A salinity-induced C3-CAM transition increases energy conservation in the halophyte Mesembryanthemum crystallinum L

A strongly increased ATP/ADP ratio was found during the nocturnal phase I in crassulacean acid metabolism (CAM)-induced Mesembryanthemum crystallinum plants. Conversely, during the daytime phase III in CAM-performing plants the ATP/ADP ratio dropped to a similar level to that of C3 plants, cytochrome c oxidase activity was stimulated and mitochondrial Mn-superoxide dismutase activity was strongly increased. The findings suggest that a salinity-induced C3-CAM transition might be an efficient energy-conserving strategy for M. crystallinum plants, in which the strong nocturnal ATP production seems to be, at least partially, independent from the coupled mitochondrial electron transport.     

Regulatory protein phosphorylation of phosphoenolpyruvate carboxylase in the facultative crassulacean-acid-metabolism plant Mesembryanthemum crystallinum L.

Phosphoenolpyruvate PyrP carboxylase (PyrPC) and PyrPC kinase were copurified from dark-adapted leaves of the common ice plant Mesembryanthemum crystallinum L. with crassulacean-acid metabolism (CAM). Purification by (NH4)2SO4 fractionation, chromatography on Fractogel-DEAE and hydroxylapatite resulted in a PyrPC preparation with a specific activity of 23-25 U/mg protein and a protein kinase activity of 255 mumol Pi.mol-1 PyrPC.s-1. After in vitro phosphorylation, the most prominently phosphorylated polypeptide was identified as PyrPC by immunoblotting and sequencing. Phosphorylation of PyrPC in vitro by incubation with 400 microM MgATP decreased its sensitivity towards malate. When purified in the absence of the protease inhibitor chymostatin, PyrPC lost an N-terminal sequence of 128 amino acids. Although the carboxylation reaction was unaffected, the truncated PyrPC could neither be phosphorylated in vitro nor inhibited by malate. This result and data obtained by limited proteolysis concur with the hypothesis [Jiao, J.A. & Chollet, R. (1989) Arch. Biochem. Biophys. 283, 300-305] that Ser11 is the phosphorylation site of the CAM PyrPC of M. crystallinum. At pH 7.0, the Km for ATP of the protein kinase was 25 microM; phosphorylation of PyrPC was maximal after 30 min at pH 7.0. The kinase showed also activity with histone III-S but not with dephosphorylated casein. It was inhibited by malate. The results show, that reversible protein phosphorylation is an important factor in the regulation of PyrPC in the facultative CAM plant M. crystallinum, similar to C4 and constitutive CAM plants.     

Influence of long photoperiods on plant development and expression of Crassulacean acid metabolism in Mesembryanthemum crystallinum

Abstract. In the natural habitat plants of Mesembryanthemum crystallinum are induced to perform Crassulacean acid metabolism (CAM) after 3 months, and reproductive growth begins after 5 months (Winter, Liittge & Winter, 1978, Oecologia (Berlin), 34, 225-237). The life cycle of M. crystallinum and the extent of growth required prior to induction of enzymes of Crassulacean acid metabolism (CAM) are dramatically shortened by growing seedlings with a long photoperiod (3=16h/8h light/dark). Reproductive growth begins as soon as five weeks after germination when plants are grown in continuous light (under 600μmol quanta m⁻² s⁻¹, 30°C). In plants grown under well-watered conditions, the activities of PEP carboxylase and NADP-malic enzyme begin increasing markedly 2 weeks after germination, with plants grown under longer photoperiods having higher enzyme activities. After 3 weeks of growth, leaves accumulated a large amount of malate, but the microequivalents of malate present were up to nine times greater than the total titratable acidities. Interestingly, plants from a 24h/0h or a 20h/4h photo-period showed no diurnal fluctuation of malate, but did produce malate in the light as a major photosynthetic end product. That is, under these environmental conditions, principal enzymes of CAM can be induced without the plants performing CAM. However, plants grown in a 16h/8h photoperiod did exhibit nocturnal accumulation of malate after 3 weeks of growth. In plants of all three growth conditions, the activities of NADP-malic enzyme and PEP carboxylase were further increased two- to live-fold by irrigating 3-week-old-plants with 350mol m⁻³ NaCl. Such early enhancement of these enzymes by salt and the shortened life cycle may be due to an accelerated development under the long photoperiods.     

The Effect of Irradiance on Carboxylating/Decarboxylating Enzymes and Fumarase Activities in Mesembryanthemum crystallinum L. Exposed to Salinity Stress

Abstract: In Mesembryanthemum crystallinum plants, treated for 9 days with 0.4 M NaCl at low light intensities (80 - 90 or 95 - 100 μE m^-2 s^-1; λ = 400 - 700 nm), no day/night malate level differences (Δmalate) were detected. At high light (385 - 400 μE m^-2 s^-1) strong stimulation of PEPC activity, accompanied by a Δmalate of 11.3 mM, demonstrated the presence of CAM metabolism. This indicates that, to evolve day/night differences in malate concentration, high light is required. Salt treatment at low light induces and increases the activity of NAD- and NADP-malic enzymes by as much as 3.7- and 3.9-fold, while at high light these values reach 6.4- and 17.7-fold, respectively. The induction of activity of both malic enzymes and PEPC (phospo enol pyruvate carboxylase) take place before Δmalate is detectable. An increase in SOD (superoxide dismutase) was observed in plants cultivated at high light in both control and salt-treated plants. However, in salt-treated plants this effect was more pronounced. Carboxylating and decarboxylating enzymes seem to be induced by a combination of different signals, i.e., salt and light intensity. Plants performing CAM, after the decrease of activity of both the decarboxylating enzymes at the beginning of the light period, showed an increase in these enzymes in darkness when the malate pool reaches higher levels. In CAM plants the activity of fumarase (Krebs cycle) is much lower than that in C₃ plants. The role of mitochondria in CAM plants is discussed.     

Expression and characterization of recombinant cytosolicNAD+-dependent malate dehydrogenase from Mesembyanthemum crystallinum

An NAD⁺-dependent cytosolic malate dehydrogenase (MDH, EC 1.1.1.37) from leaves of Mesembryanthemum crystallinum in the Crassulacean Acid Metabolism (CAM) mode was cloned, expressed in E. coli and characterized. The recombinant enzyme had a subunit molecular mass of 39.5 kDa and was recognized by antibodies raised against the cytosolic MDH from Ananas comosus . Its activity showed a maximum in the pH range of 7.5–9.5. The purified MDH is highly but not completely specific for oxaloacetate, as indicated by a low activity using various other α-ketoacids as substrates. The sequence data, subunit mass and immunoreactivity suggest that the MDH that has been cloned and characterized corresponds to the cytosolic isoform. Yet, the biochemistry of this enzyme comparative with the only other CAM plant cytosolic MDH characterized so far (that of pineapple) hints at a distinct isoform being expressed in M. crystallinum leaves.     

A CAM- and starch-deficient mutant of the facultative CAM species Mesembryanthemum crystallinum reconciles sink demands by repartitioning carbon during acclimation to salinity

In the halophytic species Mesembryanthemum crystallinum, the induction of crassulacean acid metabolism (CAM) by salinity requires a substantial investment of resources in storage carbohydrates to provide substrate for nocturnal CO(2) uptake. Acclimation to salinity also requires the synthesis and accumulation of cyclitols as compatible solutes, maintenance of root respiration, and nitrate assimilation. This study assessed the hierarchy and coordination of sinks for carbohydrate in leaves and roots during acclimation to salinity in M. crystallinum. By comparing wild type and a CAM-/starch-deficient mutant of this species, it was sought to determine if other metabolic sinks could compensate for a curtailment in CAM and enable acclimation to salinity. Under salinity, CAM deficiency reduced 24?h photosynthetic carbon gain by >50%. Cyclitols were accumulated to comparable levels in leaves and roots of both the wild type and mutant, but represented only 5% of 24?h carbon balance. Dark respiration of leaves and roots was a stronger sink for carbohydrate in the mutant compared with the wild type and implied higher maintenance costs for the metabolic processes underpinning acclimation to salinity when CAM was curtailed. CAM required the nocturnal mobilization of >70% of primary carbohydrate in the wild type and >85% of carbohydrate in the mutant. The substantial allocation of carbohydrate to CAM limited the export of sugars to roots, and the root:shoot ratio declined under salinity. The data suggest a key role for the vacuole in regulating the supply and demand for carbohydrate over the day/night cycle in the starch-/CAM-deficient mutant.     

The appearance of a new molecular species of phosphoenolpyruvate carboxylase (PEPC) and the rapid induction of CAM in Sedum telephium L.

Abstract. When detached leaves of Sedum telephium are incubated in the absence of water, a rapid switch from C₃ photosynthesis to CAM (as indicated by the onset of day-to-night fluctuations in titratable acidity. ΔH⁺) occurs within the first dark period. The C₃-CAM switch in intact plants occurs within 3 5d. Extractable activity of phospho enol pyruvate carboxylase (PEPC) increases five-fold in intact plants during CAM induction; however, during rapid CAM induction in detached leaves, there is only a very small increase in PEPC activity. Fractionation by anion exchange chromatography of crude extracts from leaves of intact plants subjected to water deficit shows that CAM induction is associated with the appearance of a molecular species of PEPC termed PEPC I. PEPC I is barely detectable in well-watered plants which are not performing CAM. The major form in these plants is termed PEPC II. In leaves from intact plants, there is a significant positive correlation between PEPC I activity and ΔH⁺ during a period of increasing water deficit. PEPC I exhibits day to night fluctuations in malate sensitivity, being less sensitive during the dark period. In contrast, PEPC II is more sensitive to inhibition by malate and has no day to night fluctuation in sensitivity. In detached leaves deprived of water, a small increase in PEPC I capacity is detected at the end of the first dark period (20 h after the start of treatment). The results suggest that PEPC I is required for attainment of maximum nocturnal malic acid synthesis. There is a significant correlation between leaf water status (relative water content), ΔH⁺, total PEPC and PEPC I activity suggesting that the internal water status of the plant may be a trigger for CAM induction. Abscisic acid applied to detached leaves does not cause nocturnal acidification.     

Mitochondrial respiration in ME-CAM, PEPCK-CAM, and C₃ succulents: comparative operation of the cytochrome, alternative, and rotenone-resistant pathways

Mitochondria are important in the function and control of Crassulacean acid metabolism (CAM) during organic acid accumulation at night and acid decarboxylation in the day. In plants of the malic enzyme-(ME) type and the phosphoenolpyruvate carboxykinase- (PEPCK) type, mitochondria may exert their role in the control of the diurnal rhythm of malic and citric acids to a differential degree. In plants of both CAM types, the oxidative capacity of mitochondria, as well as the activity of CAM-linked mitochondrial enzymes, and of the alternative and the rotenone-resistant pathways of substrate oxidation were compared. Furthermore, a C? succulent was included, as well as both C? and CAM forms of Mesembryanthemum crystallinum during a salt-induced C?-to-CAM shift. Mitochondria of PEPCK-type CAM plants exhibited a lower activity of malate oxidation, ratio of malate to succinate oxidation, and activity of mitochondrial NAD-ME. With the exception of Kalancho? daigremontiana, leaf mitochondria of all other CAM species were highly sensitive to cyanide (80-100%), irrespective of the oxidant used. This indicates that the alternative oxidase is not of general importance in CAM. By contrast, rotenone-insensitive substrate oxidation was very high (50-90%) in all CAM species. This is the first comparison of the rotenone-insensitive pathway of respiration in plants with different CAM-types. The results of this study confirm that mitochondria are involved in the control of CAM to different degrees in the two CAM types, and they highlight the multiple roles of mitochondria in CAM.     

Plasticity of crassulacean acid metabolism at subtropical latitudes: a pineapple case study

Plants with the crassulacean acid metabolism (CAM) express high‐metabolic plasticity, to adjust to environmental stresses. This article hypothesizes that irradiance and nocturnal temperatures are the major limitations for CAM at higher latitudes such as the Azores (37°45'N). Circadian CAM expression in Ananas comosus L. Merr. (pineapple) was assessed by the diurnal pattern of leaf carbon fixation into l ‐malate at the solstices and equinoxes, and confirmed by determining maximal phosphoenolpyruvate carboxylase (PEPC) activity in plant material. Metabolic adjustments to environmental conditions were confirmed by gas exchange measurements, and integrated with environmental data to determine CAM's limiting factors: light and temperature. CAM plasticity was observed at the equinoxes, under similar photoperiods, but different environmental conditions. In spring, CAM expression was similar between vegetative and flowering plants, while in autumn, flowering (before anthesis) and fructifying (with fully developed fruit before ripening) plants accumulated more l ‐malate. Below 100 µmol m^?2 s^?1, CAM phase I was extended, reducing CAM phase III during the day. Carbon fixation inhibition may occur by two major pathways: nocturnal temperature (<15°C) inhibiting PEPC activity and l ‐malate accumulation; and low irradiance influencing the interplay between CAM phase I and III, affecting carboxylation and decarboxylation. Both have important consequences for plant development in autumn and winter. Observations were confirmed by flowering time prediction using environmental data, emphasizing that CAM expression had a strong seasonal regulation due to a complex network response to light and temperature, allowing pineapple to survive in environments not suitable for high productivity.     

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.     

Distinct responses to copper stress in the halophyte Mesembryanthemum crystallinum

Selective gene expression allows the halophyte Mesembryanthemum crystallinum to survive a salt stress. To broaden our understanding of the environmental cues initiating diverse stress responses in this higher plant, unstressed and 0.4 M NaCl‐stressed plants were compared to plants treated with several concentrations of copper (CuSO₄), an increasingly relevant environmental heavy metal pollutant. Comparisons of control and copper‐stressed plants included germination, chlorophyll content, accumulation of proline, heat shock protein (HSP) 60 and a Crassulacean acid metabolism (CAM)‐specific marker enzyme, phospho enol pyruvate carboxylase (PEPCase). In germination and whole plant tests, M. crystallinum was significantly more tolerant to copper than Arabidopsis thaliana. Mature M. crystallinum plants stressed with 50 ppm CuSO₄ for 48 h became dehydrated. These plants produced a 4‐fold increase in proline concentration and accumulated both the CAM‐specific PEPCase and HSP 60 compared to controls. Higher levels of copper stress resulted in a 10‐fold increase in leaf proline content, 10‐fold HSP 60 accumulation but no detectable PEPCase protein compared to unstressed controls. HSP 60 did not accumulate under NaCl stress. Concurrent with copper‐induced genetic responses to stress, copper was accumulated and concentrated in leaves (3 500 ppm). Together, these results suggest that this halophyte copes with copper metal exposure through distinct genetic mechanisms.     

Crassulacean acid metabolism enhances underwater photosynthesis and diminishes photorespiration in the aquatic plant Isoetes australis

? Underwater photosynthesis by aquatic plants is often limited by low availability of CO(2), and photorespiration can be high. Some aquatic plants utilize crassulacean acid metabolism (CAM) photosynthesis. The benefits of CAM for increased underwater photosynthesis and suppression of photorespiration were evaluated for Isoetes australis, a submerged plant that inhabits shallow temporary rock pools. ? Leaves high or low in malate were evaluated for underwater net photosynthesis and apparent photorespiration at a range of CO(2) and O(2) concentrations. ? CAM activity was indicated by 9.7-fold higher leaf malate at dawn, compared with at dusk, and also by changes in the titratable acidity (μmol H(+) equivalents) of leaves. Leaves high in malate showed not only higher underwater net photosynthesis at low external CO(2) concentrations but also lower apparent photorespiration. Suppression by CAM of apparent photorespiration was evident at a range of O(2) concentrations, including values below air equilibrium. At a high O(2) concentration of 2.2-fold the atmospheric equilibrium concentration, net photosynthesis was reduced substantially and, although it remained positive in leaves containing high malate concentrations, it became negative in those low in malate. ? CAM in aquatic plants enables higher rates of underwater net photosynthesis over large O(2) and CO(2) concentration ranges in floodwaters, via increased CO(2) fixation and suppression of photorespiration.     

Induction of Crassulacean Acid Metabolism in the Facultative Halophyte Mesembryanthemum crystallinum by Abscisic Acid

The facultative halophyte, Mesembryanthemum crystallinum, shifts its mode of carbon assimilation from the C₃ pathway to Crassulacean acid metabolism (CAM) in response to water stress. In this study, exogenously applied abscisic acid (ABA), at micromolar concentrations, could partially substitute for water stress in induction of CAM in this species. ABA at concentrations of 5 to 10 micromolar, when applied to leaves or to the roots in hydroponic culture or in soil, induced the expression of CAM within days (as indicated by the nocturnal accumulation of total titratable acidity and malate). After applying ABA there was also an increase in phosphoenolpyruvate carboxylase and NADP-malic enzyme activities. The degree and time course of induction by ABA were comparable to those induced by salt and water stress. Electrophoretic analyses of leaf soluble protein indicate that the increases in phosphoenolpyruvate carboxylase activity during the induction by ABA, salt, and water stress are due to an increase in the quantity of the enzyme protein. ABA may be a factor in the stress-induced expression of CAM in M. crystallinum, serving as a functional link between stress and biochemical adaptation.     

Phosphoenolpyruvate Carboxylase During Development of Crassulacean Acid Metabolism and During a Diurnal Cycle in Mesembryanthemum Crystallinum

Crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinumwas induced by transfer of plants from 100 to 400 mM NaCl. Diurnalmalate fluctuations developed slowly; maximum rates of net malatesynthesis in the dark were reached only on the 10th day afterNaCl was increased to 400 mM. In contrast, phosphoenolpyruvatecarboxylase (PEPC) activity, assayed at optimum pH of 8–0,had nearly reached its maximum on the 5th day after plants weretransferred to 400 mM NaCl. Characteristics of PEPC changedduring the first 12 d of exposure of plants to 400 mM NaCl.There were increases in the ratio of PEPC activity at pH 7 0/PEPCactivity at pH 8.0, and decreases in the Km for PEP measuredat pH 7.0, and possibly in the degree of malate inhibition.All further measurements were made once CAM was well established.In vivo rates of malate synthesis were 14–18 times smallerthan PEPC activity at 2 mM PEP, both processes being measuredat 15 °C. It is suggested that the high PEPC levels favourrapid, preferential flow of carbon to malate, by maintainingvery low PEP levels in the cytoplasm. PEPC changed in characteristicsduring the diurnal cycle. During the first few minutes afterisolation, extracts made during the first hours of the day,when malate was consumed, showed very low PEPC activity at pH7.0 but high activity at pH 8.0. The activity of PEPC at pH7.0 rose gradually during storage of the extracts at 0 °C,usually reaching the activity at pH 8.0 after about 30–50min. In contrast, extracts obtained during the first hours ofthe night, when malate was synthesized, showed high PEPC activityat both pH 7.0 and 8–0 within 30–50 s after extraction.The results indicate that PEPC of M. crystallinum, performingdistinct CAM, may exist in two states. One state would favourrapid malate synthesis and transport to the vacuoles and wouldfunction during the night. The second state, with little activitybelow pH 7.5, would occur during the day, thus preventing complicationsof continued synthesis of malate while it is converted to carbohydrates.     

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

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

Gas exchange and metabolite fluctuations in green and yellow bands of variegated leaves of the monocotyledonous CAM species Agave americana

The variegated leaves of the Crassulacean acid metabolism (CAM) species Agave americana have a large central longitudinal green band with narrow yellow bands on either side. The yellow bands had 97% less pigment content, 84% lower ribulose‐1,5‐bisphosphate carboxylase/oxygenase activity, but only 20% lower phosphoenolpyruvate carboxylase activity than the green band. The green bands exhibited gas exchange typical of CAM plants, with most CO₂ uptake occurring at night, leading to a daily net CO₂ uptake of 127 mmol m⁻² day⁻¹. The yellow bands had some nighttime net CO₂ uptake but a larger loss during the daytime, indicating that they were sink tissues. Nocturnal citrate and malate accumulations for the yellow bands were 65 and 75%, respectively, of those of the green bands; sucrose supported 64‐83% of their nocturnal acid accumulation. This is the first evidence that agaves, which are malic‐enzyme‐type CAM plants, use sucrose as the carbon source for nocturnal acid accumulation. About 44% of the carbon demand of the yellow bands can be supplied by sucrose diffusing via the symplast from the adjacent green band, about 25% from fructose and glucose diffusion, and some via the apoplast.     

Daily malate fluctuations in Sedum dasyphyllum L.

Abstract

Sedum dasyphyllum L. leaves of well-watered plants kept outdoors and under controlled conditions show diurnal malic acid fluctuations. In well-watered plants growing outdoors the malate accumulation undergoes seasonal variations and seems to be inhibited by short photoperiod and/or by low temperature. The seasonal variations of CAM activity correspond to seasonal variations of mesophyll succulence. Water stress markedly depressed CAM activity. In fact, plants of S. dasyphyllum show, under controlled conditions, a decrease of malate accumulation as relative water content decreases. Recovery from water stress is fairly slow. Water potential quickly increases during rewatering and exceeds the original value after few days, suggesting a consumption of osmotic compounds during the water stress period.     

Nitrate enhancement of CAM activity in two Kalanchoë species is associated with increased vacuolar proton transport capacity

Among species that perform CAM photosynthesis, members of the genus Kalanchoë have been studied frequently to investigate the effect of environmental factors on the magnitude of CAM activity. In particular, different nitrogen sources have been shown to influence the rate of nocturnal CO₂ fixation and organic‐acid accumulation in several species of Kalanchoë. However, there has been little investigation of the interrelationship between nitrogen source (nitrate versus ammonium), concentration and the activity of the vacuolar proton pumps responsible for driving nocturnal organic‐acid accumulation in these species. In the present study with Kalanchoë laxiflora and Kalanchoë delagoensis cultivated on different nitrogen sources, both species were found to show highest total nocturnal organic‐acid accumulation and highest rates of ATP‐ and PPi‐dependent vacuolar proton transport on 2.5 mM nitrate, whereas plants cultivated on 5.0 mM ammonium showed the lowest values. In both species malate was the principal organic‐acid accumulated during the night, but the second‐most accumulated organic‐acid was fumarate for K. laxiflora and citrate for K. delagoensis. Higher ATP‐ and PPi‐dependent vacuolar proton transport rates and greater nocturnal acid accumulation were observed in K. delagoensis compared with K. laxiflora. These results show that the effect of nitrogen source on CAM activity in Kalanchoë species is reflected in corresponding differences in activity of the tonoplast proton pumps responsible for driving sequestration of these acids in the vacuole of CAM‐performing cells.