Transport of Phosphoenolpyruvate by Chloroplasts from Mesembryanthemum crystallinum L. Exhibiting Crassulacean Acid Metabolism

Chloroplasts from CAM-Mesembryanthemum crystallinum can transport phosphoenolpyruvate (PEP) across the envelope. The initial velocities of PEP uptake in the dark at 4°C exhibited saturation kinetics with increasing external PEP concentration. PEP uptake had a V_max of 6.46 (±0.05) micromoles per milligram chlorophyll per hour and an apparent K_mpep of 0.148 (±0.004) millimolar. The uptake was competitively inhibited by Pi (apparent K_i = 0.19 millimolar), by glycerate 3-phosphate (apparent K_i = 0.13 millimolar), and by dihydroxyacetone phosphate, but malate and pyruvate were without effect. The chloroplasts were able to synthesize PEP when presented with pyruvate. PEP synthesis was light dependent. The prolonged synthesis and export of PEP from the chloroplasts required the presence of Pi or glycerate 3-phosphate in the external medium. It is suggested that the transport of pyruvate and PEP across the chloroplasts envelope is required during the gluconeogenic conversion of carbon from malate to storage carbohydrate in the light.     

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.     

Intracellular Localization of Some Key Enzymes of Crassulacean Acid Metabolism in Sedum praealtum

The intracellular locations of six key enzymes of Crassulacean acid metabolism were determined using enzymically isolated mesophyll protoplasts of Sedum praealtum D.C. Data from isopycnic sucrose density gradient centrifugation established the chloroplastic location of pyruvate Pi dikinase, the mitochondrial location of NAD-linked malic enzyme, and exclusively nonparticulate (not associated with chloroplasts, peroxisomes, or mitochondria) locations of phosphoenolpyruvate carboxylase, NADP-linked malic enzyme, enolase, and phosphoglycerate mutase. The consequences of this enzyme distribution with respect to compartmentalization of the pathway and the transport of metabolites in Crassulacean acid metabolism are discussed.     

Salt Requirement for Crassulacean Acid Metabolism in the Annual Succulent, Mesembryanthemum crystallinum

In experiments with the facultative Crassulacean acid metabolism (CAM) species, Mesembryanthemum crystallinum, only plants which received high levels of inorganic salts fixed substantial amounts of CO₂ by the CAM pathway. Equivalent osmolarities of polyethylene glycol 6000 did not yield any CAM fixation. Plant water potential and turgor pressure had no detectable influence on the amount of CAM fixation. These observations rule out the possibility that the inorganic ions were acting as osmotic agents.     

Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Mitochondria isolated from leaves of Mesembryanthemum crystallinumoxidized malate by both NAD malic enzyme and NAD malate dehydrogenase.Rates of malate oxidation were higher in mitochondria from plantsgrown at 400 mil NaCl in the rooting medium and performing Crassulaceanacid metabolism (CAM) than in mitochondria from plants grownat 20 mM NaCl and exhibiting C₃-photosynthetic CO₂ fixation.The mitochondria isolated from plants both in the CAM and C₃modes were tightly coupled and gave high respiratory control.At optimum pH for malate oxidation (pH 7.0), pyruvate was themajor product in mitochondria from CAM-M. crystallinum, whereasmitochondria from C₃-M. crystallinum produced predominantlyoxaloacetate. Both the extracted NAD malic enzyme in the presenceof CoA and the oxidation of malate to pyruvate by the mitochondriafrom plants in the CAM mode had a pH optimum around 7.0 withactivity declining markedly above this pH. The activity of NAD-malicenzyme, expressed on a cytochrome c oxidase activity basis,was much higher in mitochondria from the CAM mode than the C₃mode. The results indicate that mitochondria of this speciesare adapted to decarboxylate malate at high rates during CAM. ¹Current address: Lehrstuhl für Botanik II, UniversitätWurzburg, Mittlerer Dallenbergweg 64, 8700 Würzburg, WestGermany. ²Current address: KD 120, Chemical Research Division, OntarioHydro, 800 Kipling Avenue, Toronto, Ontario M8Z5S4, Canada. ³Current address: Department of Botany, Washington State University,Pullman, Washington 99164-4230, U.S.A. (Received March 13, 1986; Accepted September 18, 1986)     

Isoforms of NADP-Malic Enzyme from Mesembryanthemum crystallinum L. That are Involved in C3 Photosynthesis and Crassulacean Acid Metabolism

Exposure of the facultative halophyte Mesembryanthemum crystallinumL. to salt stress induces a shift from C₃ photosynthesis toCrassulacean acid metabolism (CAM). During induction of CAM,the activity of NADP-malic enzyme (EC 1.1.1.40 [EC] ) increased asmuch as 12-fold in leaves, while the enzymatic activity in rootsfell to half of the original level. These changes in the activityof the enzyme corresponded to changes in levels of the enzymeprotein. NADP-malic enzymes extracted from leaves in the C₃and CAM modes could be distinguished by differences in electrophoreticmobility during electrophoresis on a non-denaturing polyacrylamidegel. NADP-malic enzyme extracted from roots in the C₃-mode andin the CAM mode migrated as fast as the enzyme extracted fromleaves in the CAM mode on the same gel. Although the patternof peptide fragments from NADP-malic enzyme from CAM-mode leaveswas similar to that from C₃-mode leaves, as indicated by peptidemapping, both immunoprecipitation and an enzyme-linked immunosorbentassay revealed some antigenic differences between the enzymesextracted from leaves in the C₃ and the CAM modes. These resultssuggest the existence of at least two isoforms of NADPmalicenzyme that differ in their levels of expression during inductionof CAM. (Received April 21, 1994; Accepted September 5, 1994)     

Diurnal Ion Fluctuations in the Mesophyll Tissue of the Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Both laboratory- and field-grown Mesembryanthemum crystallinum plants exhibited large scale diurnal ion fluctuations. In mesophyll tissue, potassium and sodium levels varied in conjunction with acid levels while chloride levels varied in opposition. Thus, dark CO₂ fixation in this Crassulacean acid metabolism species seems analogous to the common plant process of malate synthesis to balance cation surplus. Sodium levels in the epidermis appeared to fluctuate in opposition to those in the mesophyll. It is proposed that inorganic cations cycle between mesophyll and epidermal tissue to balance malate accumulation and to produce stomatal opening in the dark.     

Properties of Leaf NAD-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Species Mesembryanthemum crystallinum

NAD-malic enzyme (NAD-ME) functions to decarboxylate malatein the light in leaves of certain species displaying Crassulaceanacid metabolism (CAM). The properties of NAD-ME in desaltedextracts from the inducible CAM species, Mesembryanthemum crystallinumwere examined. The shapes of the malate saturation curve andthe activity versus pH curve at 10 mM malate were dependenton the presence of the activator CoA. The malate saturationcurve was sigmoidal in the absence of an activator and hyperbolicin the presence of CoA. The pH optimum with 10mM malate andMn²⁺ as cofactor was as low as 6.5 without an activator, andincreased to 7.2 in the presence of CoA. Fumarate activationwas synergistic with CoA above pH 7.2. The enzyme displayedhysteretic behavior under suboptimal assay conditions. Rapid extraction and desalting of the enzyme (<1.5 mim) followedimmediately by assay did not reveal any difference in the propertiesof the enzyme on a day/night basis. It is proposed that diurnalregulation of the enzyme in vivo is mediated by pH and malatelevel without a change in the oligomeric form of the enzyme.The molecular weight of the enzyme was approximately 350,000at pH 6.5 or 7.8. The enzyme obtained from M. crystallinum inthe C₃ mode was very similar to the CAM enzyme except that itdisplayed a lower V_max. ³ Current address: MSU-DOE Plant Research Lab, Michigan StateUniversity, E. Lansing, Michigan, U.S.A. 48824. (Received October 2, 1984; Accepted December 20, 1984)     

Changes in Levels of Phosphoenolpyruvate Carboxylase with Induction of Crassulacean Acid Metabolism in Mesembryanthemum crystallinum L.

Expanded leaves of Mesembryanthemum crystallinum L. performingC₃ photosynthesis were induced to perform pronounced Crassulaceanacid metabolism (CAM) by exposing the plant roots to higherNaCl concentration. Levels of phosphoenolpyruvate (PEP) carboxylaseactivity increased 10-fold during the 7-day induction period.Densitometric analysis of Coomassie-stained sodium dodecyl sulfate(SDS) polyacrylamide gradient slab gels of leaf extracts, preparedduring the course of CAM induction, revealed that at least fivebands of polypeptides increased in content (kilodalton valuesof 98, 91, 45, 41, 38). Higher levels of three additional polypeptides(kilodalton values of 102, 76, 33) became apparent after tissuehad been grown for 2 weeks at 400 mM NaCl. Of these polypeptides,that having a mass of 98 kilodaltons was identified as the subunitof PEP carboxylase by comparison with the corresponding bandfrom partially purified PEP carboxylase from the same tissue.Only a faint 98 kilodalton band was evident on SDS gels fortissue operating in the C₃ mode; staining intensity at thislocation increased with increasing NaCl-salinity in the rootingmedium until CAM was fully induced. These data provide evidencefor net synthesis of PEP carboxylase and several other proteinsduring the induction of CAM in M. crystallinum. ¹ Present address: USDA, P. O. Box 867 Airport Rd., Beckley,WV. 25801, U.S.A. ² Present address: Department of Botany, Washington State University,Pullman, Washington 99164, U.S.A. ³ Present address: Botanisches Institut der Universit?t, MittlererDallenbergweg 64, 8700 W?rzburg, W.-Germany. (Received October 27, 1981; Accepted March 15, 1982)     

Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

Induction of Crassulacean Acid Metabolism in Mesembryanthemum crystallinum by High Salinity: Mass Increase and de Novo Synthesis of PEP-Carboxylase

Intact plants of the halophilic species Mesembryanthemum crystallinum were induced to exhibit Crassulacean acid metabolism by irrigation with nutrient solution containing 500 millimolar NaCl. During the induction period, the extractable activity of phosphoenolpyruvate carboxylase (PEPcase) increased approximately 40-fold. This increase was linearly correlated with a mass increase of PEPcase protein as measured by single radial immunodiffusion. De novo synthesis of PEPcase protein was shown by immunoprecipitation of the newly synthesized, radioactively labeled protein in leaf discs from salt-treated plants. Nontreated plants were characterized by a low level of the enzyme and low rates of PEPcase synthesis. Synthesis of this enzyme in leaf discs was correlated with the concentration of NaCl in the nutrient solution during growth.     

Respiratory CO(2) as Carbon Source for Nocturnal Acid Synthesis at High Temperatures in Three Species Exhibiting Crassulacean Acid Metabolism

Temperature effects on nocturnal carbon gain and nocturnal acid accumulation were studied in three species of plants exhibiting Crassulacean acid metabolism: Mamillaria woodsii, Opuntia vulgaris, and Kalanchoë daigremontiana. Under conditions of high soil moisture, nocturnal CO₂ gain and acid accumulation had temperature optima at 15 to 20°C. Between 5 and 15°C, uptake of atmospheric CO₂ largely accounted for acid accumulation. At higher tissue temperatures, acid accumulation exceeded net carbon gain indicating that acid synthesis was partly due to recycling of respiratory CO₂. When plants were kept in CO₂-free air, acid accumulation based on respiratory CO₂ was highest at 25 to 35°C. Net acid synthesis occurred up to 45°C, although the nocturnal carbon balance became largely negative above 25 to 35°C. Under conditions of water stress, net CO₂ exchange and nocturnal acid accumulation were reduced. Acid accumulation was proportionally more decreased at low than at high temperatures. Acid accumulation was either similar over the whole temperature range (5-45°C) or showed an optimum at high temperatures, although net carbon balance became very negative with increasing tissue temperatures. Conservation of carbon by recycling respiratory CO₂ was temperature dependent. At 30°C, about 80% of the dark respiratory CO₂ was conserved by dark CO₂ fixation, in both well irrigated and water stressed plants.     

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.     

Characteristics of Crassulacean Acid Metabolism in the Succulent C(4) Dicot, Portulaca oleracea L

Crassulacean acid metabolism (CAM) was investigated in leaves and stems of the succulent C₄ dicot Portulaca oleracea L. Diurnal acid fluctuations, CO₂ gas exchange, and leaf resistance were monitored under various photoperiod and watering regimes. No CAM activity was seen in well watered plants grown under 16-hour days. Under 8-hour days, however, well watered plants showed a CAM-like pattern of acid fluctuation with amplitudes of 102 and 90 microequivalents per gram fresh weight for leaves and stems, respectively. Similar patterns were also observed in detached leaves and defoliated stems. Leaf resistance values indicated that stomata were open during part of the dark period, but night acidification most likely resulted from refixation of respiratory CO₂. In water-stressed plants maximum acid accumulations were reduced under both long and short photoperiods. At night, these plants showed short periods of net CO₂ uptake and stomatal opening which continued all night long during preliminary studies under natural environmental conditions. Greatest acid fluctuations, in P. oleracea, with amplitudes of 128 microequivalents per gram fresh weight, were observed in water-stressed plants which had been rewatered, especially when grown under short days. No net CO₂ uptake took place, but stomata remained open throughout the night under these conditions. These results indicate that under certain conditions, such as water stress or short photoperiods, P. oleracea is capable of developing an acid metabolism with many similarities to CAM.     

Ecophysiology of Crassulacean Acid Metabolism (CAM)

BACKGROUND AND SCOPE: Crassulacean Acid Metabolism (CAM) as an ecophysiological modification of photosynthetic carbon acquisition has been reviewed extensively before. Cell biology, enzymology and the flow of carbon along various pathways and through various cellular compartments have been well documented and discussed. The present attempt at reviewing CAM once again tries to use a different approach, considering a wide range of inputs, receivers and outputs. INPUT: Input is given by a network of environmental parameters. Six major ones, CO(2), H(2)O, light, temperature, nutrients and salinity, are considered in detail, which allows discussion of the effects of these factors, and combinations thereof, at the individual plant level ('physiological aut-ecology'). RECEIVERS: Receivers of the environmental cues are the plant types genotypes and phenotypes, the latter including morphotypes and physiotypes. CAM genotypes largely remain 'black boxes', and research endeavours of genomics, producing mutants and following molecular phylogeny, are just beginning. There is no special development of CAM morphotypes except for a strong tendency for leaf or stem succulence with large cells with big vacuoles and often, but not always, special water storage tissues. Various CAM physiotypes with differing degrees of CAM expression are well characterized. OUTPUT: Output is the shaping of habitats, ecosystems and communities by CAM. A number of systems are briefly surveyed, namely aquatic systems, deserts, salinas, savannas, restingas, various types of forests, inselbergs and paramós. CONCLUSIONS: While quantitative census data for CAM diversity and biomass are largely missing, intuition suggests that the larger CAM domains are those systems which are governed by a network of interacting stress factors requiring versatile responses and not systems where a single stress factor strongly prevails. CAM is noted to be a strategy for variable, flexible and plastic niche occupation rather than lush productivity. 'Physiological syn-ecology' reveals that phenotypic plasticity constitutes the ecophysiological advantage of CAM.     

Photosynthetic Carbon Assimilation in a Shootless Orchid, Chiloschista usneoides (DON) LDL: A Variant on Crassulacean Acid Metabolism

Photosynthetic carbon assimilation in the roots of a shootless orchid Chiloschista usneoides (DON) LDL involves the synthesis and accumulation of malic acid from CO₂ in darkness. Malic acid is consumed in the light.

The roots do not possess stomata or any means of diurnally regulating the diffusive conductance of the pathway between the internal gas phase of the plant and the atmosphere. Regulation of internal CO₂ concentration near to atmospheric levels avoids a large net loss of CO₂ to the atmosphere during malic acid consumption in the light.

The water-absorbing function of the velamen conflicts with the photosynthetic function of the roots. Plants with water-saturated velamina do not acquire CO₂ from the atmosphere at night.

     

Purification by Immunoadsorption and Immunochemical Properties of NADP-Dependent Malic Enzymes from Leaves of C(3), C(4), and Crassulacean Acid Metabolism Plants

NADP:malic enzyme from corn (Zea mays L.) leaves was purified by conventional techniques to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies raised against this protein in rabbits were purified, coupled covalently to protein A-Sepharose CL-4B, and used as an immunoaffinity resin to purify the NADP:malic enzymes of the C₃ plants spinach (Spinacia oleracea L.) and wheat (Triticum aestivum L.), of the Crassulacean acid metabolism (CAM) plant Bryophyllum daigremontianum R. Hamed et Perr. de la Bathie and the C₄ plants corn, sugarcane (Saccharum officinarum L.), and Portulaca grandiflora L. Such procedures yielded homogeneous protein preparations with a single protein band, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, except for P. grandiflora L. with two bands. The specific activities of the purified proteins ranged between 56 and 91 units (milligrams per protein). NADP:malic enzyme represented up to 1% of the total soluble protein in C₄ plants, 0.5% in the CAM plant, and less than 0.01% in C₃ plants. In immunotitration tests involving immunoprecipitation and immunoinhibition of activity by an antiserum against the corn leaf enzyme, the NADP:malic enzymes of corn and sugarcane showed virtually full identity of epitopes, while the NADP:malic enzymes of the C₃ and CAM plants exhibited a cross-reaction of one-twentieth and one-fourth by these tests, respectively. The NADP:malic enzyme of P. grandiflora exhibited characteristics more closely related to the enzymes of C₃ and CAM plants than to those of C₄ plants.     

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.     

Analysis of chlorophyll a fluorescence in C3 and CAM forms of Mesembryanthemum crystallinum

Comparisons of chlorophyll a fluorescence characteristics ofC₃ and CAM forms of Mesembryanthemum crystallinum were usedto identify features of the photosynthetic mechanism associatedwith CAM. The reduction status, Q, was lower and predicted PSII activityhigher in the C₃ form than in the CAM form throughout the photoperiod.These differences were particularly pronounced during the firsthour of illumination when non-photochemical quenching attributableto the intrathylakoid proton gradient was also at its highestin the CAM form. It is argued that this high proton gradientdiminishes PSII activity and serves a protective role againstphotoinhibition at a time in the CAM cycle when both CO₂ concentrationwithin the leaf, and carbon cycle enzyme activation levels arelikely to be low. Differences in fluorescence characteristics between the C₃ andCAM forms also indicate modification of the energy transductionmechanisms of the CAM form possibly related to the increasedoverall demand for ATP in CAM photosynthesis. Total non-photochemicalquenching was higher in the CAM form than in the C₃ form. Aninverse relationship between fast and slowly-relaxing componentsof non-photochemical quenching can be interpreted in terms ofthe changing demand for ATP in the different phases of CAM. Key words: C₃/CAM photosynthesis, chlorophyll fluorescence, state transitions, cyclic photophosphorylation