Crassulacean Acid Metabolism Induction Research Articles

Modulation of Light-Dependent Stomatal Opening in Isolated Epidermis Following Induction of Crassulacean Acid Metabolism in Mesembryanthemum crystallinum L.

Light quality-dependent stomatal opening in epidermal tissue isolated from Mesembryanthemum crystallinum was investigated to determine stress-induced modulation of signal transduction in guard cells. Plants were irrigated with a non-saline medium to promote C 3 photosynthesis or, alternatively, a medium containing NaCl in order to induce Crassulacean acid metabolism (CAM). For plants grown on a non-saline medium, blue light was more effective than red in causing stomatal apertures to widen. This differential response to light quality by stomata, which is typically attributed to separate photoreceptor systems in C 3 plants, was not observed for stomata in epidermal tissue isolated from CAM plants. Regardless of color, stomata prepared from saline-treated plants showed no opening response to light, but rather displayed a degree of closing (approximately 12 %) compared to aperture widths measured from dark-adapted epidermal tissue. Stomata from CAM plants also failed to show further opening when treated with fusicoccin, in contrast to a large opening response by stomata from C 3 plants. Despite a lack of opening by stomata isolated from CAM plants in response to either light or fusicoccin, apertures retained the capacity to close in response to (S)-abscisic acid. The results suggest that the lack of a photo-induced opening response by stomata following induction of CAM in M. crystallinum may be a result of inactivation of both guard-cell photoreceptors, which are functional in unstressed (C 3 ) plants.

Growth, water relations, and CAM induction inSedum album in response to water stress

Growth analysis indicated that carbon gain in the alpine succulentSedum album tended to take place early during the growing season. Leaf water potential remained unchanged for a considerable length of time after the imposition of water stress in the field. Induction of crassulacean acid metabolism (CAM) by protracted water stress occurred before any signs of stress could be observed in the leaves, and appeared to be influenced by a complex interaction of environmental conditions including temperature and duration of sunshine. Increased levels of proline and betaine towards the end of the growing season appeared to reflect seasonal changes.

Growth-specific phytomass allocation in Mesembryanthemum nodiflorum as influenced by CAM induction in the field

The implications for growth-specific resource allocation and the C 3 to Crassulacean Acid Metabolism (CAM) shift in Mesembryanthemum nodiflorum were studied in the field. The C 3 to CAM shift occurs during flowering and fruiting stages allowing for a delayed shift of phytomass from leaf to sexual organs. This behaviour enables the plants to accumulate the additional resources necessary for maximizing the reproductive output. This attribute in the life cycle of Mesembryanthemum nodiflorum allows its dominance over other annual plants growing at the study site in the deltaic Mediterranean coast of Egypt.

Effects of growth regulators on the induction of Crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum L.

The classical induction of Crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum L. by water stress is observed within one week when fourto five-week-old plants (grown under a 16/8 h photoperiod at ca. 600 μmol quanta · m−2 · s−1) are irrigated with 350 mM NaCl. The induction of CAM was evaluated by measuring phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) and NADP-malic enzyme (NADP-ME, EC 4.1.1.82) activities and nocturnal increases in malate content and titratable acidity of leaf extracts, and the daily pattern of CO2 exchange and stomatal conductance during the 7-d induction period. Three growth regulators, abscisic acid (ABA), farnesol (an antitranspirant and analog of ABA), and benzylaminopurine (BAP), were found to substitute for NaCl for induction of CAM when fed to plants in nutrient media. Daily irrigation with solutions containing micromolar levels (optimum ca. 10 micromolar) of these growth regulators led to the induction of CAM similar to that by high salt. Application of the growth regulators, like NaCl, caused large increases in the activity of NADP-ME and the activity and level of PEPCase, which are components of the biochemical machinery required for CAM. Western immunoblotting showed that the increased activity of PEPCase on addition of ABA, farnesol and BAP was mainly due to increased levels of the CAM-specific isoforms. Also, dehydration of cut leaves over 8.5 h under light resulted in a severalfold increase in PEPCase activity. An equivalent increase in PEPCase activity in excised leaves was also obtained by feeding 150 mM NaCl, or micromolar levels of ABA or BAP via the petiole, which supports results obtained by feeding the growth regulators to roots. However, the increase in PEPCase activity was inhibited by feeding high levels of BAP to cut leaves prior to dehydration, indicating a more complex response to the cytokinin. Abscisic acid may have a role in induction of CAM in M. crystallinum under natural conditions as there is previous evidence that induction by NaCl causes an increase in the content of ABA, but not cytokinins, in leaves of this species.

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

ABSTRACT 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.

Molecular cloning and expression of chloroplast NADP-malate dehydrogenase during Crassulacean acid metabolism induction by salt stress

A full-length cDNA clone for NADP(+)-dependent malate dehydrogenase (NADP-MDH; EC 1.1.1.82) from the facultative CAM plant,Mesembryanthemum crystallinum has been isolated and characterized. NADP-MDH is responsible for the reduction of oxaloacetate to malate in the chloroplasts of higher plants. The cDNA clone is 1747 bp in size and contains a single open reading frame encoding a 441 amino acid long precursor polypeptide with a predicted molecular weight of 47 949. The predicted, mature NADP-MDH polypeptide sequence fromM. crystallinum shares 82.7% to 84% amino acid identity with other known higher plant sequences. Genomic Southern blot analysis ofM. crystallinum DNA indicates that MDH is encoded by a small gene family. Steady-state transcript levels for chloroplast NADP-MDH decrease transiently in the leaves after salt stress and then increase to levels greater than two-fold higher than in unstressed plants. Transcript levels in roots are extremely low and are unaffected by salt-stress treatment. In vitro transcription run-on experiments using isolated nuclei from leaf tissue confirm that the accumulation of NADP-MDH transcripts is, at least in part, the result of increased transcription of this gene during salt stress. The salt-stress-induced expression pattern of this enzyme suggests that it may participate in the CO2 fixation pathway during CAM.

Regulation of Phosphoenolpyruvate Carboxylase and Crassulacean Acid Metabolism Induction in Mesembryanthemum crystallinum L. by Cytokinin

Phosphoenolpyruvate carboxylase (PEPCase), the key enzyme of Crassulacean acid metabolism, is induced by water stress in leaves of Mesembryanthemum crystallinum. In water-stressed plants or excised leaves, exogenous cytokinin suppresses PEPCase transcript accumulation in the leaves. Cytokinin (6-benzylaminopurine) used in concentrations from 5 to 500 micromolar (a) inhibits the upregulation of PEPCase transcripts, enzyme activity, and Crassulacean acid metabolism induction in salt-stressed intact plants when sprayed once daily during the stress period, (b) inhibits the accumulation of PEPCase mRNA in leaves from well-watered plants, (c) down-regulates PEPCase transcripts within 8 hours in prestressed, intact plants after a single spraying of an individual leaf, (d) inhibits accumulation of PEPCase transcripts in excised, wilting leaves, and (e) accelerates the net decrease of PEPCase transcripts in excised leaves from prestressed plants under rehydration conditions. When roots, the main site of cytokinin biosynthesis, are excised, PEPCase induction under drought stress is intensified. We propose that roots, acting as sensors of soil water status, may regulate PEPCase gene expression in the leaves with cytokinin as a signal transducer.

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 C(3) 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.

Dynamics of tonoplast proton pumps and other tonoplast proteins of Mesembryanthemum crystallinum L. during the induction of Crassulacean acid metabolism

In plants of Mesembryanthemum crystallinum the activities of the two proton pumps on the tonoplast, i.e. the ATPase and the pyrophosphatase, and the gelelectrophoretic pattern of the total tonoplast proteins were analyzed during the transition of the metabolic state from C3 photosynthesis to Crassulacean acid metabolism (CAM). In one series, CAM was induced by watering the plants with NaCl. In another series, the change of the metabolic state to CAM was a consequence of the aging of the plants. No significant differences in the specific activities of ATP hydrolysis were found in plants performing C3 photosynthesis and CAM, respectively. However, with both series the protein content of tonoplast preparations and, in parallel, the total ATP hydrolytic activity of the tonoplast ATPase were higher after the change to CAM. In contrast, the specific activity of pyrophosphate hydrolysis was maximum in the preparations of young plants and diminished after the induction of CAM in both series. Therefore the tonoplast ATPase seems to be the main enzyme responsible for the energization of malate accumulation in CAM. The tonoplast pyrophosphatase is important in the early stages of plant growth and plays a minor role in CAM. With M. crystallinum the change from C3 photosynthesis to CAM is accompanied by de-novo synthesis of tonoplast proteins. Several polypeptides with relative molecular masses (Mrs) of 55, 41, and 36 kDa were clearly more pronounced in the gel-electrophoretic pattern of the total tonoplast protein after CAM induction. These changes were independent of the CAM-inducing salt treatment or aging. Moreover, two subunits of the tonoplast ATPase with Mrs of about 27 and 31 kDa showed particularly high intensities only in the CAM state. It is assumed that the subunit composition of the tonoplast ATPase differs in the two metabolic states and that the two subunits induced modify the regulation of the ATPase in CAM. In addition, the reaction of the plants to the NaCl treatment per se was the induction at the tonoplast of a polypeptide with an Mr of 24 kDa.

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

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 δ13C values than seeds from plants kept in normal air.

Environmental Control of Phosphoenolpyruvate Carboxylase Induction in Mature Mesembryanthemum crystallinum L.

Mesembryanthemum crystallinum L. plants shift the mode of carbon assimilation from C(3) to Crassulacean acid metabolism when stressed by high salinity. A prerequisite for Crassulacean acid metabolism induction is the synthesis of phosphoenolpyruvate carboxylase (PEPCase). A moderate increase in the abundance of PEPCase transcripts and activity is observed in 7-week-old, well-watered plants. This increase in PEPCase coincides in time with a decrease in the growth rate of the shoots. The steady-state level of PEPCase activity is uniform along the leaves of well-watered plants, as can be shown by comparing leaves of different age from individual 7-week-old plants. In contrast, the rate of induction in response to salt stress varies with the age of plants and to a lesser extent with the age of the leaves. Two-week-old seedlings induce PEPCase slowly under a moderate salt stress regimen, whereas older plants induce faster. When individual leaves from a seven-week-old plant are compared with respect to induction velocity, no clear-cut correlation with leaf age is apparent. The highest induction rate is observed in leaves from node five that are about 2 weeks old at the beginning of the experiment. PEPCase transcripts are readily down-regulated to minute levels when detached leaves are hydrated. The levels reached after 8 hours of rehydration are very similar, regardless of whether the leaves were cut from young or old plants or whether the plants were previously salt-stressed or well-watered. It is concluded that environmental rather than developmental factors are predominant in determining abundance of PEPCase activity and transcripts in leaves of mature M. crystallinum plants.

Daily Changes in CO2 and Water Vapor Exchange, Chlorophyll Fluorescence, and Leaf Water Relations in the Halophyte Mesembryanthemum crystallinum during the Induction of Crassulacean Acid Metabolism in Response to High NaCl Salinity

Simultaneous measurements of net CO(2) exchange, water vapor exchange, and leaf water relations were performed in Mesembryanthemum crystallinum during the development of crassulacean acid metabolism (CAM) in response to high NaCl salinity in the rooting medium. Determinations of chlorophyll a fluorescence were used to estimate relative changes in electron transport rate. Alterations in leaf mass per unit area, which-on a short-term basis-largely reflect changes in water content, were recorded continuously with a beta-gauge. Turgor pressure of mesophyll cells was determined with a pressure probe. As reported previously (K Winter, DJ von Willert [1972] Z Pflanzenphysiol 67: 166-170), recently expanded leaves of plants grown under nonsaline conditions showed gas-exchange characteristics of a C(3) plant. Although these plants were not exposed to any particular stress treatment, water content and turgor pressure regularly decreased toward the end of the 12 hour light periods and recovered during the following 12 hours of darkness. When the NaCl concentration of the rooting medium was raised to 400 millimolar, in increments of 100 millimolar given at the onset of the photoperiods for 4 consecutive days, leaf water content and turgor pressure decreased by as much as 30 and 60%, respectively, during the course of the photoperiods. These transient decreases probably triggered the induction of the biochemical machinery which is required for CAM to operate. After several days at 400 millimolar NaCl, when leaves showed features typical of CAM, overall turgor pressure and leaf mass per unit area had increased above the levels before onset of the salt treatment, and diurnal alterations in leaf water content were reduced. Net carbon gain during photoperiods and average intercellular CO(2) partial pressures at which net CO(2) uptake occurred, progressively decreased upon salinization. Reversible diurnal depressions in leaf conductance and net CO(2) uptake, with minima recorded in the middle of the photoperiods, preceded the occurrence of nocturnal net CO(2) uptake. During these reductions, intercellular CO(2) partial pressure and rates of photosynthetic electron transport decreased. With advancing age, leaves of plants grown under nonsaline conditions exhibited progressively greater diurnal reductions in turgor pressure and developed a low degree of CAM activity.

Changes in activities and levels of pyruvate, orthophosphate dikinase with induction of Crassulacean acid metabolism in mesembryanthemum crystallinum L.

In Mesembryanthetnum crystallinum L.leaves exhibiting C3 photosynthesis, Crassulacean acid metabolism (CAM), as characterized by CO2 uptake during the dark period, was induced by exposing the plant roots to high NaCl concentration over a week. The increase in malate content in the leaves at the end of the dark period correlated with the increases in the activities of phosphoenolpyruvate carboxylase (PEPCase), NADP malic enzyme (NADP-ME) and pyruvate, orthophosphate dikinase (PPDK) which exhibited a maximum about a week after NaCl treatment. It was found that PPDK existed in C3-M. ctystallinum and the increases in the activities of PEPCase and PPDK were accompanied by the de novo synthesis of these proteins

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 C(3) 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.

Phytochrome-mediated responses of cells and protoplasts of green calli obtained from the leaves of a CAM plant

Green callus obtained from leaves of the CAM-inducible plant Kalanchoe blossfeldiana cv. Montezuma has previously been shown to perform C3-type photosynthesis under 16-h days and to shift to crassulacean acid metabolism (CAM) under 9-h days. The utilization of photoperiodic regimes (i.e. night interruptions by 30 min red light) established that CAM induction in the callus was under the control of phytochrome, as shown by measurements of CAM criteria: phosphoenolpyruvate carboxylase activity and malic acid pools. Short-term responsiveness of the callus cells to phytochrome modulations by monochromatic radiations was also established by the rapid changes observed in the diameter of the callus-derived protoplasts. These results provide further evidence that whole plant correlations are not necessary for phytochrome operativity.

Membrane Particles, Proteins and ATPase Activity of Tonoplast Vesicles of Mesembryanthemum crystallinum in the C‐3 and CAM State*

AbstractDimensions and area densities of membrane particles were studied by electron microscopy of replicas of freeze‐fractured suspensions of tonoplast vesicles of Mesembryanthemum crystallinum L. in the C‐3 state and after induction of crassulacean acid metabolism (CAM) by salinity. The results are compared with the relative contribution of tonoplast‐ATPase protein to total membrane protein obtained from integration of elution profiles in size‐exclusion chromatography. Coverage of tonoplast area by globular membrane particles was 20% and 36 % and ATPase in relation to total membrane protein was 33 % and 35 % in C‐3 and CAM M. crystallinum, respectively. Thus, by order of magnitude, it is most likely that the particles represent the ATPase. In Kalanchoë daigremontiana Hamet et Perrier de la Bâthie the ATPase also constituted 36% of total tonoplast protein. Induction of CAM in M. crystallinum was associated with an increase in specific ATPase activity of the tonoplast and an increase of the size and area coverage of tonoplast particles, whereas the relative contribution of ATPase protein to total tonoplast protein and the molecular mass of the ATPase holoenzyme, as seen in size‐exclusion chromatography, remained almost unchanged.

Salt stress leads to differential expression of two isogenes of phosphoenolpyruvate carboxylase during Crassulacean acid metabolism induction in the common ice plant.

The common ice plant is a facultative halophyte in which Crassulacean acid metabolism, a metabolic adaptation to arid environments, can be induced by irrigating plants with high levels of NaCl or by drought. This stress-induced metabolic transition is accompanied by up to a 50-fold increase in the activity of phosphoenolpyruvate carboxylase (PEPCase). To analyze the molecular basis of this plant response to water stress, we have isolated and characterized two members of the PEPCase gene family from the common ice plant. The PEPCase isogenes, designated Ppc1 and Ppc2, have conserved intron-exon organizations, are 76.4% identical at the nucleotide sequence level within exons, and encode predicted polypeptides with 83% amino acid identity. Steady-state levels of mRNAs from the two genes differ dramatically when plants are salt-stressed. Transcripts of Ppc1 increase about 30-fold in leaves within 5 days of salt stress. In contrast, steady-state levels of Ppc2 transcripts decrease slightly in leaf tissue over the same stress period. Steady-state levels of transcripts of both genes decrease in roots over 5 days of salt stress. We have used in vitro transcription assays with nuclei isolated from leaves to demonstrate that the increased expression of Ppc1 caused by water stress occurs in part at the transcriptional level.

Salt Stress Leads to Differential Expression of Two Isogenes of Phosphoenolpyruvate Carboxylase during Crassulacean Acid Metabolism Induction in the Common Ice Plant

Salt Stress Leads to Differential Expression of Two Isogenes of Phosphoenolpyruvate Carboxylase during Crassulacean Acid Metabolism Induction in the Common Ice Plant

Time Course of mRNA Induction Elicited by Salt Stress in the Common Ice Plant (Mesembryanthemum crystallinum)

In the facultative halophyte Mesembryanthemum crystallinum (common ice plant), irrigation with solutions containing NaCl induces an alternate mode of carbon dioxide fixation, Crassulacean acid metabolism (CAM). The salt stress protocol which we have established facilitates the study of CAM induction and the correlation of changes in metabolism and gene expression. We have studied the time course of mRNA induction for phosphoenolpyruvate carboxylase (PEPCase) (gene: ppc) and several other enzymes of carbon metabolism during stress. While CAM is not fully established for at least 10 days after the start of stress, mRNA amounts for PEPCase and for other CAM enzymes, such as Pyruvate orthophosphate dikinase, increase between day 2 and 3 after stress induction. Increases continue for at least 5 days. Concomitant with the increase of CAM transcripts, fluctuations in the mRNA amounts for genes rbcS and cab were observed. Transcript levels for these proteins decreased several-fold during a 3 to 4 day period.

Expression of the CAM-form of phospho(enol)pyruvate carboxylase and nucleotide sequence of a full length cDNA from Mesembryanthemum crystallinum.

We have determined the complete nucleotide sequence of a full length cDNA encoding the Crassulacean acid metabolism (CAM) isogene of phospho(enol)pyruvate carboxylase (PEPCase). The cDNA clone, 3348 bp in length, was obtained from mRNA isolated from Mesembryanthemum crystallinum (common ice plant) which had undergone salt stress and subsequent induction of CAM. The long open reading frame encodes PEPCase (EC 4.1.1.31) with a predicted molecular mass of 110533 daltons. The deduced amino acid sequence of the ice plant PEPCase is most similar to that from maize having an amino acid identity of 74.9%. Sequence identity in corresponding regions of the PEPCase proteins from Escherichia coli and the cyanobacterium Anacystis nidulans are 41.4% and 33.5%, respectively. A compilation of the four amino acid sequences permitted the identification of phylogenetically conserved regions within the proteins which may play a role in the function of this important enzyme in plant metabolism. Gene specific probes from 3' coding and noncoding regions of the cDNA clone used to probe genomic Southern blots established that this PEPCase gene is present in one copy in the nuclear genome of M. crystallinum. Transcripts arising from this gene increase dramatically when M. crystallinum is irrigated with 0.5 M NaCl, a stress which induces this plant to switch the primary fixation of CO2 from C3 (Calvin cycle) to CAM mode. The salt-induced mRNA encodes a PEPCase isoform which is undetectable in plants in the C3 mode as demonstrated by Northern hybridization.