Crassulacean Acid Metabolism Cycle Research Articles

Correlation between citric acid and nitrate metabolisms during CAM cycle in the atmospheric bromeliad Tillandsia pohliana

Crassulacean acid metabolism (CAM) confers crucial adaptations for plants living under frequent environmental stresses. A wide metabolic plasticity can be found among CAM species regarding the type of storage carbohydrate, organic acid accumulated at night and decarboxylating system. Consequently, many aspects of the CAM pathway control are still elusive while the impact of this photosynthetic adaptation on nitrogen metabolism has remained largely unexplored. In this study, we investigated a possible link between the CAM cycle and the nitrogen assimilation in the atmospheric bromeliad Tillandsia pohliana by simultaneously characterizing the diel changes in key enzyme activities and metabolite levels of both organic acid and nitrate metabolisms. The results revealed that T. pohliana performed a typical CAM cycle in which phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase phosphorylation seemed to play a crucial role to avoid futile cycles of carboxylation and decarboxylation. Unlike all other bromeliads previously investigated, almost equimolar concentrations of malate and citrate were accumulated at night. Moreover, a marked nocturnal depletion in the starch reservoirs and an atypical pattern of nitrate reduction restricted to the nighttime were also observed. Since reduction and assimilation of nitrate requires a massive supply of reducing power and energy and considering that T. pohliana lives overexposed to the sunlight, we hypothesize that citrate decarboxylation might be an accessory mechanism to increase internal CO 2 concentration during the day while its biosynthesis could provide NADH and ATP for nocturnal assimilation of nitrate. Therefore, besides delivering photoprotection during the day, citrate might represent a key component connecting both CAM pathway and nitrogen metabolism in T. pohliana; a scenario that certainly deserves further study not only in this species but also in other CAM plants that nocturnally accumulate citrate.

Discrimination in the dark. Resolving the interplay between metabolic and physical constraints to phosphoenolpyruvate carboxylase activity during the crassulacean acid metabolism cycle.

A model defining carbon isotope discrimination (delta13C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO2 response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance, and online delta13C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of delta13C were higher than predicted from the ratio of intercellular to external CO2 (p(i)/p(a)) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, g(i), was 0.044 mol m(-2) s(-1) bar(-1). A higher estimate of g(i) (0.085 mol m(-2) s(-1) bar(-1)) was needed to account for the modeled and measured delta18O discrimination throughout the dark period. The differences in estimates of g(i) from the two isotope measurements, and an offset of -5.5 per thousand between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase delta13C discrimination during nighttime CO2 fixation while reducing delta13C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.

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

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

The "Kluge-Lüttge Kammer": A Preliminary Evaluation of an Enclosed, Crassulacean Acid Metabolism (CAM) Mesocosm that Allows Separation of Synchronized and Desynchronized Contributions of Plants to Whole System Gas Exchange

Crassulacean acid metabolism (CAM) is recognized as a photosynthetic adaptation of plants to arid habitats. This paper presents a proof-of-concept evaluation of partitioning net CO2 exchanges for soil and plants in an arid, exclusively CAM mesocosm, with soil depth and succulent plant biomass approximating that of natural Sonoran Desert ecosystems. We present the first evidence that an enclosed CAM-dominated soil and plant community exposed to a substantial day/night temperature difference (30/20 degrees C), exhibits a diel gas exchange pattern consisting of four consecutive phases with a distinct nocturnal CO2 uptake. These phases were modulated by plant assimilation and soil respiration processes. Day-time stomatal closure of the CAM cycle during phase III was used to eliminate aboveground photosynthetic assimilation and respiration and thereby to estimate belowground plant plus soil respiration. Rapid changes in temperature appeared to synchronize single plant gas exchange but individual plant gas exchange patterns were desynchronized at constant day/night temperatures (25 degrees C), masking the distinct mesocosm pattern. Overall, the mean carbon budget of this CAM model Sonoran Desert system was negative, releasing an average of 22.5 mmol CO2 m-2 d-1. The capacity for nocturnal CO2 assimilation in this exclusively CAM mesocosm was inadequate to recycle CO2 released by plant and soil respiration.

The Occurrence of Crassulacean Acid Metabolism in Epiphytic Ferns, with an Emphasis on the Vittariaceae

The potential for nighttime CO2 uptake and/or increases in tissue acidity characteristic of crassulacean acid metabolism (CAM) was investigated, to varying degrees, in 12 species of Vittariaceae as well as in seven species in four other families of tropical epiphytic ferns. Evidence of CAM (actually CAM cycling, i.e., diel changes in tissue acidity without nighttime atmospheric CO2 uptake), though highly variable, was found in two species of Vittariaceae. The ecophysiological significance of this finding is puzzling, because the occurrence of these plants in deeply shaded, extremely moist habitats is rare, if not unique, among plants expressing any degree of CAM. The results of this study confirm that CAM among the ferns is not limited to the Polypodiaceae, and they emphasize the polyphyletic nature of the evolution of CAM among higher plants.

Lateral diffusion of CO2 in leaves of the crassulacean acid metabolism plant Kalancho� daigremontiana Hamet et Perrier

Dynamic patchiness of photosystem II (PSII) activity in leaves of the crassulacean acid metabolism (CAM) plant Kalanchoe daigremontiana Hamet et Perrier, which was independent of stomatal control and was observed during both the day/night cycle and circadian endogenous oscillations of CAM, was previously explained by lateral CO2 diffusion and CO2 signalling in the leaves [Rascher et al. (2001) Proc Natl Acad Sci USA 98:11801-11805; Rascher and Luttge (2002) Plant Biol 4:671-681]. The aim here was to actually demonstrate the importance of lateral CO2 diffusion and its effects on localized PSII activity. Covering small sections of entire leaves with silicone grease was used for local exclusion of a contribution of atmospheric CO2 to internal CO2 via transport through stomata. A setup for combined measurement of gas exchange and chlorophyll fluorescence imaging was used for recording photosynthetic activity with a spatiotemporal resolution. When remobilization of malic acid from vacuolar storage and its decarboxylation in the CAM cycle caused increasing internal CO2 concentrations sustaining high PSII activity behind closed stomata, PSII activity was also increased in adjacent leaf sections where vacuolar malic acid accumulation was minimal as a result of preventing external CO2 supply due to leaf-surface greasing, and where therefore CO2 could only be supplied by diffusion from the neighbouring malic acid-remobilizing leaf tissue. This demonstrates lateral CO2 diffusion and its effect on local photosynthetic activity.

Expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase genes. Implications for genotypic capacity and phenotypic plasticity in the expression of crassulacean acid metabolism.

In plants with crassulacean acid metabolism (CAM), dark CO2 uptake is mediated by phosphoenolpyruvate carboxylase (PEPC), an enzyme that can be regulated at transcriptional and posttranslational levels. Reversible phosphorylation of PEPC is catalyzed by a dedicated PEPC kinase, which in turn is regulated at the transcriptional level over the 24-h cycle in CAM plants. PEPC kinase controls the day/night regulation of PEPC during the CAM cycle, thus facilitating plasticity for optimizing CO2 uptake under different environmental conditions. To understand the importance of PEPC kinase in relation to its target PEPC in terms of CAM performance, the expression of the genes encoding the two enzymes was investigated in four species of Clusia that have photosynthetic patterns ranging from C3 photosynthesis to constitutive CAM. By linking changes in the expression of PEPC and PEPC kinase to day/night patterns of leaf gas exchange, organic acid, and soluble sugar contents under different environmental conditions, the genetic and metabolic limitations to CAM plasticity were assessed. The results indicate that PEPC expression is a major factor underpinning the genotypic capacity for CAM and that PEPC kinase expression does not appear to limit CAM. The day/night regulation of Ppck transcript abundance was found to be a consequence of CAM and the day/night cycling of associated metabolites, rather than the primary controlling factor for the temporal separation of carboxylation processes.

Two functionally different vacuoles for static and dynamic purposes in one plant mesophyll leaf cell

It is a common belief that plant mesophyll cells are occupied up to 95% by a single multipurpose vacuole. The common ice plant, Mesembryanthemum crystallinum L., however, requires two contrasting functions of the vacuole under salt stress. Large amounts of NaCl have to be sequestered permanently for osmotic purpose and for protecting the cytoplasm from NaCl toxicity. A dynamic exchange with the cytoplasm is required because photosynthesis proceeds under these conditions via the metabolic cycle of crassulacean acid metabolism (CAM). Nocturnally acquired CO2 must be kept as malate in the vacuole and re-mobilized in the daytime. Here, we show that two large independent types of vacuoles with different transport properties meet the requirements for the contrasting functions within the same cell.

Identification of rhythmic subsystems in the circadian cycle of crassulacean acid metabolism under thermoperiodic perturbations.

Leaves of the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier de la Bâthie show overt circadian rhythms in net CO2 uptake, leaf conductance to water and intercellular CO2 concentration, which are entrained by periodic temperature cycles. To probe their sensitivity to thermoperiodic perturbations, intact leaves were exposed to continuous light intensity and temperature cycles with a period of 16 h, applying a set of different baseline temperatures and thermodriver amplitudes. All three overt rhythms were analyzed with respect to their frequency spectra and their phase relations with the thermodriver. For most stimulation protocols, stomatal conductance and net CO2 change were fully or partially entrained by the temperature pulses, while the internal CO2 concentration remained dominated by oscillations in the circadian range. Prolonged time series recorded for up to 22 d in continuous light underline the robustness of these circadian oscillations. This suggests that the overt circadian rhythm of net CO2 uptake in CAM results from the interaction of two coupled original systems: (i) an endogenous cycle of CO2 fixation in the mesophyll, showing very robust periodic activity, and (ii) stomatal movements that respond to environmental stimuli independently of rhythmic processes in the mesophyll, and thus modulate the gas exchange amplitude.

Non-linear regulation in the circadian rhythm of the metabolic cycle of crassulacean acid metabolism (CAM) in higher plants

Non-linear regulation in the circadian rhythm of the metabolic cycle of crassulacean acid metabolism (CAM) in higher plants

A phylogenetic view of low‐level CAM in Pelargonium (Geraniaceae)

Crassulacean acid metabolism (CAM) is common in several plant families and is often associated with succulence. Few studies have examined the occurrence of CAM from a phylogenetic perspective. The genus Pelargonium is promising for such a study because members are characterized by dramatic variation in growth form (including geophytes, shrubs, and stem succulents) and because growth form diversity is expressed to the greatest extent in a monophyletic group comprising 80% of Pelargonium species. This clade, predominantly from the winter rainfall region of southern Africa, likely proliferated in response to Miocene or Pliocene aridification. We present a survey for CAM across Pelargonium, emphasizing the winter rainfall clade. Dawn/dusk fluctuations in titratable acidity were examined in 41 species, with detailed measurements of carbon uptake and stomatal conductance under progressive water stress in four species. No species exhibited obligate CAM. When well-watered, most species exhibited stomatal conductances and acid fluctuations characteristic of C(3) photosynthesis, though some exhibited more pronounced increases in nocturnal acidity, suggesting CAM cycling. In four species examined during dry-down, water stress led to increased nighttime acid levels and decreased daytime stomatal conductance. Ultimately, stomata closed and external carbon uptake ceased, consistent with CAM idling. These results are discussed from the perspective of the evolution of CAM flexibility.

Mode of photosynthesis during different life stages of hemiepiphytic Clusia species.

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. Carbon isotope fractionation and nocturnal acid accumulation in Clusia osaensis Hammel-ined., C. peninsulae Hammel-ined. and C. valerii Standl. were investigated during the seedling, epiphytic and hemiepiphytic phases in a lowland tropical forest in Costa Ricato study photosynthetic adaptations of different plant growth stages to their habitat. Foliar δ 13C values around -24 to -32‰ indicate predominant C3 fixation of CO2 and low crassulacean acid metabolism (CAM) activity in all three Clusia species. Only terrestrially rooted plants of C. osaensis showed increased CAM expression. However, all developmental stages exhibited significant CAM cycling as shown by significant day-night fluctuations of titratable protons and of malic and citric acid. In C. valerii and C. peninsulae, an increase in CAM expression with plant development was not observed, and CAM cycling in hemiepiphytic-stage plants was completely repressed during the high rainfall season. The expression of CAM in the three Clusia species is therefore not developmentally controlled but triggered by environmental factors such as water availability and light intensity. These factors remain relatively stable in this ecosystem and CAM is therefore not fully expressed. However, CAM cycling may be of ecophysiological significance in all life stages as it serves as a mechanism to improve carbon economy by reducing respiratory CO2 losses.

Photosynthetic and anatomical characteristics in the C4–crassulacean acid metabolism-cycling plant Portulaca grandiflora

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. Portulaca grandiflora (Lind.) is a succulent species with C4 photosynthesis and crassulacean acid metabolism (CAM) cycling in leaves, and CAM-idling type photosynthesis in stems. We investigated the level and localization of carbon fixation enzymes and photosynthetic activity of leaves and stems of P. grandiflora under well-watered and drought conditions. As CAM activity increased during water stress, the leaf water-storage tissue collapsed, presumably transferring water to the bundle sheath and mesophyll cells, and so maintaining the C4 photosynthetic pathway. Tissue prints indicated an increase in phosphoenolpyruvate carboxylase (PEPC) in the water-storage tissue of leaves and the cortex of stems. Immunoblot analyses after 10 d of water stress showed that leaves had a slight decrease in the proteins of the C4-CAM pathway, while at the same time a new isoform of NADP-malic enzyme (NADP-ME) appeared. In contrast, the stem showed increases in proteins of the CAM pathway when water stressed. Under water stress, diurnal fluctuation in acidity in leaves was not accompanied by a net gain or loss of CO2 at night, and there was sustained, but decreased, fixation of CO2 during the day, characteristic of CAM cycling. High gross rates of O2 evolution were maintained during the day under water stress, suggesting induction of alternative electron sinks. With induced diurnal fluctuations in acidity in stems, there was no net carbon gain during the day or night. These results demonstrate, for the first time, that the stem of P. grandiflora is an inducible CAM-idling tissue. Our results also indicate that the C4 and CAM pathways operate independently of one another in P. grandiflora.

Sucrose transport across the vacuolar membrane of Ananas comosus.

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. In Ananas comosus L. (Merr.) (pineapple), a widely cultivated bromeliad that exhibits crassulacean acid metabolism (CAM), much of the carbohydrate synthesized during the daytime appears to accumulate as soluble sugars in the vacuole. To investigate the mechanism of sugar transport into the vacuole, microsomal extracts were prepared from deacidifying leaves harvested during Phase III of the CAM cycle. The vesicle preparations exhibited features expected for a fraction highly enriched in vacuolar membranes (tonoplast), i.e. the ATPase activity of 16 ±2nkat mg-1 protein was inhibited 96% by 50 mm KNO3, an inhibitor of vacuolar ATPases, and was only 7% inhibited by 100μm NaN3 plus 100μm Na3VO4, inhibitors of mitochondrial and plasma membrane ATPases, respectively. Further, the microsomal ATPase activity showed a pH optimum between 7.0 and 8.0, typical of a vacuolar ATPase. When presented with Mg-ATP, vesicles established H+ gradients that could be maintained for at least 25 min. The vesicles were able to take up [14C]sucrose from an external medium. Sucrose uptake exhibited saturable kinetics with an apparent Km of 50 m sucrose and apparent Vmax of 171 ± 5 pkat mg-1 protein. Sucrose uptake was not dependent upon, nor stimulated by, Mg-ATP, suggesting that the mechanism of sucrose transport into the vacuole in A. comosus does not involve H+-coupled cotransport. However, the initial rates of sucrose uptake from the external medium were stimulated when vesicles were preloaded with sucrose. This trans-stimulation is consistent with characteristics expected for a sucrose uniporter capable of operating in an exchange mode. It is proposed that the accumulation of glucose and fructose in leaf vacuoles of Ananas during the light period involves at least two steps - transport of sucrose into the vacuole by a mechanism exhibiting characteristics of a sucrose uniporter, followed by cleavage of sucrose by a vacuolar acid invertase to form glucose and fructose.

Regulation of Rubisco activity in crassulacean acid metabolism plants: better late than never

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. The diurnal regulation of Rubisco was compared for a range of crassulacean acid metabolism (CAM) species in the context of high carboxylation and electron transport capacities, which may be an order of magnitude greater than rates of net CO2 uptake. Early in the light period, Rubisco activity and electron transport were limited when phosphoenolpyruvate carboxylase (PEPC) may have been operating, and maximal extractable activities and activation state for Rubisco were achieved at the end of Phase III, prior to the direct atmospheric uptake of CO2 during Phase IV. The delayed activation was associated with levels of Rubisco activase protein, which reached a maximum at midday, and may account for this pattern of Rubisco activation. This regulation may be modified by environmental conditions - processes that tend to restrict PEPC activity, such as drought stress or incubation of leaves overnight in an oxygen-free atmosphere, release Rubisco from inhibition early in the light period. The quantum yield of light use also tracks Rubisco carboxylation, being particularly low at dawn when PEPC is active. The plasticity in expression of the CAM cycle is therefore matched by the regulation of key carboxylases, with extractable Rubisco activity maximal when drawdown of atmospheric CO2 to cells in succulent CAM tissues is most likely to limit photon utilization shortly after midday, during Phase IV.

Metabolic control of photosynthetic electron transport in crassulacean acid metabolism-induced Mesembryanthemum crystallinum.

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. We investigated photosynthetic electron transport in leaves of the facultative crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum L. After CAM induction, electron transport exhibited variable redox kinetics during the diurnal CAM cycle. In CAM Phase IV, most of PSI (P700) and chlorophyll a fluorescence relaxed with a halftime of 20 ms after a saturating light pulse. This time-constant may reflect the overall linear electron flux from PSII to PSI in saturating light. Comparable relaxation kinetics were also determined for C3 plants. At the end of CAM Phase I and during Phase II, slow components (> 50% of signal amplitude) appeared in both P700 reduction and fluorescence relaxation. They displayed halftimes > 250 ms and > 1 s, suggesting a strong restriction of the linear electron flux from H2O to NADP. The appearance of the slow redox components was accompanied by a decrease in the Fv/Fm ratio of chlorophyll a fluorescence, suggesting a reversible detachment of light-harvesting complex (LHC) II from PSII. The slow redox fractions and the depression of Fv/Fm disappeared again in parallel to malate decarboxylation during CAM Phase III. We discuss a reversible downregulation of linear electron flux during CAM Phases II and III, due to a reversible deprivation of cytochrome-b6f complexes (cyt-bfs) and PSI from the linear system. In parallel, a redistribution of some LHCIIs could also occur. This could be an adaptive response to a reduced metabolic demand for NADPH due to a limited carbon flux through the Calvin cycle, resulting from low Rubisco activation. Furthermore, the cyt-bfs and PSIs deprived of linear electron transport could support cyclic electron flux to cover an increased ATP demand during gluconeogenesis in CAM Phase III.

Plastidic metabolite transporters and their physiological functions in the inducible crassulacean acid metabolism plant Mesembryanthemum crystallinum.

The inducible crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum accumulates malic acid during the night and converts it to starch during the day via a pathway that, because it is located in different subcellular compartments, depends on specific metabolite transport across membranes. The chloroplast glucose transporter (pGlcT) and three members of the phosphate translocator (PT) family were isolated. After induction of CAM, transcript amounts of the phosphoenolpyruvate (PEP) phosphate translocator (PPT) and the glucose-6-phosphate (Glc6P) phosphate translocator (GPT) genes were increased drastically, while triose phosphate (TP) phosphate translocator (TPT) and the pGlcT transcripts remained unchanged. PPT- and GPT-specific transcripts and transporter activities exhibited a pronounced diurnal variation, displaying the highest amplitude in the light. pGlcT transcripts were elevated towards the end of the light period and at the beginning of the dark period. These findings, combined with diurnal variations of enzyme activities and metabolite contents, helped to elucidate the roles of the PPT, GPT, TPT and pGlcT in CAM. The main function of the PPT is the daytime export from the stroma of PEP generated by pyruvate orthophosphate:dikinase (PPDK). The increased transport activity of GPT in the light suggests a higher requirement for Glc6P import for starch synthesis rather than starch mobilization. Most likely, Glc6P rather than 3-phosphoglycerate or triose phosphates is the main substrate for daytime starch biosynthesis in M. crystallinum plants in which CAM has been induced (CAM-induced), similar to non-green plastids. In the dark, starch is mobilized both phosphorylytically and amylolytically and the products are exported by the GPT, TPT and pGlcT. The transport activities of all three phosphate translocators and the transcript amounts of the pGlcT adapt to changing transport requirements in order to maintain high metabolic fluxes during the diurnal CAM cycle.

Oscillatory model of crassulacean acid metabolism: structural analysis and stability boundaries with a discrete hysteresis switch

We propose a simple oscillatory model of crassulacean acid metabolism (CAM) describing the CO2 uptake and nocturnal acidification of CAM plants by a system of coupled non‐linear differential equations. Large differences in the content of metabolite pools are treated using a pseudo‐steady‐state approach. For the first time, simulations of the CAM cycle investigate its dependence on all three major control parameters simultaneously: temperature, photon flux density and external CO2 concentration. Under stationary conditions in time the model shows either endogenous rhythmicity or two distinct steady states. Stability boundaries are calculated in parameter space.

Malate transport and vacuolar ion channels in CAM plants

Malate is a ubiquitous vacuolar anion in terrestrial plants that plays an important role in carbon metabolism and ionic homeostasis. In plants showing crassulacean acid metabolism (CAM), malate is accumulated as a central intermediary in the process of photosynthetic carbon assimilation, and it is also one of the major charge-balancing anions present in the vacuole. During the CAM cycle, malic acid produced as a result of dark CO(2) fixation accumulates in the vacuole at night (2 H(+) per malate), and is remobilized from the vacuole in the following light period. CAM plants thus provide a good model for studying both the mechanism and control of malate transport across the tonoplast. Thermodynamic considerations suggest that malate(2-) (the anionic species transported out of the cytosol) is passively distributed across the tonoplast. Malic acid accumulation could thus be explained by malate(2-) transport into the vacuole occurring electrophoretically in response to the transmembrane electrical potential difference established by the tonoplast H(+)-ATPase and/or H(+)-PPase. Recent studies using the patch-clamp technique have provided evidence for the existence of a vacuolar malate-selective anion channel (VMAL) in both CAM species and C(3) species. The VMAL current has a number of distinctive properties that include strong rectification (opening only at cytosolicside negative membrane potentials that would favour malate uptake into the vacuole), lack of Ca(2+) dependence, and slow activation kinetics. The kinetics of VMAL activation can be resolved into three components, consisting of an instantaneous current and two slower components with voltage-independent time constants of 0.76 s and 5.3 s in Kalanchoë daigremontiana. These characteristics suggest that the VMAL channel represents the major pathway for malate transport into the vacuole, although the strong rectification of the channel means there may be a separate, still-to-be-identified, transport mechanism for malate efflux.

Fluorescence Quenching in the Varied Photosynthetic Modes of Portulacaria afra (L.) Jacq.

The kinetics of chlorophyll fluorescence were measured in Portulacaria afra (L.) Jacq. when the plants were functioning in either Crassulacean acid metabolism (CAM) or C(3)/CAM cycling (called cycling) modes, as determined by fluctuation in titratable acidity and gas exchange properties. Cycling plants showed primarily daytime CO(2) uptake typical of C(3) plants, but with a slight diurnal acid fluctuation, whereas CAM plants showed nocturnal CO(2) uptake, daytime stomatal closure, and a large diurnal acid fluctuation. Results from fluorescence measurements indicated no significant differences in photochemical quenching between cycling and CAM plants; however, sizable differences were detected in nonphoto-chemical quenching (q(n)), with the largest differences being observed during the middle of the day. Cycling plants had lower q(n) than CAM plants, indicating altered photosynthetic regulation processes. This q(n) difference was believed to be related to reduced internal CO(2) concentration in the CAM plants because of daytime stomatal closure and reduced deacidification rates in the late afternoon when most of the malic acid has been utilized. Experimentally, higher external CO(2) given to plants in the CAM mode resulted in a decline in q(n) in comparison to that measured in plants in the cycling mode. No changes were observed in photochemical quenching when CO(2) was added.