Adaptation of the tonoplast V-type H+-ATPase of Mesembryanthemum crystallinum to salt stress, C3–CAM transition and plant age

Plants of the facultative halophyte and CAM species Mesembryanthemum crystallinum L. (Aizoaceae) were stressed for 8 d with 400 mol m⁻³ NaCl in the root medium. NaCl was then removed from the substratum, and the plants were watered again with NaCl-free solution. A second set of plants was maintained as controls. A small degree of CAM, as indicated by day-night changes in malate levels, was expressed during ageing of the plants. Salinity-stress-dependent CAM induction was reversible by the removal of salt, as indicated by similar Δ malate levels in previously salt-stressed plants and in non-stressed plants on day 19 of the experiment. Tonoplast vesicles were isolated from leaves during the time-course of stress application, stress removal and ageing. Parameters of the tonoplast H⁺-ATPase were correlated to the application of salinity, the expression of CAM and ageing. It was concluded, first, that a pronounced increase in the amount of tonoplast H⁺-ATPase is related to salinity per se and a smaller increase to ageing; secondly, that there is an increase in the specific activity of the enzyme related to ageing; thirdly, that the induction of two new polypeptides with molecular masses of 32 and 28 kDa is correlated in time with the expression of CAM, and, fourthly, that the two new polypeptides are part of the tonoplast H⁺-ATPase holoenzyme.     

Salt Stress Increases the Level of Translatable mRNA for Phosphoenolpyruvate Carboxylase in Mesembryanthemum crystallinum

Mesembryanthemum crystallinum responds to salt stress by switching from C₃ photosynthesis to Crassulacean acid metabolism (CAM). During this transition the activity of phosphoenolpyruvate carboxylase (PEPCase) increases in soluble protein extracts from leaf tissue. We monitored CAM induction in plants irrigated with 0.5 molar NaCl for 5 days during the fourth, fifth, and sixth week after germination. Our results indicate that the age of the plant influenced the response to salt stress. There was no increase in PEPCase protein or PEPCase enzyme activity when plants were irrigated with 0.5 molar NaCl during the fourth and fifth week after germination. However, PEPCase activity increased within 2 to 3 days when plants were salt stressed during the sixth week after germination. Immunoblot analysis with anti-PEPCase antibodies showed that PEPCase synthesis was induced in both expanded leaves and in newly developing axillary shoot tissue. The increase in PEPCase protein was paralleled by an increase in PEPCase mRNA as assayed by immunoprecipitation of PEPCase from the in vitro translation products of RNA from salt-stressed plants. These results demonstrate that salinity increased the level of PEPCase in leaf and shoot tissue via a stress-induced increase in the steady-state level of translatable mRNA for this enzyme.     

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.     

Differential effects of drought and light levels on accumulation of citric and malic acids during CAM in Clusia

Night-time citrate accumulation has been proposed as a response to stress in CAM plants. To address this hypothesis, gas exchange patterns and nocturnal acid accumulation in three species of Clusia were investigated under controlled conditions with regard to water stress and responses to low and high photosynthetic photon flux density (PPFD). Under high PPFD, leaves of Clusia nocturnally accumulated large amounts of both malic and citric acids. Under low PPFD and well-watered conditions, substantial night-time citrate accumulation persisted, whereas malate accumulation was close to zero. Malate accumulation and night-time CO₂ uptake from the atmosphere declined in all three species during prolonged drought periods, whereas citrate accumulation remained similar or increased. Recycling of respiratory CO₂ was substantial for both well-watered and water-stressed plants. The suggestion that citrate accumulation is energetically more favourable than malate accumulation is not supported if the source of CO₂ for the formation of malate is respiratory CO₂. However, the breakdown of citric acid to pyruvate in the light period releases three molecules of CO₂, while the breakdown of malic acid releases only one CO₂ per pyruvate formed. Thus, citric acid should be more effective than malic acid as a mechanism to increase CO₂ concentration in the mesophyll and may help to prevent photoinhibition. Organic acid accumulation also affected the vacuolar pH, which reached values of 2·6–3·0 at dawn. At these pH values, the transport of 2H⁺/ATP is still feasible, suggesting that it is the divalent form of citrate which is being transported in the vacuoles. Since citrate is a well-known buffer, and Clusia spp. show the largest day-night changes in organic acid levels measured in any CAM plant, it is possible that citrate increases the buffer capacity of the vacuoles. Indeed, malate and titratable acidity levels are positively related to citrate levels. Moreover, Clusia species that show the highest nocturnal accumulation of organic acids are also the ones that show the greatest changes in citric acid levels.     

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.     

Increased Expression of a myo-Inositol Methyl Transferase in Mesembryanthemum crystallinum Is Part of a Stress Response Distinct from Crassulacean Acid Metabolism Induction

The facultative halophyte Mesembryanthemum crystallinum responds to osmotic stress by switching from C₃ photosynthesis to Crassulacean acid metabolism (CAM). This shift to CAM involves the stress-initiated up-regulation of mRNAs encoding CAM enzymes. The capability of the plants to induce a key CAM enzyme, phosphoenolpyruvate carboxylase, is influenced by plant age, and it has been suggested that adaptation to salinity in M. crystallinum may be modulated by a developmental program that controls molecular responses to stress. We have compared the effects of plant age on the expression of two salinity-induced genes: Gpdl, which encodes the photosynthesis-related enzyme glyceraldehyde 3-phosphate dehydrogenase, and Imtl, which encodes a methyl transferase involved in the biosynthesis of a putative osmoprotectant, pinitol. Imtl mRNA accumulation and the accompanying increase in pinitol in stressed Mesembryanthemum exhibit a pattern of induction distinct from that observed for CAM-related genes. We conclude that the molecular mechanisms that trigger Imtl and pinitol accumulation in response to salt stress in M. crystallinum differ in some respects from those that lead to CAM induction. There may be multiple signals or pathways that regulate inducible components of salinity tolerance in this facultative halophyte.     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Investigation of phosphoeno/pyruvate carboxylase (PEPCase) in Mesembryanthemum crystallinum L. in C3 and CAM photosynthetic states

When exposed to osmotic stress, Mesembryanthemum crystallinum plants switch from C₃ to CAM photosynthesis. Phosphoenolpyruvate carboxylase (PEPCase) is a key enzyme in CAM plants, being responsible for the initial fixation of CO₂. In C₃ plants the enzyme has been shown to be involved in the replenishing of TCA cycle intermediates and in the operation of stomatal guard cells. Multiple PEPCase isoforms were observed in C₃-performing leaves with four isoelectric points of 5.2, 5.5, 5.6 and 5.9 and four apparent subunit molecular masses of 105, 108, 113 and 116 kDa. In some instances, subunits of different size possessed exactly the same pI. The induction of CAM led to the predominance of a new isoform of pI 6.5 with subunit molecular mass of 108 kDa, but in addition, changes were observed in some of the isoforms present in the C₃ plant. PEPCase subunits were purified from the C₃ and CAM forms of M. crystallinum and subjected to pep-tide mapping. Two distinct though similar sets of maps were obtained, one from the CAM isoform (pI 6.5) and C₃-associated subunits of pi 5.9 and another for C₃ subunits of pI 5.2 and 5.5. It was inferred from these data that the C₃ isoforms expressed in the leaf were derived from at least two genes. The C₃ isoform (pI 5.9) showing greatest similarity to the CAM isoform in terms of peptide mapping also increased in response to salt stress. It is speculated that the CAM isoform may have evolved from this enzyme.     

The effects of salinity,crassulacean acid metabolism and plant age on the carbon isotope composition of <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L., a halophytic C<Subscript>3</Subscript>-CAM species

The carbon isotope composition of the halophyte Mesembryanthemum crystallinum L. (Aizoaceae) changes when plants are exposed to environmental stress and when they shift from C₃ to crassulacean acid metabolism (CAM). We examined the coupling between carbon isotope composition and photosynthetic pathway by subjecting plants of different ages to salinity and humidity treatments. Whole shoot ¹³C values became less negative in plants that were exposed to 400 mM NaCl in the hydroponic solution. The isotopic change had two components: a direct NaCl effect that was greatest in plants still operating in the C₃ mode and decreased proportionally with increasing levels of dark fixation, and a second component related to the degree of CAM expression. Ignoring the presumably diffusion-related NaCl effect on carbon isotope ratios results in an overestimation of nocturnal CO₂ gain in comparison to an isotope versus nocturnal CO₂ gain calibration established previously for C₃ and CAM species grown under well-watered conditions. It is widely taken for granted that the shift to CAM in M. crystallinum is partially under developmental control and that CAM is inevitably expressed in mature plants. Plants, cultivated under non-saline conditions and high relative humidity (RH) for up to 63 days, maintained diel CO₂ gas-exchange patterns and ¹³C values typical of C₃ plants. However, a weak CAM gas-exchange pattern and an increase in ¹³C value were observed in non-salt-treated plants grown at reduced RH. These observations are consistent with environmental control rather than developmental control of the induction of CAM in mature M. crystallinum under non-saline conditions.     

Induction of crassulacean acid metabolism in Mesembryanthemum crystallinum increases reproductive success under conditions of drought and salinity stress

Summary Mesembryanthemum crystallinum L., an inducible crassulacean acid metabolism (CAM) plant, was grown for approximately 5 weeks following germination in well-watered, non-saline soil in a controlled-environment chamber. During this time, plants were characterized by C₃ photosynthetic carbon metabolism. After the initial 5 weeks, CAM was induced by a combination of high soil salinity and reduced soil water content. One group of plants was allowed to engage in CAM by being continuously exposed to normal CO₂-containing air (about 350–400 ppm). A second group of plants was deprived of ambient CO₂ each night (12 h dark period) until completion of their life cycle, thereby minimizing potential carbon gain via dark CO₂ fixation. The capacity to express CAM under conditions of drought and salinity stress markedly improved reproductive success: plants kept in normal CO₂-containing air produced about 10 times more seeds than plants kept in CO₂-free air during dark periods. Seeds from plants deprived of ambient CO₂ overnight had more negative ¹³C values than seeds from plants kept in normal air.     

Na+/H+-transporter, H+-pumps and an aquaporin in light and heavy tonoplast membranes from organic acid and NaCl accumulating vacuoles of the annual facultative CAM plant and halophyte Mesembryanthemum crystallinum L.

Crassulacean acid metabolism (CAM) was induced in Mesembryanthemum crystallinum L. by either NaCl- or high light (HL)- stress. This generated in mesophyll cells predominantly of NaCl-stressed plants two different types of vacuoles: the generic acidic vacuoles for malic acid accumulation and additionally less acidic (“neutral”) vacuoles for NaCl sequestration. To examine differences in the tonoplast properties of the two types of vacuoles, we separated microsomal membranes of HL- and NaCl-stressed M. crystallinum plants by centrifugation in sucrose density gradients. Positive immunoreactions of a set of antibodies directed against tonoplast specific proteins and tonoplast specific ATP- and PP_i-hydrolytic activity were used as markers for vacuolar membranes. With these criteria tonoplast membranes were detected in both HL- and NaCl-stressed plants in association with the characteristic low sucrose density but also at an unusual high sucrose density. In HL-stressed plants most of the ATP- and PP_i-hydrolytic activity and cross reactivity with antibodies including that directed against the Na⁺/H⁺-antiporter from Arabidopsis thaliana was detected with light sucrose density. This relationship was inverted in NaCl-stressed plants; they exhibited most pump activity and immunoreactivity in the heavy fraction. The relative abundance of the heavy membrane fraction reflects the relative occurrence of “neutral” vacuoles in either HL- or NaCl-stressed plants. This suggests that tonoplasts of the “neutral” vacuoles sediment at high sucrose densities. This is consistent with the view that this type of vacuoles serves for Na⁺ sequestration and is accordingly equipped with a high capacity of proton pumping and Na⁺ uptake via the Na⁺/H⁺-antiporter.     

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.     

Changes in Properties of Phosphoenolpyruvate Carboxylase with Induction of Crassulacean Acid Metabolism (CAM) in the C4 Plant Portulaca Oleracea

Aiming at understanding the odd case of CAM expression by a C₄ plant, some properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, orthophosphate: oxaloacetate carboxylyase, phosphorylating) were comparatively studied in leaves of CAM-expressing and non-expressing Portulaca oleracea L. plants. CAM expression was induced by growing plants under an 8-h photoperiod and under water-stress. CAM induction in leaves of these plants (designated as CAM) is indicated by the nocturnal acidification and by the clear diurnal oscillation pattern and amplitude of acidity, malic acid, and PEPC activity characteristic of CAM plants. Treatment of the other plant group (designated as C₄) by growth under a 16-h photoperiod and well-watered conditions did not induce expression of the tested criteria of CAM in plants. In these C₄ plants, the mentioned CAM criteria were undetectable. PEPC from CAM and C₄ Portulaca responded differently to any of the studied assay conditions or effectors. For example, extent and timing of sensitivity of PEPC to pH change, inhibition by malate, activation by glucose-6-phosphate or inorganic phosphate, and the enzyme affinity to the substrate PEP were reversed with induction of CAM from the C₄-P. oleracea. These contrasting responses indicate distinct kinetic and regulatory properties of PEPC of the two modes. Thus by shifting to CAM in the C₄ Portulaca a new PEPC isoform may be synthesised to meet CAM requirements. Simultaneous occurrence of both C₄ and CAM is suggested in P. oleracea when challenged with growth under stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.