CO(2)-concentrating: consequences in crassulacean acid metabolism

The consequences of CO(2)-concentrating in leaf air-spaces of CAM plants during daytime organic acid decarboxylation in Phase III of CAM (crassulacean acid metabolism) are explored. There are mechanistic consequences of internal CO(2) partial pressures, p(i)(CO(2)). These are (i) effects on stomata, i.e. high p(i)(CO(2)) eliciting stomatal closure in Phase III, (ii) regulation of malic acid remobilization from the vacuole, malate decarboxylation and refixation of CO(2) via Rubisco (ribulose bisphosphate carboxylase/oxygenase), and (iii) internal signalling functions during the transitions between Phases II and III and III and IV, respectively, in the natural day/night cycle and in synchronizing the circadian clocks of individual leaf cells or leaf patches in the free-running endogenous rhythmicity of CAM. There are ecophysiological consequences. Obvious beneficial ecophysiological consequences are (i) CO(2)-acquisition, (ii) increased water-use- efficiency, (iii) suppressed photorespiration, and (iv) reduced oxidative stress by over-energization of the photosynthetic apparatus. However, the general potency of these beneficial effects may be questioned. There are also adverse ecophysiological consequences. These are (i) energetics, (ii) pH effects and (iii) Phase III oxidative stress. A major consequence of CO(2)-concentrating in Phase III is O(2)-concentrating, increased p(i)(CO(2)) is accompanied by increased p(i)(O(2)). Do reversible shifts of C(3)/CAM-intermediate plants between the C(3)-CAM-C(3) modes of photosynthesis indicate that C(3)-photosynthesis provides better protection from irradiance stress? There are many open questions and CAM remains a curiosity.     

Carbon dioxide signalling in plant leaves

The role of carbon dioxide (CO(2)) as a signal in biochemical regulation networks of plants is fathomed. Transport mechanisms of CO(2) and HCO3- are surveyed, which are the prerequisite for signalling. A CO(2) sensor is not known to date, but any reaction where CO(2)/HCO3- is a substrate can be a candidate. Carbon concentrating mechanisms, e.g., in higher plants C(4)-photosynthesis and crassulacean acid metabolism (CAM), generate high internal CO(2) concentrations, important for photosynthesis, but also as a basis for signalling via diffusion of CO(2). Spatiotemporal dynamics of desynchronization/synchronization of photosynthetic activity over leaves can be followed by chlorophyll fluorescence imaging. One example of desynchronization is based on patchiness of stomatal opening/closing in heterobaric leaves due to anatomic constraints of lateral CO(2) diffusion. During CAM, largely different internal CO(2) concentrations prevail in the leaves, offering opportunities to study the effect of lateral diffusion of CO(2) in synchronizing photosynthetic activity over the entire leaves.     

Influence of Photoperiod and Leaf Age on Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq

The possibility that Crassulacean acid metabolism (CAM) is subject to long day photoperiodic control in Portulacaria afra (L.) Jacq., a facultative CAM plant, was studied. Periodic measurements of ¹⁴CO₂ uptake, stomatal resistance, and titratable acidity were made on plants exposed to long and short day photoperiods. Results indicates that waterstressed P. afra had primarily nocturnal CO₂ uptake, daytime stomatal closure, and a large diurnal acid fluctuation in either photoperiod. Mature leaf tissue from nonstressed plants under long days exhibited a moderate diurnal acid fluctuation and midday stomatal closure. Under short days, there was a reduced diurnal acid fluctuation in mature leaf tissue. Young leaf tissue taken from nonstressed plants did not utilize the CAM pathway under either photoperiod as indicated by daytime CO₂ uptake, lack of diurnal acid fluctuation, and incomplete daytime stomatal closure.

The induction of CAM in P. afra appears to be related to the water status of the plant and the age of the leaf tissue. The photosynthetic metabolism of mature leaves may be partly under the control of water stress and of photoperiod, where CAM is favored under long days.

     

Diel leaf growth cycles in Clusia spp. are related to changes between C3 photosynthesis and crassulacean acid metabolism during development and during water stress

This study reports evidence that the timing of leaf growth responds to developmental and environmental constraints in Clusia spp. We monitored diel patterns of leaf growth in the facultative C₃-crassulacean acid metabolism (CAM) species Clusia minor and in the supposedly obligate CAM species Clusia alata using imaging methods and followed diel patterns of CO₂ exchange and acidification. Developing leaves of well-watered C. minor showed a C₃-like diel pattern of gas exchange and growth, with maximum relative growth rate (RGR) in the early night period. Growth slowed when water was withheld, accompanied by nocturnal CO₂ exchange and the diel acid change characteristic of CAM. Maximum leaf RGR shifted from early night to early in the day when water was withheld. In well-watered C. alata , similar changes in the diel pattern of leaf growth occurred with the development of CAM during leaf ontogeny. We hypothesize that the shift in leaf growth cycle that accompanies the switch from C₃ photosynthesis to CAM is mainly caused by the primary demand of CAM for substrates for nocturnal CO₂ fixation and acid synthesis, thus reducing the availability of carbohydrates for leaf growth at night. Although the shift to leaf growth early in the light is presumably associated with the availability of carbohydrates, source–sink relationships and sustained diurnal acid levels in young leaves of Clusia spp. need further evaluation in relation to growth processes.     

Responses of succulents to plant water stress

Experiments were performed to test the hypothesis that succulents “shift” their method of photosynthetic metabolism in response to environmental change. Our data showed that there were at least three different responses of succulents to plant water status. When plant water status of Portulacaria afra (L.) Jacq. was lowered either by withholding water or by irrigating with 2% NaCl, a change from C₃-photosynthesis to Crassulacean acid metabolism (CAM) occurred. Fluctuation of titratable acidity and nocturnal CO₂ uptake was induced in the stressed plants. Stressed Peperomia obtusifolia A. Dietr. plants showed a change from C₃-photosynthesis to internal cycling of CO₂. Acid fluctuation commenced in response to stress but exogenous CO₂ uptake did not occur. Zygocactus truncatus Haworth plants showed a pattern of acid fluctuation and nocturnal CO₂ uptake typical of CAM even when well irrigated. The cacti converted from CAM to an internal CO₂ cycle similar to Peperomia when plants were water-stressed. Reverse phase gas exchange in succulents results in low water loss to carbon gain. Water is conserved and low levels of metabolic activity are maintained during drought periods by complete stomatal closure and continual fluctuation of organic acids.     

Age-specific changes of acidity, phosphoenolpyruvate carboxylase, ribulose-1,5-bisphosphate carboxylase/oxygenase, abscisic acid and leaf water potential in Mesembryanthemum nodiflorum

Age-induced changes in 1) nocturnal and diurnal acidity fluctuations that coincide with the ongoing environmental conditions, 2) the build up of abscisic acid (ABA) in plant roots and leaves during sunrise, midday, and sunset in all growing stages, 3) the changes in phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activities as key enzymes of the photosynthetic pathways of C3 and CAM, 4) leaf water potential (ψ1), and 5) Km and Vmax for PEPC to express its activity and affinity, were studied in Mesembryanthemum nodiflorum during transition from C3 to CAM mode of CO2 fixation. The acidity during sunset in mature stage was higher than in earlier stages and reflected the impact of environmental conditions on physiological and metabolic changes. Moreover, the higher acidity during sunrise and sunset was observed during the senescence than the mature stage; this might be due to CO2 release and oxygen intake during senescence induced ethylene formation that lead to increased malic acid formation. The ABA concentration was high in M. nodiflorum leaves, but stomatal closure was insensitive to elevated ABA concentrations recorded. Vmax of PEPC, Km, and the affinity of PEPC during later stages indicated the ability of PEPC to fix CO2 taking up at night in CAM cycle of M. nodiflorum. Less affinity during sunrise indicated inhibitory effect of malate on PEPC during the release of CO2. The second peak of PEPC activity before sunset caused CO2 fixation. The RuBPCO was inactive at night. Slight increase in ABA during sunset, and night drop in air temperature and increase in relative humidity reduced markedly transpiration rate without decreasing ψ1. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Induction of CAM in Mesembryanthemum crystallinum Abolishes the Stomatal Response to Blue Light and Light-Dependent Zeaxanthin Formation in Guard Cell Chloroplasts

Facultative CAM plants such as Mesembryanthemum crystallinum(ice plant) possess C3 metabolism when unstressed but developCAM under water or salt stress. When ice plants shift from C3metabolism to CAM, their stomata remain closed during the dayand open at night. Recent studies have shown that the stomatalresponse of ice plants in the C3 mode depends solely on theguard cell response to blue light. Recent evidence for a possiblerole of the xanthophyll, zeaxanthin in blue light photoreceptionof guard cells led to the question of whether changes in theregulation of the xanthophyll cycle in guard cells parallelthe shift from diurnal to nocturnal stomatal opening associatedwith CAM induction. In the present study, light-dependent stomatalopening and the operation of the xanthophyll cycle were characterizedin guard cells isolated from ice plants shifting from C3 metabolismto CAM. Stomata in epidermis detached from leaves with C3 metabolismopened in response to white light and blue light, but they didnot open in response to red light. Guard cells from these leavesshowed light-dependent conversion of violaxan-thin to zeaxanthin.Induction of CAM by NaCI abolished both white light- and bluelight-stimulated stomatal opening and light-dependent zeaxanthinformation. When guard cells isolated from leaves with CAM weretreated with 100 mM ascorbate, pH 5.0 for 1 h in darkness, guardcell zeaxanthin content increased at rates equal to or higherthan those stimulated by light in guard cells from leaves inthe C3 mode. The ascorbate effect indicates that chloroplastsin guard cells from leaves with CAM retain their competenceto operate the xanthophyll cycle, but that zeaxanthin formationdoes not take place in the light. The data suggest that inhibitionof light-dependent zeaxanthin formation in guard cells mightbe one of the regulatory steps mediating the shift from diurnalto nocturnal stomatal opening typical of plants with CAM. (Received July 5, 1996; Accepted December 12, 1996)     

Chlorophyll Fluorescence and Organic Acid Oscillations during Transition from CAM to C3-photosynthesis inClusia minor L. (Clusiaceae)

In species of Clusia, switching from C₃-photosynthesis (C₃-PS)to crassulacean acid metabolism (CAM) may be a means of optimizingwater use, plant carbon balance and photon utilization duringperiods of stress. We ask whether, in perennial species of Clusia,the switch from CAM back to C₃-PS is also of ecophysiologicalsignificance. Our objective was to investigate the performanceof C. minor L. during a short-term shift from CAM to C₃-PS.During the transition from CAM to C₃-PS, nocturnal malate andcitrate accumulation decreased whereas CO₂uptake increased duringthe daytime. However, after 7 d, marked nocturnal accumulationof citrate and 24 h CO₂uptake occurred. In contrast to C₃-likephotosynthesis, a pronounced reduction in the effective quantumyield of photosystem II,     

The stomata of the fern Adiantum capillus-veneris do not respond to CO2 in the dark and open by photosynthesis in guard cells

The stomata of the fern Adiantum capillus-veneris lack a blue light-specific opening response but open in response to red light. We investigated this light response of Adiantum stomata and found that the light wavelength dependence of stomatal opening matched that of photosynthesis. The simultaneous application of red (2 micromol m(-2) s(-1)) and far-red (50 micromol m(-2) s(-1)) light synergistically induced stomatal opening, but application of only one of these wavelengths was ineffective. Adiantum stomata did not respond to CO2 in the dark; the stomata neither opened under a low intercellular CO2 concentration nor closed under high intercellular CO2 concentration. Stomata in Arabidopsis (Arabidopsis thaliana), which were used as a control, showed clear sensitivity to CO2. In Adiantum, stomatal conductance showed much higher light sensitivity when the light was applied to the lower leaf surface, where stomata exist, than when it was applied to the upper surface. This suggests that guard cells likely sensed the light required for stomatal opening. In the epidermal fragments, red light induced both stomatal opening and K+ accumulation in guard cells, and both of these responses were inhibited by a photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The stomatal opening was completely inhibited by CsCl, a K+ channel blocker. In intact fern leaves, red light-induced stomatal opening was also suppressed by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that Adiantum stomata lack sensitivity to CO2 in the dark and that stomatal opening is driven by photosynthetic electron transport in guard cell chloroplasts, probably via K+ uptake.     

Clusia: Holy Grail and enigma

Clusia is the only genus with bona fide dicotyledonous trees performing Crassulacean acid metabolism (CAM). Clusia minor L. is extraordinarily flexible, being C(3)/CAM intermediate and expressing the photosynthetic modes C(3), CAM, CAM-cycling, and CAM-idling. C(3) photosynthesis and CAM can be observed simultaneously in two opposite leaves on a node and possibly even within the same leaf in the interveinal lamina and the major vein tissue, respectively. The relative activity of photosystem II (PhiPSII) indicating photosynthetic energy use, is larger under photorespiratory than under non-photorespiratory conditions due to the particular energy demand of photorespiration. The heterogeneity of PhiPSII over the leaves as visualized by chlorophyll fluorescence imaging in the C(3) mode is larger under non-photorespiratory conditions than under photorespiratory conditions. These observations indicate that photorespiration, presumably by its particular energy demand, synchronizes photosynthetic activity over the leaves. In the CAM mode, the heterogeneity of PhiPSII is more dependent on the transitions between CAM phases. Free-running circadian oscillations of photosynthesis are strongly dampened in both the C(3) and the CAM mode. Photorespiration is under circadian clock control in both the C(3) and the CAM mode. PhiPSII and the heterogeneity of PhiPSII oscillate in phase with CO(2) uptake and photorespiration only under non-photorespiratory conditions. Under photorespiratory conditions, PhiPSII does not oscillate and there is no heterogeneity, again indicating the stabilizing function of photorespiration. Plants acclimatized to perform CAM switch to C(3) photosynthesis during free-running oscillations while subjected to constant illumination.     

Effects of Various Levels of CO(2) on the Induction of Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq

In response to water stress, Portulacaria afra (L.) Jacq. (Portulacaceae) shifts its photosynthetic carbon metabolism from the Calvin-Benson cycle for CO₂ fixation (C₃) photosynthesis or Crassulacean acid metabolism (CAM)-cycling, during which organic acids fluctuate with a C₃-type of gas exchange, to CAM. During the CAM induction, various attributes of CAM appear, such as stomatal closure during the day, increase in diurnal fluctuation of organic acids, and an increase in phosphoenolpyruvate carboxylase activity. It was hypothesized that stomatal closure due to water stress may induce changes in internal CO₂ concentration and that these changes in CO₂ could be a factor in CAM induction. Experiments were conducted to test this hypothesis. Well-watered plants and plants from which water was withheld starting at the beginning of the experiment were subjected to low (40 ppm), normal (ca. 330 ppm), and high (950 ppm) CO₂ during the day with normal concentrations of CO₂ during the night for 16 days. In water-stressed and in well-watered plants, CAM induction as ascertained by fluctuation of total titratable acidity, fluctuation of malic acid, stomatal conductance, CO₂ uptake, and phosphoenolpyruvate carboxylase activity, remained unaffected by low, normal, or high CO₂ treatments. In well-watered plants, however, both low and high ambient concentrations of CO₂ tended to reduce organic acid concentrations, low concentrations of CO₂ reducing the organic acids more than high CO₂. It was concluded that exposing the plants to the CO₂ concentrations mentioned had no effect on inducing or reducing the induction of CAM and that the effect of water stress on CAM induction is probably mediated by its effects on biochemical components of leaf metabolism.     

Photosynthetic flexibility in Pedilanthus tithymaloides poit,a CAM plant

The induction of CAM in Pedilanthus tithymaloides (Euphorbiaceae) under water-limited conditions was evaluated by following diurnal oscillations of CO2 fixation, titratable acidity and malic acid content in the leaf extracts. CAM induction was assessed by measuring the activities of phosphoenolpyruvate carboxylase (PEPC), NADH-malate dehydrogenase (MDH) and phosphoenolpyruvate caroxykinase (PEPCK) in the leaves as well. Drought resulted in large increases in the nocturnal acid accumulation and rates of CO2 uptake in the leaves of P. tithymaloides. The drought-induced CAM activity tended to be reversible after re-watering. Nevertheless, under well-watered conditions, plants of P. tithymaloides showed day time CO2 uptake patterns with less pronounced diurnal oscillations of organic acids. Our data indicate that although P. tithymaloides is a CAM plant, environmental variables like drought induce photosynthetic flexibility in this species. This type of plasticity in CAM and metabolic versatility in P. tithymaloides should be an adaptation for prolonged survival under natural adverse edaphic and microclimate situations.     

Nocturnal sap flow in the C<Subscript>3</Subscript>-CAM species, <Emphasis Type="Italic">Clusia minor</Emphasis>

Clusia minor L. is a C₃-CAM species in which Crassulacean acid Metabolism (CAM) is induced, among other factors, by water deficit. We propose that CAM induction by natural drought in C. minor shifts the sap flow pattern from daytime to a night-time one, and that the decreased osmotic potential due to increased malate content in droughted plants aids in the increase in nocturnal sap flow. In order to test these hypotheses, we followed for 2 years the seasonal changes in parameters of water relationships and sap flow velocity in one single, freestanding tree growing in Caracas. Leaf water and osmotic potential were measured psychrometrically, nocturnal proton accumulation by titration of aqueous leaf extracts and sap flow density with thermal dissipation probes. Leaf water, osmotic and turgor potential remained relatively high throughout the seasons. Nocturnal proton accumulation was nil under extreme drought or after frequent and heavy rains, and high after moderate rainfall. Estimated malate and citrate concentrations contributed up to 80 and 60%, respectively, of the value of osmotic potential. The shape of the daily courses of sap flow velocity varied seasonally, from mostly diurnal during the dry season to mostly nocturnal after a short dry spell during the rainy season, when nocturnal acid accumulation attained high values. There was a strong positive relationship between the proportion of the integrated sap flow courses corresponding to the night and dawn [H⁺] (r ² = 0.88). Increased nocturnal sap flow in the CAM stage of the tree of C. minor may be explained by a lower osmotic potential due to an increased acid concentration, together with increased stomatal aperture, as suggested by increased nocturnal acid accumulation probably due to nocturnal CO₂ fixation.     

Cool-night temperature induces spike emergence and affects photosynthetic efficiency and metabolizable carbohydrate and organic acid pools in <Emphasis Type="Italic">Phalaenopsis aphrodite</Emphasis>

After being acclimated to constant warm (28 degrees C day/28 degrees C night) and cool-night temperature (28 degrees C day/20 degrees C night) regimes in growth chambers for 2 weeks, the two groups of mature Phalaenopsis aphrodite subsp. formosana plants both clearly exhibited a diurnal oscillation of stomatal conductance, net CO(2) uptake rate, malate and starch levels, and the phosphoenolpyruvate carboxylase (EC 4.1.1.31) and NAD(+)-malic enzyme (EC 1.1.1.39) activities. Hence, P. aphrodite is an obligate crassulacean acid metabolism plant. Nevertheless, different night temperature greatly affected both the stomatal conductance and the contribution of ambient and respiratory CO(2) to the nocturnal accumulation of malate. However, the amounts of nocturnal accumulated malate and daily deposited starch appeared to have no significant difference between the two groups. These results demonstrate that P. ahrodite is congruent with the characteristics of CAM plants having great flexibility and plasticity in response to changes in environmental conditions. In addition, the formation of reproductive stem, viz. spike, was noticeably inhibited by a constant warm temperature, but induced by a fluctuating warm day and cool night condition. The relationship between the metabolic pool variation and spike induction of Phalaenopsis is also discussed.     

Ecological physiology of Pereskia guamacho, a cactus with leaves

The specialized physiology of leafless, stem-succulent cacti is relatively well understood. This is not true, however, for Pereskia (Cactaceae), the 17 species of leafy trees and shrubs that represent the earliest diverging lineages of the cacti. Here we report on the water relations and photosynthesis of Pereskia guamacho, a small tree of the semiarid scrubland of Venezuela's Caribbean coast. Sapwood-specific xylem conductivity (Ksp) is low when compared to other vessel-bearing trees of tropical dry systems, but leaf-specific xylem conductivity is relatively high due to the high Huber value afforded by P. guamacho's short shoot architecture. P. guamacho xylem is not particularly vulnerable to drought-induced cavitation, especially considering the high leaf water potentials maintained year round. This is confirmed by the lack of significant variation exhibited in Ksp between wet and dry seasons. In the rainy season, P. guamacho exhibited C3-like patterns of stomatal conductance, but during a prolonged drought we documented nocturnal stomatal opening with a concomitant accumulation of titratable acid in leaves. This suggests that P. guamacho can perform drought-induced crassulacean acid metabolism (CAM photosynthesis), although delta 13C values imply that most carbon is assimilated via the C3 pathway. P. guamacho leaves display very low stomatal densities, and maximum stomatal conductance is low whether stomata open during the day or night. We conclude that leaf performance is not limited by stem hydraulic capacity in this species, and that water use is conservative and tightly regulated at the leaf level.     

Isolation and characterization of mutants of common ice plant deficient in crassulacean acid metabolism

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.     

CO2 provides an intermediate link in the red light response of guard cells

Guard cells in intact leafs display light-induced membrane potential changes, which alter the direction of K+-transport across the plasma membrane (Roelfsema et al., 2001). A beam of blue light, but not red light, directed at the impaled guard cell triggers this response, while both light qualities induce opening of stomata. To gain insight into this apparent contradiction, we explored the possible interaction between red light and CO2. Guard cells in the intact plant were impaled with double-barrelled electrodes and illuminated with red light. Cells that were hyperpolarized in CO2-free air, depolarized after a switch to air with 700 micro l l(-1) CO2, in a reversible manner. As a result, K+-fluxes across the plasma membrane changed direction, to favour K+ extrusion and stomatal closure in the presence of CO2. Concurrent with the depolarization, an inward current across the plasma membrane appeared, most likely due to activation of anion channels. Guard cell responses to CO2 could be recorded in darkness as well as in red light. However, in darkness some cells spontaneously depolarized, these cells hyperpolarized again in red light. Here, red light was projected on a large area of the leaf and decreased the intracellular CO2 concentration by about 250 micro l l(-1), as measured with a miniature CO2 sensor placed in the substomatal cavity. We conclude, that in intact leaves the red light response of guard cells is mediated through a decrease of the intercellular CO2 concentration.     

Water stress and light intensity effects on growth and nocturnal acid accumulation in a terrestrial CAM bromeliad (Bromelia humilis Jacq.) under natural conditions

Summary Seasonal variations in CAM performance of sunexposed and partially shaded populations of Bromelia humilis were measured under natural conditions in a semi-arid region in northern Venezuela. The sun population consisted of smaller plants, with lower chlorophyll and total nitrogen contents per unit leaf area compared with plants from the partial-shade population. During the dry season CAM activity, assessed as nocturnal acid accumulation, was higher in the partial-shade population. Acid accumulation was stimulated by irrigation in both populations within 24 h after treatment. Daily changes in concentration of soluble sugars were opposite to leaf acidity indicating their role as carbon source for acid synthesis during the night. The change in nocturnal sugar concentration was always more than the amount required for acid accumulation, suggesting other carbohydrate-consuming processes such as transportation of sugars out of the leaf. CAM activity was higher during the rainy season, and differences between populations were smaller. At the end of the rainy season reduction of CAM activity caused by drought was first detected in the sun population. Measured ratios of glucan/soluble sugar show a higher proportion of readily utilizable sugars during periods of active CAM and growth. Under conditions of continuous high light intensity and air temperature leading to all year round high potential evaporation in semiarid tropical regions, fully exposed populations of B. humilis show a pronounced reduction of metabolic activity. Partial shade favours growth and CAM activity in this constitutive CAM species. It is concluded that water stress, and not light intensity, is the predominant limiting factor for growth of this species under natural conditions.