Spectral dependence of flavonol and betacyanin accumulation in Mesembryanthemum crystallinum under enhanced ultraviolet radiation

Mesembryanthemum crystallinum L. (Aizoaceae) is a drought‐ and salt‐tolerant halophyte that is able to endure harsh environmental conditions. Upon irradiation with high light irradiance (1200–1500 µmol m^?2 s^?1) it displays a rapid cell‐specific accumulation of plant secondary metabolites in the upper leaf epidermis; a phenomenon that is not detectable with salt or drought treatment. The accumulation of these compounds, the betacyanins and acylated flavonol glycosides, increases if the plants are exposed to polychromatic radiation with a progressively decreasing short‐wave cut‐off in the ultraviolet range. The response is localized in the epidermal bladder cells on the tips of young leaves and epidermal layers of fully expanded leaves. It is demonstrated that the accumulation of flavonols and betacyanins can be described by a weakly sigmoid dose function in combination with an exponential decrease of the response function of the plant with increasing wavelength.     

Ultrastructure of Chloroplasts and Their Storage Inclusions in the Primary Leaves of Mesembryanthemum crystallinum Affected by Putrescine and NaCl

The effects of salinity (300 mM NaCl), putrescine (Put), and the combination of two agents on the structure of chloroplasts and storage deposits were studied in the third leaf pair of a facultative halophyte Mesembryanthemum crystallinum. Within 6 days, the common ice plants responded to NaCl and Put treatments by diminished chloroplast volumes and swollen grana. Different effects of the experimental treatments were primarily manifested in the chloroplast storage inclusions. Under the salinity conditions, the starch content dropped down almost threefold as compared to untreated plants (control), whereas the number of plastoglobules did not change. Put and Put + NaCl treatments further decreased the starch content per unit section area; in contrast, the plastoglobule area per chloroplast section increased eightfold and tenfold in Put and Put + NaCl treatments, respectively. The morphology and electronic density of plastoglobules changed in all treatments. In both Put treatments there ware no destructive changes in the chloroplasts, and therefore the authors presume that the increase in the numbers plastoglobules was related to the redirection of cell metabolism towards the products of the higher reduction potential. The ferritin deposits in the chloroplasts were observed in all treatments they were more abundant in the vascular parenchyma cells, especially under salinity. The ability of the common ice plants to accumulate large Fe quantities in their chloroplasts and the characteristic pectin-filled pockets, which were observed earlier, and intercellular spaces are probably related to the genetically determined traits of plant adaptation to salinity and water deficit.     

Salt-induced changes in protein composition in light-grown callus of Mesembryanthemum crystallinum

The halophyte Mesembryanthemum crystallinum (ice plant) has been suggested as a model for salt-tolerance in higher plants. To investigate salt-induced changes in polypeptide patterns at the cellular level, a light-grown callus of M. crystallinum with substantial chlorophyll content, was established and the effect of NaCl on the composition of phenol-extracted protein was examined by SDS- and 2D-polyacrylamide gel electrophoresis (PAGE). SDS-PAGE showed the accumulation of five polypeptides with estimated molecular masses of 40, 34, 32, 29 and 14 kDa was enhanced by the addition of 200 m M NaCl to the culture media. The addition of ABA (10 μ M ) or mannitol (400 m M ) did not elicit the same degree of accumulation of these salt-specific proteins. These polypeptides were classified into two groups according to their course of induction: early responsive (40, 34, 29 kDa) and late-responsive (32, 14 kDa) proteins. In addition, two polypeptides (20, 18 kDa) were transiently accumulated during salt treatment. Further separation of soluble proteins by 2-D gel electrophoresis, either isoelectric focusing (IEF) or non-equilibrium pH-gradient electrophoresis (NEPHGE) followed by SDS-PAGE, showed more alterations in accumulation of polypeptides by NaCl than 1-D gel electrophoresis. Overall, levels of more than 30% of basic polypeptides, detected by NEPHGE/SDS-PAGE, were altered by 200 m M NaCl treatment, while only 10% of neutral and acidic polypeptides, detected by IEF/SDS-PAGE, were changed. The enhanced expression of these proteins by salt in cultured cells is most likely related to the cellular responses to salinity, and not to the mechanism of CAM induction in this facultative halophyte.     

Oxidative stress and fluctuations of free and conjugated polyamines in the halophyte Mesembryanthemum crystallinum L. under NaCl salinity

The accumulation of conjugated and free polyamines in plants is very important for their protection against oxidative stress induced by abiotic factors. In the present study, the species halophytic plant Mesembryanthemum crystallinum L. was used as a model system in which the process of Crassulacean Acid Metabolism induction is linked with oxidative stress, especially under salinity conditions. A comparative analysis of the content of free polyamines, perchloric (PCA)-soluble and PCA-insoluble conjugated polyamines in mature leaves and roots was carried out with plants exposed to salinity. It was found that adult leaves and roots under normal conditions or salinity (400 mM NaCl) contained all types of free polyamines (putrescine, spermidine, spermine, and cadaverine). In leaves only PCA-insoluble conjugates were found, which showed a tendency to grow with increased duration of salt action (1.5–48 h). In contrast to leaves, in roots all forms of polyamine conjugates (PCA-soluble and -insoluble) were detected. However, the formation of all conjugates, especially PCA-soluble forms in roots, was sharply inhibited by salt shock (400 mM NaCl, 1.5 h) or exogenous cadaverine (1 mM) treatment. PCA-soluble conjugates of cadaverine in roots were found only when the treatment was carried out in combination with aminoguanidine (1 mM), as a result of diamine oxidase inhibition and consequently a decreasing of H₂O₂ production in plant cells. The activation of diamine oxidase and guaiacol peroxidase by NaCl or exogenous cadaverine was observed in leaves and roots. Thus, the activation of oxidative degradation of polyamines combined with H₂O₂–peroxidase reaction in cells are involved in the regulation of free and conjugated polyamines titers under salinity.     

Oxidative stress and fluctuations of free and conjugated polyamines in the halophyte Mesembryanthemum crystallinum L. under NaCl salinity

The accumulation of conjugated and free polyamines in plants is very important for their protection against oxidative stress induced by abiotic factors. In the present study, the species halophytic plant Mesembryanthemum crystallinum L. was used as a model system in which the process of Crassulacean Acid Metabolism induction is linked with oxidative stress, especially under salinity conditions. A comparative analysis of the content of free polyamines, perchloric (PCA)-soluble and PCA-insoluble conjugated polyamines in mature leaves and roots was carried out with plants exposed to salinity. It was found that adult leaves and roots under normal conditions or salinity (400 mM NaCl) contained all types of free polyamines (putrescine, spermidine, spermine, and cadaverine). In leaves only PCA-insoluble conjugates were found, which showed a tendency to grow with increased duration of salt action (1.5–48 h). In contrast to leaves, in roots all forms of polyamine conjugates (PCA-soluble and -insoluble) were detected. However, the formation of all conjugates, especially PCA-soluble forms in roots, was sharply inhibited by salt shock (400 mM NaCl, 1.5 h) or exogenous cadaverine (1 mM) treatment. PCA-soluble conjugates of cadaverine in roots were found only when the treatment was carried out in combination with aminoguanidine (1 mM), as a result of diamine oxidase inhibition and consequently a decreasing of H₂O₂ production in plant cells. The activation of diamine oxidase and guaiacol peroxidase by NaCl or exogenous cadaverine was observed in leaves and roots. Thus, the activation of oxidative degradation of polyamines combined with H₂O₂–peroxidase reaction in cells are involved in the regulation of free and conjugated polyamines titers under salinity.     

Starch-degrading enzymes during the induction of CAM in Mesembryanthemum crystallinum

Mesembryanthemum crystallinum plants were irrigated with 400 mol m^?3 NaCl to induce CAM and levels of leaf starch, and activities of starch-degrading enzymes were measured. During Crassulacean acid metabolism (CAM) induction, daily starch turnover gradually became more pronounced and was three- to four-fold greater than in leaves of C₃ plants after 3 weeks. Activities of α- and β-amylase, D-enzyme and starch phosphorylase all increased 10- to 20-fold within 3 weeks of the start of salt treatment. Activities of α- and β-amylase increased more than fourfold within the first 24 h of salt treatment, which is the fastest increase in enzyme activities so far measured during the induction of CAM with salt solution in intact plants of this species. Most enzyme activities were partially chloroplastic; however, the principal starch-degrading activity was constituted by an extra-chloroplastic β-amylase. CAM starch-phosphorylase activity, which was mainly chloroplastic, exhibited a two- to three-fold diurnal change in parallel with starch content. CAM induction in M. crystallinum is clearly associated with greater starch turnover and enhanced starch-degrading enzyme activities, which as catalysts of the initial reaction to release carbon for synthesis of phosphoenolpyruvate (PEP) appear highly significant for the functioning of the CAM pathway. The diurnal rhythm of phosphorylase activity may be of particular significance.     

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 Putrescine on the Apoplast Ultrastructure in the Leaf Mesophyll of Mesembryanthemum crystallinum under Salinity Stress

The effects of NaCl, putrescine (Put), and the combination of two agents on the contents of free polyamines (PA), peroxidase activity, and the ultrastructure of the mesophyll apoplast were studied in the third leaf pair of Mesembryanthemum crystallinum L. plants. The NaCl solution was added to soil daily for three days (100 ml of the 100 mM solution), and plants were sprayed with the 1 mM Put solution twice per day for a six-day period. The accumulation of Put and especially spermidine (Spd) by day 3 of salinization was followed by a dramatic drop in Put and Spd contents by day 6. In contrast, the activities of soluble and ion-bound peroxidases increased following a long lag-period. Treatments with Put and NaCl plus Put considerably enhanced this rise in two peroxidase activities. An electron microscopic examination of cell walls in the control and stressed plants demonstrated that a gap developed at the middle lamella with pockets filled with amorphous polysaccharides (AP), presumably pectins. At the maximum gap width, the pockets fused with the intercellular spaces, and, in this case, the intercellular spaces also contained AP. Following salinization, AP in the apoplast swelled and expanded. Apparently the genetic determination of high AP content in M. crystallinum plants is the basis for the ability of juvenile plants to bind Na⁺ and Cl^– ions and excess PA and also to accumulate water. In the plants treated with NaCl plus Put, the number of pockets and their volume increased, and the surface of some cell walls became plated with suberin, thus providing an additional barrier for ion transport from the pockets and intercellular spaces into cells. The formation of suberin plates was correlated with the high activity of ion-bound peroxidase essential for suberin deposition. The authors presume that H₂O₂ results from PA oxidation and, in its turn, induces the activity of peroxidase involved in the suberin plate formation.     

Photoautotrophic Cell Suspension Cultures from Mesembryanthemum crystallinum and their Response to Salt Stress

For the first time, photoautotrophic cell suspension cultures of Mesembryanthemum crystallinum have been established. The cells are growing in a sugar-free culture medium in the presence of 2 % (v/v) CO₂ as the sole carbon source. A 16 h light photoperiod is applied. Increase in fresh and dry weight during a 21 days growth cycle was more than 3-fold. Treatment of the cells with 200 mM NaCl from day 10 to day 21 of subculture stimulated cell culture growth, enhanced CO₂ fixation and elicited an increase in the extractable activities of enzymes related to CO₂ fixation (RubisCO; PEP carboxylase) and malic acid metabolism (NAD / NADP dependent malic enzyme and malic acid dehydrogenase). The cells performed osmotic adjustment to high salinity by uptake of K⁺, Na⁺, Cl^? and formation of proline as well as by a reduction in cell size. Although sugar and starch content of the cells changed during light/dark transition, a CAM-related diurnal fluctuation of malic acid was not observed.     

Compartmentation of Cadmium and Iron in Mesembryanthemum crystallinum Plants during the Adaptation to Cadmium Stress

The common ice plants (Mesembryanthemum crystallinum) at the stage of five leaf pairs were exposed to cadmium chloride solutions (1, 0.1, and 0.01 mM) under the conditions of water culture. After five days, the partition of cadmium and iron in the plant organs and in the cell structures of the apical root region were investigated. Plant adaptation to excess cadmium in the environment was assessed by an increase in the leaf and root weight, a change in peroxidase activity, and an accumulation of proline. The common ice plant accumulated cadmium mainly in the root system. At a high concentration of cadmium in the nutrient solution (1 mM), its content in the root exceeded 2 g/kg fr wt, while at a concentration of 0.01 mM, it was as low as 10 mg/kg. Dithizone staining of transverse sections of the root apical region showed that, after a 48-h-long exposure of plants to 0.1 mM cadmium chloride, cadmium was localized in the cell walls of endodermis and metaxylem. The level of cadmium in leaves varied from 0.5 to 18 mg/kg fr wt. However, there was only a weak correlation between cadmium accumulation and the extent of a biomass decrease in the leaves of various stories, when cadmium concentration in the medium (1 mM cadmium chloride) was toxic. This fact could be related to a marked efflux of endogenous iron from old leaves into the young ones and to a change in the cadmium/iron ratio in the tissues. Proline accumulation in the third leaf pair and in the roots occurred at a relatively low cadmium content (10–12 mg/kg fr wt) in these organs. Maxima of activity of all three forms of peroxidase, viz., soluble, ionically-bound, and covalently-bound peroxidases, in roots were found at a high accumulation of cadmium in these organs (45 mg/kg fr wt). These maxima exceeded 3–4-fold the activity in aging leaves containing 5 mg cadmium/kg fr wt. A decrease in peroxidase activity in leaves was accompanied by a 3.3-fold decrease in iron content; thus, it could be caused by a deficiency of available iron necessary for the enzyme functioning. It was concluded that the resistance of Mesembryanthemum crystallinum, a halophyte, to excess cadmium content in the medium was achieved by its predominant accumulation in roots, where excess cadmium is compartmentalized in the apoplast and seems to be subjected to detoxification through pectate formation. Moreover, the leaves and, particularly, the roots are characterized by a high activity of the antioxidant systems, such as guaiacol-dependent peroxidases, and an occurrence of proline at modest cadmium concentrations.     

盐条件下不同植物生长调节剂对冰菜种子萌发的影响

冰菜是一种具有极强耐盐性的新型蔬菜,具有极高的营养价值和保健作用。本文针对冰菜种子萌发率低的问题,采用不同浓度的赤霉素(GA_3)、水杨酸(SA)和抗坏血酸(Vc)等3种植物生长调节剂在盐条件下处理冰菜种子。研究结果表明:冰菜种子萌发的最适盐浓度为10 g·L^-1。在此盐度下,若添加10 mg·L^-1的GA_3,冰菜种子的发芽率最高可达77.3%;也可添加20 mg·L^-1的水杨酸或20 mg·L^-1的抗坏血酸,都会对冰菜种子萌发具有明显的促进效果。     

Efficient plant regeneration of Mesembryanthemum crystallinum via somatic embryogenesis

 An efficient plant regeneration procedure has been established from hypocotyl explants of the common ice plant, Mesembryanthemum crystallinum L, a halophytic leaf succulent that exhibits a stress-induced switch from C3 photosynthesis to crassulacean acid metabolism (CAM). Somatic embryos were initiated and developed up to globular and heart stages in Murashige and Skoog (MS) media supplemented with 3% sucrose, 0.6% bacto-agar, 80 mM NaCl, 5 μM 2,4-D and 1 μM kinetin. High frequency regeneration occurred when somatic embryos were germinated on media that lacked 2,4-D. High cytokinin treatment suppressed normal growth of embryos and favored abnormal embryo proliferation. Without growth regulators, regenerated plants rooted on MS medium with 100% efficiency. Mature, regenerated plants were fertile and morphologically identical to seed-derived plants. Received: 29 April 1999 / Revision received: 2 July 1999 · Accepted: 12 July 1999     

Proline accumulation in two bean cultivars under salt stress and the effect of polyamines and ornithine

Proline accumulation in two different bean (Phaseolus vulgaris L.) cultivars, one drought-sensitive (Canario 60) and one drought-resistant (Pinto Villa) was investigated. Both tolerated salt concentrations up to 150 mM NaCl, but the sensitive Canario 60 did not survive at 400 mM NaCl. In response to salt stress, both cvs. accumulated proline in all the analyzed tissues, the lowest contents were detected in roots. Pinto Villa accumulated higher proline concentrations than Canario 60 only at 400 mM NaCl. The addition of polyamines or ornithine increased proline content in plant tissues without stress, while they decreased it under salt stress.     

Subcellular localization and stress responses of superoxide dismutase isoforms from leaves in the C3-CAM intermediate halophyte Mesembryanthemum crystallinum L.

BSA, bovine serum albumin
CAM, Crassulacean acid metabolism
DTT, dithiothreitol
EDTA, ethylenediaminetetraacetic acid
FPLCfast protein liquid chromatography
HEPES, N-(2-hydroxyethyl)piperazine-?-(ethanesulphonic acid)
ME, β-mercaptoethanol
NBT, nitro blue tetrazolium
PAGE, polyacrylamide gel electrophoresis
SDS, sodium dodecyl sulphate
SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis
Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39)
SOD, superoxide dismutase (EC 1.15.1.1)
TEMED, N,N,?,?-tetramethylethylenediamine
Tris, Tris (hydroxymethyl) aminomethane
Tricine, N-Tris(hydroxymethyl)methylglycine

Treatment of Mesembryanthemum crystallinum for several days with 0·4 kmol m^–3 NaCl in the root medium, in parallel to an increase of the cell sap osmolarity enhances activity of important antioxidative enzymes, such as superoxide dismutases (SODs). M. crystallinum is equipped with three SOD isoforms. These isoforms were identified as Mn-, Fe-, and Cu/Zn-SODs, respectively. Mn-SOD was found in the mitochondrial fraction, Fe-SOD in the chloroplast fraction, and Cu/Zn-SOD is probably localized in the cytosol. The Fe-SOD found in M. crystallinum is the first iron-containing SOD enzyme to be characterized in the plant family Aizoaceae. Salt treatment increases the activity of this isoform earlier than the other SODs. Molecular masses of SOD isoforms were determined as 82, 48 and 34 kDa for Mn-, Fe-, Cu/Zn-SODs, respectively. Native Mn-SOD seems to be a tetramer, while Fe-SOD and Cu/Zn-SOD are dimers. All SOD isoforms show high thermal stability. Mn-SOD is active even after short heating at 90 °C and Fe-SOD at 70 °C. Moreover, high concentrations of β-mercaptoethanol used as a reducing agent did not destroy the function of all isoforms. With the salinity treatment in M. crystallinum, Crassulacean acid metabolism (CAM) is induced. Build-up of large stationary O₂ concentrations in the leaf air spaces is associated with the photosynthetic CO₂ reduction behind closed stomata in phase III of CAM. This illustrates why M. crystallinum may require higher antioxidative activities under NaCl stress and also explains earlier findings that CAM plants are more resistant than C₃ plants to environmental stress as imposed by, for example, SO₂ and O₃.     

Evidence that the induction of crassulacean acid metabolism by water stress in Mesembryanthemum crystallinum (L.) involves root signalling

The aim of this study was to investigate whether the root system of Mesembryanthemum crystallinum (L.) plays a role in triggering the induction of crassulacean acid metabolism (CAM) during water stress. Depriving well-irrigated plants of water, by allowing the soil surrounding the roots to dry, caused increased daily losses in leaf relative water content (RVVC) and mesophyll cell turgor pressure. The RWC of the roots also declined. Subsequently plants exhibited physiological characteristics of CAM photosynthesis (i.e. diurnal fluctuations in leaf titratable acidity and nocturnal net CO₂ fixation). When the root system of plants was divided equally between two soil compartments and one half deprived of water, plants exhibited physiological characteristics of CAM without prior changes in leaf RWC content or mesophyll cell turgor pressure. Only the RWC of the water-stressed portion of the roots was reduced. These data suggest that in water-stressed plants daily changes in leaf water relations greater than those observed in well-irrigated plants, are not essential to trigger CAM expression. It is probable that a reduction in soil water availability can be perceived by the roots of M. crystallinum and that this information is conveyed to the leaves triggering the transition from C₃ to CAM photosynthesis.     

Salt tolerance, salt accumulation, and ionic homeostasis in an epidermal bladder-cell-less mutant of the common ice plant Mesembryanthemum crystallinum

The aerial surfaces of the common or crystalline ice plant Mesembryanthemum crystallinum L., a halophytic, facultative crassulacean acid metabolism species, are covered with specialized trichome cells called epidermal bladder cells (EBCs). EBCs are thought to serve as a peripheral salinity and/or water storage organ to improve survival under high salinity or water deficit stress conditions. However, the exact contribution of EBCs to salt tolerance in the ice plant remains poorly understood. An M. crystallinum mutant lacking EBCs was isolated from plant collections mutagenized by fast neutron irradiation. Light and electron microscopy revealed that mutant plants lacked EBCs on all surfaces of leaves and stems. Dry weight gain of aerial parts of the mutant was almost half that of wild-type plants after 3 weeks of growth at 400 mM NaCl. The EBC mutant also showed reduced leaf succulence and leaf and stem water contents compared with wild-type plants. Aerial tissues of wild-type plants had approximately 1.5-fold higher Na(+) and Cl(-) content than the mutant grown under 400 mM NaCl for 2 weeks. Na(+) and Cl(-) partitioning into EBCs of wild-type plants resulted in lower concentrations of these ions in photosynthetically active leaf tissues than in leaves of the EBC-less mutant, particularly under conditions of high salt stress. Potassium, nitrate, and phosphate ion content decreased with incorporation of NaCl into tissues in both the wild type and the mutant, but the ratios of Na(+)/K(+) and Cl(-)/NO(3)(-)content were maintained only in the leaf and stem tissues of wild-type plants. The EBC mutant showed significant impairment in plant productivity under salt stress as evaluated by seed pod and seed number and average seed weight. These results clearly show that EBCs contribute to succulence by serving as a water storage reservoir and to salt tolerance by maintaining ion sequestration and homeostasis within photosynthetically active tissues of M. crystallinum.     

Biochemical and Immunological Properties of Solubilized Tonoplast ATPase of the Facultative CAM Plant Mesembryanthemum crystallinum in the C3 and CAM States

A nitrate-sensitive, azide-insensitive ATPase isolated from M. crystallinum in the C₃ and in the CAM state has been solubilized in active form using octylglucoside and Zwittergent 3–14. Like the membrane-bound tonoplast ATPase, the solubilized ATPase showed an increase in ATP-hydrolysis activity after transition from the C₃ to the CAM mode of photosynthesis. The characteristics of the membrane-bound and the solubilized tonoplast ATPase were comparable with respect to salt stimulation, inhibitor effects, and MgATP^2–-concentration dependence. Differing from the membrane-bound ATPases, the solubilized ATPase from C₃- and CAM-M. crystallinum showed a pH optimum between pH 6.5 and 7.0. In order to compare the solubilized ATPases immunologically, antibodies were prepared against the tonoplast fraction of C₃- and CAM-M. crystallinum. A cross-reaction was observed between antibodies against the tonoplast ATPase from C₃- and CAM-M. crystallinum and the solubilized ATPase from C₃- and CAM-M. crystallinum. A cross-reaction was also observed between antibodies against the tonoplast ATPase from C₃- and CAM-M. crystallinum and the solubilized tonoplast ATPase from Kalanchoë daigremontiana. However, there was no cross-reaction with the solubilized plasmalemma ATPase from Festuca rubra.