on the basis of waterdisplacement measurements. A third way of estimation is from measurements of turgor changes and cellular water-storage capacity using the pressure probe, cytomorphometry and the Scholander pressure chamber. An extension of the interpretation of results reported in the literature shows that for leaf succulent CAM plants the three different approaches give similar values of ΔV ranging between 2.3 and 10.7% (v/v). It is evident that nocturnal malic-acid accumulation osmotically drives significant water storage in CAM leaf tissue.

Characterization of the vacuolar ATPase activity of the crassulacean-acid-metabolism plant Kalanchoë daigremontiana. Receptor modulating.

Plants performing crassulacean acid metabolism show a large nocturnal accumulation of malic acid in the vacuole of the photosynthetic cells. It has been postulated that an H+-translocating ATPase energizes the transport of malic acid across the tonoplast into the vacuole. In the present work we have characterized the ATPase activity associated with vacuoles of the crassulacean-acid-metabolism plant Kalanchoë daigremontiana and compare it with other phosphohydrolases. Vacuoles were isolated by polybase-induced lysis of mesophyll-cell protoplasts. The vacuoles had a high activity of unspecific acid phosphatase (pH optimum 5.3). The acid phosphatase was strongly inhibited by ammonium molybdate (with 50% inhibition at about 0.5 mmol m-3), but was not completely inhibited even at much higher ammonium-molybdate concentrations. In contrast, the vacuolar ATPase activity, assayed in the presence of 100 mmol m-3 ammonium molybdate, had a pH optimum of 8.0. ATP was the preferred substrate, but GTP, ITP and ADP were hydrolyzed at appreciable rates. The mean ATPase activity at pH 8.0 was 14.5 nmol h-1 (10(3) vacuoles)-1, an average 13% of which was attributable to residual acid-phosphatase activity. Inorganic-pyrophosphatase activity could not be demonstrated unambiguously. The vacuolar ATPase activity was Mg2+-dependent, had an apparent Km for MgATP2- of 0.31 mol m-3, and was 32% stimulated by 50 mol m-3 KCl. Of the inhibitors tested, oligomycin slightly inhibited the vacuolar ATPase activity and diethylstilbestrol and NO-3 were both markedly inhibitory. Dicyclohexylcarbodiimide and tributyltin were also strongly inhibitory. Tributyltin caused a 50% inhibition at about 0.3 mmol m-3. This is taken as evidence that the vacuolar ATPase might function as an H+-translocating ATPase. It is shown that the measured activity of the vacuolar ATPase would be of the right order to account for the observed rates of nocturnal malic-acid accumulation in K. daigremontiana.

Adenine-nucleotide levels during crassulacean acid metabolism and the energetics of malate accumulation in Kalanchoë tubiflora

The contention that nocturnal malic-acid accumulation in the vacuoles of crassulacean acid metabolism (CAM) plants is an energy-dependent process was confirmed by experiments on excised phyllodia of Kalanchoë tubiflora with the inhibitors 2,4-dinitrophenol (DNP), m-chlorocarbonylcyanide phenylhydrazone (CCCP) and diethylstilbestrol (DES). Levels of ATP, ADP and P i were also measured in bulk-tissue extracts of phyllodia during the normal CAM rhythm. ATP levels increased during the dark period and decreased during the light period, whereas ADP and P i levels did not show any significant changes. In some plants the ATP levels started to decrease before the end of the dark period. It is suggested that the changes in ATP levels are a reflection of the changing flux through glycolysis towards malate in the dark period. The results were used to estimate the free energy available from ATP hydrolysis ( ΔG ATP), which was compared with the H +-electrochemical potential difference ( Δ μ H + ) across the tonoplast. This permitted consideration of the stoichiometry of the H +-ATPase that is postulated to be responsible for malic-acid accumulation in the vacuole.

Nocturnal Accumulation of Malic Acid Occurs in Mesophyll Tissue without Proton Transport to Epidermal Tissue in the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

The inducible Crassulacean acid metabolism plant, Mesembryanthemum crystallinum, accumulates malic acid, i.e. equivalent amounts of malate anions and protons in the mesophyll cells at night. Levels of malate and titratable acidity are low in the epidermal tissue and do not change significantly during the day/night cycle. This result is in contrast to a recent report (Bloom 1979 Plant Physiol 64: 919-923) that the synthesis of malic acid during dark CO(2) fixation is associated with an equivalent exchange of inorganic cations from epidermal tissue with protons in the mesophyll cells.

2H+:1 malate2− stoichiometry during Crassulacean Acid Metabolism is unaffected by lipophilic cations

Abstract. Lipophilic cations inhibit nocturnal malic acid accumulation in leaf cells of the Crassulacean Acid Metabolism plant Kalanchoë tubiflora. perhaps by interacting directly or indirectly with active malic acid transport into the vacuoles. Lipophilic cations do not affect passive efflux of malic acid from the vacuoles. Membrane potentials are depolarized, oxygen uptake is stimulated by lipophilic cations and there may also be stomatal responses. Thus it is striking that lipophilic cations do not alter the stoichiometry of 2 titratable H : 1 enzymatically‐determined malate2− during diurnal malic acid oscillations of Crassulacean Acid Metabolism in Kalanchoë. This suggests that coupling between protons and malate during transport into the vacuole must be tight. Transport as undissociated acid is unlikely because the dissociation equilibrium in the cytoplasm is largely on the side of malate2−. These results appear to suggest an intimate molecular interaction between a proton pump and a presumed malate2− translocator at the tonoplast of leaf cells with Crassulacean Acid Metabolism.

Electrochemical investigation of active malic acid transport at the tonoplast into the vacuoles of the CAM plantKalanchoë daigremontiana

The membrane potential of cells in leaf slices of the CAM plantKalanchoe daigremontiana Hamet et Perrier in the light and in the dark is −200 mV on the average; it is reversibly depolarized by the metabolic inhibitors FCCP (5×10−6 m) and CN− (5×10−3 m); it shows the light-dependent transient oscillations ubiquitously observed in green cells; it is independent of the amount of malic acid accumulated in the cells (in a tested range between 30 and 140mm); and it is considerably hyperpolarized by the fungal toxin fusicoccin (30×10−6 m). Fusicoccin inhibits nocturnal malic acid accumulation in intact isolated phyllodia of the CAM plantKalanchoe tubiflora (Harv.) Hamet but does not affect remobilization of malic acid during the day. Electrochemical gradients for the various ions resulting from dissociation of malic acid, i.e., H+, Hmal− and mal2−, were calculated using the Nernst equation. With a very wide range of assumptions on cytoplasmic pH and malate concentration results of calculations suggest uphill transport of H+ and Hmal− from the cytoplasm into the vacuole, while mal2− might be passively distributed at the tonoplast. On the basis of the present data the most likely mechanism of active malic acid accumulation in the vacuoles of CAM plants appears to be an active H+ transport at the tonoplast coupled with passive movement of mal2− possibly mediated by a translocator (“catalyzed diffusion”), with subsequent formation of Hmal− (2 H++mal2−→H++Hmal−) at vacuolar pH's.