Crassulacean Acid Metabolism Rhythm Research Articles

Proton and anion transport across the tonoplast vesicles in bromeliad species

Crassulacean acid metabolism (CAM) is one of the key innovations in the Neotropical family Bromeliaceae that has enabled many of its species to occupy seasonally water-limited terrestrial environments or microclimatically arid epiphytic niches. However, the relationship between CAM activity and the transport processes responsible for vacuolar organic-acid accumulation at night has not been systematically explored in this family. In the present investigation, ATP- and PPi-dependent proton transport rates were studied in tonoplast membrane vesicles isolated from leaves of six CAM and one C3 species of bromeliads. A consistent feature of these species was the high activity of the tonoplast ATP-driven H+ pump, which, when averaged across the seven species tested, showed a higher specific activity than the tonoplast PPi-driven H+ pump. For all CAM species, the rate of ATP-dependent proton transport into the tonoplast vesicles was strongly influenced by the nature of the balancing organic-acid anion, which displayed the following order of effectiveness: fumarate>malate>citrate. Measurements of leaf organic-acid content in six CAM bromeliads at dusk and dawn showed that nocturnal accumulation of malate exceeded citrate by a factor of ~2.4-20.0-fold in five of six bromeliad species used in this study, demonstrating a close correlation between the CAM rhythm and the intrinsic properties of the vacuolar membrane across which these organic acids are transported.

Responses to different external light rhythms by the circadian rhythm of Crassulacean acid metabolism in Kalanchoe daigremontiana

AbstractTime series of net CO2 exchange (JCO2) and leaf conductivity for water vapour (gH2O) were measured and subsequently analysed mathematically in the Crassulacean acid metabolism (CAM) plant Kalanchoe daigremontiana (Hamet et Perrier de la Bâthie) under constant environmental conditions and under imposed external rhythms of lower and higher light intensity. The time series were analysed by Fourier methods and a correlation analysis considering the first time derivatives of JCO2, gH2Oand photosynthetically active photon flux density (PPFD). The ratio of internal to external CO2 (ci/ca) was also considered in the analysis, leading to a discussion of the interaction of stomata and carbon assimilation under periodic stimulation. It is suggested that for stimulation with frequencies close to the endogeneous circadian period, stomatal conductance and carbon assimilation oscillate synchronously, guard‐cell movements trailing behind changes in internal CO2 with a delay of 10–15 min. For stimulation frequencies far shorter than the endogeneous period, this synchrony can be disturbed due to independent responses of stomata and assimilation to light pulses, leading to an arrhythmic gas exchange pattern. These results are discussed in the context of understanding circadian oscillations as the output of a multioscillator, multisignalling pathway system on the organismic and metabolic level.

Effects of temperature on circadian rhythm of crassulacean acid metabolism

Crassulacean acid metabolism (CAM) is known to exhibit endogenous circadian rhythm under continuous light conditions. The rhythm, however, disappears at higher temperatures above approximately 30°C. We investigated effects of the temperature on the rhythm of phosphorylation of phosphoenolpyruvate carboxylase (PEP-C), the key enzyme in the regulation of CAM, with a CAM plant Graptopetalum paraguayense. We also analyzed expression of chlorophyll a/b binding protein gene (CAB) as an indicator of the state of the central oscillator, because CCA1 is a transcription factor of CAB in Arabidopsis thaliana and is thought to be a component of the oscillator or a component closely associated with it. The expression of CAB is expected to be under direct control of the clock. Under the continuous light condition, the rhythm in the phosphorylation of PEP-C, which was estimated by measuring the inhibition by 2 mM malate of the activity in leaf extract, did not persist at 32°C. The rhythm in the mRNA level of CAB, which persisted at 20°C, also disappeared at 32°C, suggesting that the circadian clock stops at high temperatures. Decreasing the temperature from 32°C to 25°C reset the rhythm of the phosphrylation of PEP-C. On the other hand, temperature shift from 27°C, at which the rhythm persisted, to 20°C had no effect on the phase of the rhythm.

Generation of rhythmic and arrhythmic behaviour of Crassulacean acid metabolism in Kalancho� daigremontiana under continuous light by varying the irradiance or temperature: Measurements in vivo and model simulations

Leaves of Kalanchoe daigremontiana Hamet et Perr. at a photon flux density (PFD) above 220 μmol·m−2s−1 (400–700 nm) or at leaf temperatures above 27.0 °C showed a rapid loss of rhythmicity, and a more or less pronounced damping-out of the endogenous circadian rhythm of CO2 exchange under continuous illumination. This rhythm was reinitiated after reduction of the PFD by 90–120 μmol·m−2·s−1 or reduction of leaf temperature by 3.5–11.0 °C under otherwise unchanged external conditions. The reduction in the magnitude of the external control parameter of the Crassulacean acid metabolism (CAM) rhythm (i.e. PFD or leaf temperature) set the phase of the new rhythm. The maxima of CO2 uptake occurred about 5, 28, 51, 75 h after the reduction. Simulations with a CAM model under comparable conditions showed a similar behaviour. The influence of temperature on the endogenous CAM rhythm observed in K. daigremontiana in vivo could be simulated by incorporating into the model temperature-dependent switch modes for passive efflux of malate from the vacuole to the cytoplasm. Thus, the model indicates that tonoplast function plays an important role in regulation of the endogenous CAM rhythm in K. daigremontiana.

Day-night cycles of net CO2 exchange in crassulacean acid metabolism as related to day-night changes of abscisic-acid levels

Eight to nine months old seedlings of the Cactaceae Gereus validus HAWORTH grown in soil culture were chosen to study day-night cycles of net CO2 exchange, indicating the stomatal rhythm of crassulacean acid metabolism, CAM, in relation to day-night changes of abscisic-acid levels. Drought stress was imposed by repotting the seedlings in dry sand and keeping them without watering for up to 37–39 days while control plants were watered regularly. Abscisicacid levels were higher in the stressed plants than in the controls and higher in the light period aa compared to the dark period. In the stressed plants abscisic-acid levels increased throughout the light period. Abscisic acid reached particularly high levels in the late afternoon in plants stressed for 37 days. It is conceivable that stomatal closure during the first part of the light period of CAM is elicited by high internal partial pressures of CO2 built up by decarboxylation of nocturnally stored malic acid. However, the elimination of late-afternoon stomatal opening and the reduction of stomatal opening during the dark period of CAM observed under conditions of drought stress must have other reasons. The analyses of abscisic acid presented allow the conclusion that this stress hormone is involved in stomatal regulation of CAM under such conditions.

Water flow and water storage in Agave deserti: osmotic implications of crassulacean acid metabolism

Abstract Water flow and water storage were investigated for Agave deserti, a desert succulent showing crassulacean acid metabolism (CAM). The anatomy and water relations of the peripheral chlorenchyma, where CAM occurs, and the central water‐storage parenchyma were investigated for its massive leaves so that these tissues could be incorporated as discrete elements into an electrical‐circuit analogue of the whole plant. The daily cycling of osmotic pressure was represented by voltage sources in series with the storage capacitors. With soil water potential and leaf transpiration rate as input variables, axial water flow through the vascular bundles and radial flows into and out of storage during the day/night cycle were determined.The predominantly nocturnal transpiration was coincident with increases in cell osmotic pressure and in titratable acid of the leaf chlorenchyma. In the outer layers of the chlorenchyma, water potential was most negative at the beginning of the night when transpiration was maximum, while the water‐storage parenchyma reached its minimal water potential 9 h later. The roots plus stem contributed 7% and the leaves contributed 50% to the total water flow during maximal transpiration; peak water flow from the soil to the roots occurred at dawn and was only 58% of the maximal transpiration rate. Over each 24‐h period, 39% of the water lost from the plant was derived from storage, with flow into storage occurring mainly during the daytime. Simulations showed that the acid accumulation rhythm of CAM had little impact on water uptake from the soil under the conditions employed. In the outer chlorenchyma, water potential and water flows were more sensitive to the day/night changes in transpiration than in osmotic pressure. Nevertheless, cell osmotic pressure had a large influence on turgor pressure in this tissue and determined the extent to which storage was recharged during the latter part of the night.

Day-night changes in leaf water relations associated with the rhythm of crassulacean acid metabolism in Kalanchoë daigremontiana

A study was made of the day-night changes under controlled environmental conditions in the bulk-leaf water relations of Kalanchoë daigremontiana, a plant showing Crassulacean acid metabolism. In addition to nocturnal stomatal opening and net CO2 uptake, the leaves of well-watered plants showed high rates of gas exchange during the whole of the second part of the light period. Measurements with the pressure chamber showed that xylem tension increased during the night and then decreased towards a minimum at about midday; a significant increase in xylem tension was also seen in the late afternoon. Cell-sap osmotic pressure paralleled leaf malate content and was maximum at dawn and minimum at dusk. The relationship between these two variables indicated that the nocturnally synthesized malate was apparently behaving as an ideal osmoticum. To estimate bulk-leaf turgor pressure, values for water potential were derived by correcting the pressurechamber readings for the osmotic pressure of the xylem sap. This itself was found to depend on the malate content of the leaves. Bulk-leaf turgor pressure changed rhythmically during the day-night cycle; turgor was low during the late afternoon and for most of the night, but increased quickly to a maximum of 0.20 MPa around midday. In water-stressed plants, where net CO2 uptake was restricted to the dark period, there was also an increase in bulk-leaf turgor pressure at the start of the light period, but of reduced magnitude. Such changes in turgor pressure are likely to be of considerable ecological importance for the water economy of crassulacean-acid-metabolism plants growing in their natural habitats.

Mechanism of passive malic-acid efflux from vacuoles of the CAM plantKalanchoë daigremontiana

An analysis was carried out of the mechanism of malic-acid efflux from vacuoles of mesophyll cells of the crassulacean acid metabolism (CAM) plantKalanchoe daigremontiana. Following its accumulation in the vacuole as a result of nocturnal CO2 fixation, the malic acid is passively transported back across the tonoplast in the subsequent light period and is decarboxylated in the cytoplasm. Malic-acid efflux was studied using leaf slices in solution or by following malic-acid utilization (deacidification) in leaves of intact plants. Samples of leaf-cell sap were taken at different times during the day-night rhythm to establish the relation between cell-sap pH and malate content. From the empirically determined pK values for malic acid in the cell sap, it was then possible to calculate the proportion of malate existing as the undissociated acid (H2mal0) and in the anionic forms (Hmal1− and mal2−) for all times during the CAM rhythm. In leaf-slice experiments it has been found that the rate of malic-acid efflux increases exponentially with the malic-acid content of the tissue. This is shown to be related to the increasing amounts of H2mal0 present at high malic-acid contents. At low malic-acid contents (<65 mol m−3), when H2mal0 is not present in significant amounts, efflux must be in the form of Hmal−1 and/or mal2−. At high malic-acid contents it is suggested that efflux occurs predominantly in the form of passive, noncatalyzed diffusion of H2mal0 across the tonoplast by a ‘lipid-solution’ mechanism. This is supported by the fact that the slope of the curve relating efflux to H2mal0 concentration, when corrected for the presumed contributions from Hmal1− and mal2− transport and plotted on a log-log basis, approaches 1.0 at the highest malic-acid contents. Moreover, the permeability coefficient required to be consistent with such a mechanism\((P_{H_2 mal^0 } = 1.0to2.0 \times 10^{ - 8} m\sec ^{ - 1} )\) is similar to that estimated from a Collander plot, using the partition coefficient of malic acid between ether and water. We suggest that\(P_{H_2 mal^0 } \) may be important in determining the maximum amounts of malic acid that can be accumulated during the CAM rhythm.

Cytoplasmic pH and the Control of Crassulacean Acid Metabolism

Summary Isolated phyllodia of Kalanchoe tubiflora were treated in various ways to elucidate the involvement of cytoplasmic pH in crassulacean acid metabolism (CAM). Solutes were applied via the cut ends of phyllodia and distributed in the tissue by transpiration. Relative bulk cytoplasmic pH was determined using the 14 C-DMO technique. Large concentrations of the weak acid DMO (50 mM) reduced cytoplasmic pH and inhibited malic-acid mobilization during the light period. Conversely, weak bases increased cytoplasmic pH and inhibited malic-acid accumulation during the dark period. Fusicoccin had similar effects as the weak bases. Treatments inhibiting or preventing malate mobilization during the normal light period, such as prolonged darkness, DCMU or reduced turgor due to application of 200 mM mannitol, eliminated the changes of cytoplasmic pH characteristic of CAM leaf cells during the light period. The uncoupler and protonophore CCCP stimulated passive malic-acid efflux from slices of phyllodia, but other metabolic inhibitors were without effect. The results are discussed in terms of the influence of cytoplasmic pH on the control of the CAM rhythm.

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.

Effects of Osmotic Gradients on Vacuolar Malic Acid Storage

Malate synthesis by CO(2) dark fixation and malate accumulation in the vacuoles of leaf slices of Kalanchoë daigremontiana Hamet et Perrier, a plant performing crassulacean acid metabolism, occurs only in external solutions where the osmotic pressure difference between the cells and the medium is low. Conversely, malate loss from the vacuoles depends on a high osmotic pressure difference between the cells and the medium and is observed in media of low osmotic pressure. This suggests that the diurnal oscillations of malate levels in crassulacean acid metabolism leaf cells are regulated by osmotic gradients. These findings support a model which is introduced to explain how the rhythm of crassulacean acid metabolism may function in the intact plant.

Potassium Independence of Osmoregulated Oscillations of Malate2- Levels in the Cells of CAM -Leaves

On an equivalent basis equal amounts of H + and malate 2- are stored and mobilized, respectively, in the diurnal rhythm of CAM (crassulacean acid metabolism). In intact plants of Bryophyllum daigremontianum this is independent of the levels of K + (between about 10 and 100 mM) and Cl - (between about 10 and 50 mM) in the leaves. Valinomycin, a K + ionophore, does not affect this rhythm. In salinity induced CAM in Mesembryanthemum crystallinum the stoichiometry of diurnal changes of H + : 1/2mal 2- of unity is even observed in the presence of very large levels of salt. Leaf slices of Bryophyllum daigremontianum obtained from leaves harvested at the end of the dark phase when leaves have large levels of malic acid, loose H + and malate 2- to external media, again with a stoichiometry of unity on an equivalent basis. The malic acid movement and the H + : 1/2mal 2- = 1 stoichiometry is independent of concomitant K + movements and of K + levels in the tissue. Valinomycin has no effect on these movements. Experiments with an uncoupler (FCCP) and with varied temperature suggest that loss to the medium is a passive process. It is concluded that the diurnal rhythm in H + and malate 2- levels and the associated movements of H + and malate 2- into and out of the vacuoles L ÜTTGE and B ALL , 1974a; L ÜTTGE et al., 1975), that 2H + + malate 2- transport at the tonoplast of CAM leaf cells is under osmoregulatory control. The results described here are compared with other osmoregulatory and turgor dependent processes where K + does play a role.