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.Abbreviation and symbols CAM Crassulacean acid metabolism - P turgor pressure - osmotic pressure - water potential Dedicated to Professor Dr. H. Ziegler on the occasion of his 60th birthday     

Circadian rhythms in Kalanchoë: effects of irradiance and temperature on gas exchange and carbon metabolism

Gas exchange in K. blossfeldiana shows a circadian rhythm in net CO₂ uptake and transpiration when measured under low and medium irradiances. The period length varies between 21.4 h at 60 W m^-2 and 24.0 h at 10 W m^-2. In bright light (80 W m^-2) or darkness there are no rhythms. High leaf temperatures result in a fast dampening of the CO₂-uptake rhythm at moderate irradiances, but low leaf temperatures can not overcome the dampening in bright light. The rhythm in CO₂ uptake is accompanied by a less pronounced and more rapidly damped rhythm in transpiration and by oscillations in malate levels with the amplitude being highly reduced. The oscillations in starch content, usually observed to oscillate inversely to the acidification in light-dark cycles, disappear after the first cycle in continuous light. The balance between starch and malate levels depends in continuous light on the irradiance applied. Leaves show high malate and low starch content at low irradiance and high starch and low malate in bright light. During the first 12 h in continuous light replacing the usual dark period, malate synthesis decreases with the increasing irradiance. Up to 50 W m^-2 starch content decreases; at higher irradiances it increases above the values usually measured at the end of the light period of the 12:12 h light-dark cycle.Abbreviations CAM Crassulacean acid metabolism - FW fresh weight - PEP phosphoenolpyruvate     

ATPase activity associated with isolated vacuoles of the crassulacean acid metabolism plant Kalanchoë daigremontiana

A technique is described that allows a relatively rapid and controlled isolation of vacuoles from leaves of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana. The method involves polybase-induced lysis of mesophyllcell protoplasts and isolation of vacuoles on a discontinuous density gradient. ATPase activity is associated with the isolated vacuoles and is not attributable to contamination by cytoplasmic constituents. It is suggested that this ATPase is responsible for the energization of malic-acid accumulation in the vacuole in CAM plants.Abbreviation CAM crassulacean acid metabolism Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday     

Effects of water stress on gas exchange and water relations of a succulent epiphyte,Kalanchoë uniflora

Summary Measurements of gas exchange, xylem tension and nocturnal malate synthesis were conducted with well-watered and droughted plants of Kalanchoë uniflora. Corresponding results were obtained with plants grown in 9 h and 12 h photoperiods. In well-watered plants, 50 to 90% of total CO₂-uptake occurred during the light period. Nocturnal CO₂-uptake and malate synthesis were higher and respiration rate was lower in old leaves (leaf pairs 6 to 10) compared to young leaves (leaf pairs 1 to 5). Within four days of drought distinct physiological changes occurred. Gas exchange during the light period decreased and CO₂-uptake during the dark period increased. Nocturnal malate synthesis significantly increased in young leaves.Respiration rate decreased during periods of drought, this decrease being more pronounced in young leaves compared to old leaves. Restriction of gas exchange during the light period resulted in a decrease of transpiration ratio from more than 100 to about 20. The difference between osmotic pressure and xylem tension decreased in young leaves, indicating a reduction in bulk leaf turgor-pressure.We conclude that both the CAM-enhancement in young leaves and the decrease of respiration rate are responsible for the increase of nocturnal CO₂-uptake during water stress. During short drought periods, which frequently occur in humid habitats, the observed physiological changes result in a marked reduction of water loss while net CO₂-uptake is maintained. This might be relevant for plant growth in the natural habitat.Abbreviations LP light period - DP dark period - CAM crassulacean acid metabolism     

Interaction of hydrolytic and phosphorolytic enzymes of starch metabolism in Kalanchoë daigremontiana

The degradation of starch by a protein fraction of Kalanchoë daigremontiana Hamet et Perrier, obtained by ammoniumsulfate precipitation (30–70%), was found to be catalyzed by -and -amylase (EC 3.2.1.1 and EC 3.2.1.2, respectively) and by starch phosphorylase (EC 2.4.1.1). The activity of these enzymes was determined by chromatographic analysis of the reaction products; separation and identification of -amylase was accomplished by heat-inactivation of -amylase and -glucosidase. When the interaction of amylolytic and phosphorolytic enzymes was comparatively studied, it was found that without inorganic phosphorus in the reaction mixture, ¹⁴C-starch was converted predominantly to maltose and glucose; supplementation with 1–10 mM orthophosphate (Pi) resulted in an increase in glucose-1-phosphate formation and a concomitant reduction of maltose production. Since the total volume of starch degradation remained approximately constant, Pi apparently inhibits -amylase (Ki about 3 mM Pi). Thus, free Pi in the cell participates in the regulation of starch catabolism, serving as a substrate for starch phosphorylase while simultaneously reducing the production of maltose. With respect to glucan synthesis, adenosinediphosphoglucose--1,4-glucosyltransferase (EC 2.4.1.22), maltose phosphorylase and maltoseglucosyltransferase were also found to be active. The last-named enzyme catalyzes an exchange between dextrins and is considered to provide primer carbohydrates for the synthesis of polyglucans.Abbreviations ADPG adenosinediphosphoglucose - G1P glucose-1-phosphate - PEG polyethylenglycol - PEP phosphoenolpyruvate - Pi orthophosphate     

Malate relations of plants and crassulacean acid metabolism: protons,carbon dioxide and water.—A review

Abstract

This review discusses the central role of malate in plant metabolism and in a large array of physiological processes in general. This is related to the function of crassulacean acid metabolism (CAM) as an ecophysiological adaptation to limited water supply.     

Perturbations of malate accumulation and the endogenous rhythms of gas exchange in the Crassulacean acid metabolism plant<Emphasis Type="Italic"> Kalanchoë daigremontiana</Emphasis>: testing the tonoplast-as-oscillator model

In continuous light, leaves of the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier exhibit a circadian rhythm of CO₂ uptake, stomatal conductance and leaf-internal CO₂ pressure. According to a current quantitative model of CAM, the pacemaking mechanism involves periodic turgor-related tension and relaxation of the tonoplast, which determines the direction of the net flux of malate between the vacuole and the cytoplasm. Cytoplasmic malate, in turn, through its inhibitory effect on phosphoenolpyruvate carboxylase, controls the rate of CO₂ uptake. According to this mechanism, when the accumulation of malate is disrupted by removing CO₂ from the ambient air, the induction of a phase delay with respect to an unperturbed control plant is expected. First, using the mathematical model, such phase delays were observed in numerical simulations of three scenarios of CO₂ removal: (i) starting at a trough of CO₂ uptake, lasting for about half a cycle (ca. 12 h in vivo); (ii) with the identical starting phase, but lasting for 1.5 cycles (ca. 36 h); and (iii) starting while CO₂ increases, lasting for half a cycle again. Applying the same protocols to leaves of K. daigremontiana in vivo did not induce the predicted phase shifts, i.e. after the end of the CO₂ removal the perturbed rhythm adopted nearly the same phase as that of the control plant. Second, when leaves were exposed to a nitrogen atmosphere for three nights prior to onset of continuous light to prevent malate accumulation, a small, 4-h phase advance was observed instead of a delay, again contrary to the model-based expectations. Hence, vacuolar malic acid accumulation is ruled out as the central pacemaking process. This observation is in line with our earlier suggestion [T.P. Wyka, U. Lüttge (2003) J Exp Bot 54:1471–1479] that in extended continuous light, CO₂ uptake switches gradually from a CAM-like to a C3-like mechanism, with oscillations of the two CO₂ uptake systems being tightly coordinated. It appears that the circadian rhythm of gas exchange in this CAM plant emerges from one or several devices that are capable of generating temporal information in a robust manner, i.e. they are protected from even severe metabolic perturbations.Abbreviations CAM Crassulacean acid metabolism - c_ia Ratio of mesophyll CO₂ concentration to external CO₂ concentration - J_C Rate of carbon dioxide uptake - J_W Transpiration rate - g_W Stomatal conductance - LL Continuous light conditions - PEPC Phosphoenolpyruvate carboxylase - Rubisco d-Ribulose-1,5-bisphosphatecarboxylase/oxygenase - Effective quantum yield of photosystem II     

Leaf anatomy,water relations and crassulacean acid metabolism in the chlorenchyma and colourless internal water-storage tissue of Carpobrotus edulis and Senecio ?mandraliscae

Both Carpobrotus edulis and Senecio ?mandraliscae possess leaves with a peripheral chlorenchyma and colourless internal water-storage tissue. Water stress in C. edulis growing under semi-natural conditions resulted in the induction of weak Crassulacean acid metabolism (CAM) whereas well-watered plants of S. ?mandraliscae exhibited a similar degree of CAM. Titratable acidity in the separated water-storage tissue was substantially lower than in the chlorenchyma in both species but, nevertheless, increased during the night and decreased during the day either when sampled from the intact plant or from incubated tissue slices. Indeed, the increase in nocturnal titratable acidity produced by the water-storage tissue in situ accounted for approx. 30% of total acidification on a per-leaf basis. It appears that during the night the water-storage tissue in these species is able to fix CO₂ which is subsequently released during the day to enter the photosynthetic carbon-reduction cycle of the chlorenchyma. Diurnal rhythms of water potential (Ψ) and osmotic potential (Ψs) were measured in separated chlorenchyma and water-storage tissue by thermocouple psychrometry. Both parameters increased during the latter part of the daytime and initial nocturnal period and decreased during the rest of the night and into the post-dawn period. The chlorenchyma of water-stressed plants of C. edulis appeared to possess a marked negative turgor pressure (as determined from Ψ-Ψs) but this was caused by a severe underestimation in the measurement of the chlorenchyma Ψ. It is suggested that this artefact arose from release of colloidal polysaccharide mucilage, or possibly tannins, from broken tannin cells producing a lowering of water activity when measured using thermocouple psychrometry.     

Crassulacean acid metabolism (CAM) in Kalanchoë daigremontiana: Temperature response of phosphoenolpyruvate (PEP)-carboxylase in relation to allosteric effectors

Net CO₂ dark fixation of Kalanchoë daigremontiana varies with night temperature. We found an optimum of fixation at about 15° C; with increasing night temperature fixation decreased. We studied the temperature dependence of the activity of phosphoenolpyruvate (PEP)-carboxylase, the key enzyme for CO₂ dark fixation. We varied the pH, the substrate concentration (PEP), and the L-malate and glucose-6-phosphate (G-6-P) concentration in the assay. Generally, lowering the pH and reducing the amount of substrate resulted in an increase in activation by G-6-P and in an increase in malate inhibition of the enzyme. Furthermore, malate inhibition and G-6-P activation increased with increasing temperature. Activity measurements between 10° C and 45°C at a given concentration of the effectors revealed that the temperature optimum and maximum activities at that optimum varied with the effector applied. Under the influence of 5 mol m^-3 L-malate the temperature optimum and maximum activity dropped drastically, especially when the substrate level was low (at 0.5 mol m^-3 PEP from 32° C to 20° C). G-6-P raised the temperature optimum and maximum activity when the substrate level was low. If both malate and G-6-P were present, intermediate values were measured. We suggest that changes in metabolite levels in K. daigremontiana leaves can alter the temperature features of PEP-carboxylase so that the observed in vivo CO₂ dark fixation can be explained on the basis of PEP-carboxylase activity.Abbreviations PEP-c phosphoenolpyruvate carboxylase - CAM crassulacean acid metabolism - PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate     

A comparative study on the regulation of C3 and C4 carboxylation processes in the constitutive crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana and the C3-CAM intermediate Clusia minor

A comparison of carbon metabolism in the constitutive crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perr. and the C₃-CAM intermediate Clusia minor L. was undertaken under controlled environmental conditions where plants experience gradual changes in light intensity, temperature and humidity at the start and end of the photoperiod. The magnitude of CAM activity was manipulated by maintaining plants in ambient air or by enclosing leaves overnight in an atmosphere of N₂ to suppress C₄ carboxylation. Measurements of diel changes in carbonisotope discrimination and organic acid content were used to quantify the activities of C₃ and C₄ carboxylases in vivo and to indicate the extent to which the activities of phosphoenolpyruvate carboxylase (PEPCase), ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and decarboxylation processes overlap at the start and end of the photoperiod. These measurements in vivo were compared with measurements in vitro of changes in the diel sensitivity of PEPCase to malate inhibition. The results demonstrate fundamental differences in the down-regulation of PEPCase during the day in the two species. While PEPCase is inactivated within the first 30 min of the photoperiod in K. daigremontiana, the enzyme is active for 4 h at the start and 3 h at the end of the photoperiod in C. minor. Enclosing leaves in N₂ overnight resulted in a two-to threefold increase in PEPCase-mediated CO₂ uptake during Phase II of CAM in both species. However, futile cycling of CO₂ between malate synthesis and decarboxylation does not occur during Phase II in either species. In terms of overall carbon balance, C₄ carboxylation accounted for ≈ 20% of net daytime assimilation in both species under control conditions, increasing to 30–34% after a night in N₂. Although N₂-treated leaves of K. daigremontiana took up 25% more CO₂ than control leaves during the day this was insufficient to compensate for the loss of CO₂ taken up by CAM the previous night. In contrast, in N₂-treated leaves of C. minor, the twofold increase in daytime PEPCase activity and the increase in net CO₂ uptake by Rubisco during Phase III compensated for the inhibition of C₄ carboxylation at night in terms of diel carbon balance.     

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

Leaves of Kalanchoë daigremontiana Hamet et Perr. at a photon flux density (PFD) above 220 mol·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 mol·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.Abbreviations CAM Crassulacean acid metabolism - PAR photosynthetically active radiation - PFD photon flux density This work was supported by a grant to F.B. and U.L. from Teilprojekt B5 in the Sonderforschungsbereich 199 of the Deutsche Forschungsgemeinschaft (Bonn, Germany) and by a grant to T. E. E. G. from the Sudienstiftung des deutschen Volkes (Bonn, Germany). Erika Ball is thanked for processing of time-course data for the analysis of Fourier spectra.     

A comparative study on diurnal changes in metabolite levels in the leaves of three crassulacean acid metabolism (CAM) species, Ananas comosus, Kalancho? daigremontiana and K. pinnata.

A comparative study on diurnal changes in metabolite levels associated with crassulacean acid metabolism (CAM) in the leaves of three CAM species, Ananas comosus (pineapple), a hexose-utilizing species, and Kalancho? daigremontiana and K. pinnata, two starch-utilizing species, were made. All three CAM species showed a typical feature of CAM with nocturnal malate increase. In the two Kalancho? species, isocitrate levels were higher than citrate levels; the reverse was the case in pineapple. In the two Kalancho? species, a small nocturnal citrate increase was found and K. daigremontiana showed a small nocturnal isocitrate increase. Glucose 6-phosphate (G-6-P), fructose 6-phosphate (F-6-P) and glucose 1-phosphate (G-1-P) levels in the three CAM species rose rapidly during the first part of the dark period and decreased during the latter part of the dark period. The levels of the metabolites also decreased during the first 3 h of the light period, then, remained little changed through the rest of the light period. Absolute levels of G-6-P, F-6-P and G-1-P were higher in pineapple than in the two Kalancho? species. Fructose 1,6-bisphosphate (F-1,6-P(2)) levels in the three CAM species increased during the dark period, then dramatically decreased during the first 3 h of the light period and remained unchanged through the rest of the light period. The extent of nocturnal F-1,6-P(2) increase was far greater in the two Kalancho? species than in pineapple. Absolute levels of F-1,6-P(2) were higher in the two Kalancho? species than in pineapple, especially during dark period. Diurnal changes in oxaloacetate (OAA), pyruvate (Pyr) and phosphoenolpyruvate (PEP) levels in the three CAM species were similar.     

Contribution of C3 carboxylation to the circadian rhythm of carbon dioxide uptake in a Crassulacean acid metabolism plant Kalanchoë daigremontiana

During the endogenous circadian rhythm of carbon dioxide uptake in continuous light by a Crassula cean acid metabolism plant, Kalancho? daigremontiana, the two carboxylating enzymes, phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco), are active simultaneously, although, until now, only the role of PEPC in generating the rhythm has been acknowledged. According to the established model, the rhythm is primarily regulated at the PEPC activity level, modulated by periodic compartmentation of its inhibitor, malate, in the vacuole and controlled by tension/relaxation of the tonoplast. However, the circadian accumulation of malic acid (the main indicator of PEPC activity) dampened significantly within the first few periods without affecting the rhythm's amplitude. Moreover, the amount of malate accumulated during a free-running oscillation was several-fold lower than the amount expected if PEPC were the key carboxylating enzyme, based on a 1:1 stoichiometry of CO(2) and malate. Together with the observation that rates of CO(2) uptake under continuous light were higher than in darkness, the evidence shows that C(3) carboxylation greatly contributes to the generation of rhythmic CO(2) uptake in continuous light in this 'obligate' CAM plant. Because the shift from predominantly CAM to predominantly C(3) carboxylation is smooth and does not distort the trajectory of the rhythm, its control probably arises from a robust network of oscillators, perhaps also involving stomata.     

The relationship between leaf water potential ψ leaf and the levels of abscisic acid and ethylene in excised wheat leaves

The amount of diffusible ethylene from excised wheat leaves (Triticum aestivum L. cv. Eclipse) increased when they were subjected to water stress. The quantity of ethylene produced was related to the severity of the stress, reaching a maximum at a leaf water potential _leaf of approximately-12 bars. Irrespective of the severity of the stress, the maximum rate of ethylene production usually occurred between 135–270 min after applying the stress and then the rate declined. Part of the decline may have been due to an oxygen deficiency in the leaf chambers. In excised water-stressed leaves there was a sigmoid relationship between increasing ethylene and abscisic acid (ABA) levels and decreasing leaf water potential values. The two curves were displaced from each other by approximately 1 bar, with ethylene evolution leading that of ABA accumulation. The maximum rate of increase in ethylene occurred between-8 and-9 bars and for ABA between-9 and-10 bars. A significant increase in the levels of these two plant growth regulators was found when the _leaf decreased outside the normal diurnal _leaf range by 1 bar for ethylene and 2 bars for ABA. Because of the sigmoid nature of the curves there was no distinct threshold _leaf value triggering-off an increase in ethylene or ABA, but with ABA the curve became very steep at a _leaf value of-9.3 bars and this could be looked upon as a kind of threshold value.It seems unlikely that the stress-induced ethylene evolution in excised wheat leaves stimulated the accumulation of ABA, because when the leaves were subjected to a substantial water stress (e.g. _leaf bars) ABA increased immediately and at a faster rate than ethylene.Abbreviations ABA abscisic acid - GLC gas-liquid chromatography - RWC relative water content - TLC thin-layer chromatography - _leaf leaf water potential     

Proton and anion transport at the tonoplast in crassulacean-acid-metabolism plants: specificity of the malate-influx system in Kalanchoë daigremontiana

Tonoplast vesicles were prepared from leaf mesophyll homogenates of the crassulacean-acid-metabolism plant Kalanchoë daigremontiana Hamet et Perrier de la Bâthie to study the effects of anions on ATP- and inorganic-pyrophosphate (PPi)-dependent H⁺ transport. In the presence of gramicidin, substrate hydrolysis by the tonoplast ATPase was characteristically stimulated by chloride and inhibited by nitrate, but was unaffected by malate and a wide range of other organic-acid anions; the PPiase was anion-insensitive. Malate was more effective than chloride both in stimulating ATP- and PPi-dependent vesicle acidification (measured as quinacrine-fluorescence quenching) and in dissipating a pre-existing inside-positive membrane potential (measured as oxonol-V-fluorescence quenching), indicating that malate was more readily transported across the tonoplast. Certain other four-carbon dicarboxylates also supported high rates of vesicle acidification, their order of effectiveness being fumarate malate -succinate > oxalacetate - tartrate; the five-carbon dicarboxylates 2-oxoglutarate and glutarate were also transported, although at lower rates. Experiments with non-naturally occurring anions indicated that the malate transporter was not stereospecific, but that it required the trans-carboxyl configuration for transport. Shorter-chain or longer-chain dicarboxylates were not transported, and neither were monocarboxylates, the amino-acid anions aspartate and glutamate, nor the tricarboxylate isocitrate. The non-permeant anions maleate and tartronate appeared to be competitive inhibitors of malate transport but did not affect chloride transport, indicating that malate and chloride influx at the tonoplast might be mediated by separate transporters.Abbreviations BTP 1,3-bis-[tris(hydroxymethyl)methylamino]-propane - CAM crassulacean acid metabolism - oxonol V bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol - pH transmembrane pH difference - PPi inorganic pyrophosphate - PPiase inorganic pyrophosphatase - quinacrine 6-chloro-9-{[4-(diethylamino)-1-methylbutyl]amino}-2-methoxyacridine dihydrochloride - transmembrane electrical potential difference     

Effects of high irradiances on photosynthesis,growth and crassulacean acid metabolism in the epiphyteKalanchoö uniflora

Summary Kalancho? uniflora was grown in the glasshouse with and without shading. Chlorophyll content, area/FW ratio and specific leaf area were higher in leaves of shaded as compared to unshaded plants. Light saturation curves and continuous gas exchange measurements showed that the apparent quantum yield and the light-saturated photosynthetic rate were higher in shaded plants. Shaded plants had lower “mesophyll resistances” than unshaded plants, indicating that the different photosynthetic capacities reflected different contents of ribulose biphosphate carboxylase-oxygenase. Highlight treatment of plants grown in the shade resulted in a decreased photosynthetic efficiency, showing that these plants were sensitive to photoinhibition. However, dry matter production was higher in unshaded than in shaded plants. Obviously the difference in irradiance between the two growth regimes did more than offset the differences in photosynthetic efficiency. Applying additional nutrients did not alter the effects of high PFDs. The results are discussed in respect to photosynthetic performence and plant distribution in the epiphytic habitat.     

Changes in lipid composition, water relations and gas exchange in leaves of two young ‘Chemlali’ and ‘Chetoui’ olive trees in response to water stress

The comparative responses of two young olive trees (Olea europaea L. ‘Chemlali’ and ‘Chetoui’) to drought stress were investigated during 1 month. Three-month-old own-rooted plants were subjected to two irrigation treatments: WW (well watered plants that were irrigated with fresh water to maintain a soil water content close to field capacity), and WS (water stressed plants by withholding water). Leaf water potential, gas exchange and leaf lipid composition were studied. ‘Chemlali’ was able to maintain higher leaf CO₂ assimilation rate and leaf stomatal conductance throughout the drought cycle compared to ‘Chetoui’. Water stress induced a larger decrease in the total lipid content in ‘Chetoui’ than in ‘Chemlali’. Interestingly, the constitution of different lipid classes was highly altered in ‘Chetoui’. Lipid changes in Chemlali, a drought tolerant cultivar, revealed more stability of its cellular membranes to drought stress as compared to the drought susceptible olive cultivar, Chétoui. Furthermore, in comparison to the controls, drought stressed plants showed an increase in the degree of unsaturation of leaf lipids in the two olive cultivars. Moreover, the results observed in Chemlali showed that besides changes in lipids composition this cultivar may have an efficient defence strategy which can be related on antioxidative production against oxidative stress.     

Characteristics,partial purification and reconstitution of the vacuolar malate transporter of the CAM plant Kalanchoë daigremontiana Hamet et Perrier de la Bâthie

When native tonoplast vesicles of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie were energized by an artificial K⁺ gradient establishing only an inside-positive electrical membrane potential (), it was shown that was sufficient as the sole driving force and that a proton gradient (pH) is not required for malate uptake. Following [¹⁴C]malate uptake, K _m-malate of the malate transporter was estimated as 2.7–3.0 mM, a value that would allow malate synthesis via phosphoenolpyruvate carboxylase and malate accumulation in vivo in view of the feed-back inhibition of cytosolic phosphoenolpyruvate carboxylase by malate. The maximum reaction velocity (V _max) was found to be between 30 and 85 nmol malate·min^–1·mg _protein ^–1 , a value that would explain nocturnal malate accumulation in K. daigremontiana even if the transporter were operating below substrate saturation. Citrate (50 mM at pH 7) inhibited transport by 78%. The malate-transport protein of the tonoplast of K. daigremontiana may be a carboxylate uniporter with strong affinities for malate and citrate. From total tonoplast proteins solubilized from native tonoplast vesicles the malate transporter was functionally reconstituted into phospholipid liposomes. The malate transporter was purified and separated from the tonoplast H⁺-ATPase by hydroxyapatite chromatography, but not from the tonoplast H⁺-pyrophosphatase. The partially purified malate-transport protein was functionally reconstituted into phospholipid liposomes. In these final proteoliposomes, 0.6% of the protein of the initial tonoplast-vesicle preparation used for solubilization of membrane proteins was recovered. Using the specific rates of malate transport as a reference, i.e. rates of transport related to protein in the preparations, enrichment of the malate transporter in the final proteoliposomes obtained with the reconstitution of the hydroxyapatite eluate was 44-fold compared to the initial native tonoplast vesicles and 2000-fold compared to the liposomes reconstituted from solubilized tonoplast proteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the peptides from the final proteoliposomes, which were functional in malate transport, showed only a few polypeptide bands among which the malate transporter must be found.Abbreviations and Symbols CAM Crassulacean acid metabolism - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - Triton X-100 polyoxyethylene(9,10)p-t-octylphenol - pH proton gradient at the tonoplast - membrane potential at the tonoplast This work was supported by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie and is now funded in SFB 199 (Teilprojekt B2) of the Deutsche Forschungsgemeinschaft. We thank Dr. Elke Fischer-Schliebs for valuable discussions and Dr. E. Martinoia for making us acquainted with his experimental approaches in his laboratory in Zürich, Switzerland, and for much valuable exchange. Dr. D.P.S. Verma, Ohio, USA, kindly provided Nod-26 antibodies, and the tonoplast H⁺-pyrophosphatase antibodies were a generous gift of Dr. M. Maeshima, Sapporo, Japan.     

Crassulacean acid metabolism in Kalanchoë species collected in various climatic zones of Madagascar: a survey by δ13C analysis

Summary The carbon isotope compositions of samples of Kalanchoë species collected at the natural stands in Madagascar were determined. The results suggest that all species of the genus Kalanchoë are capable of crassulacean acid metabolism. The observed ¹³C values cover the whole range from –10 to –30. This high diversity of the ¹³C values was found among the species of the genus as well as, in certain cases, within a single species. This suggest that the CAM patterns in Kalanchoë are generally very flexible. The ¹³C values show a clear correlation with the climate of the habitats from where the samples derived. Values indicative of CO₂ fixation taking place exclusively during the night were found in the dry regions of Madagascar, whereas ¹³C values indicative of mixed CO₂ fixation during night and day or of CO₂ fixation entirely during the day are distributed in the humid zones.