Changes of the chlorophyll fluorescence induction kinetics of C3 and CAM plants during day/night cycles

The induction kinetics of the 680 nm chlorophyll fluorescence were measured on attached leaves of Kalanchoë daigremontiana R. Hamet et Perr. (CAM plant), Sedum telephium L. and Sedum spectabile Bor. (C₃ plant in spring, CAM plant in summer) and Raphanus sativus L. (C₃ plant) at three different times during a 12/12 h day/night cycle. During the fluorescence transient the fluorescence intensity at the O, P and T-level (f_O, f_max, f_st,) was different for the plant species tested; this may be due to their different leaf structure, pigment composition and organization of their photosystems. The kinetics of the fluorescence induction depended on the time of preillumination or dark adaptation during the light/dark cycle but not on the type of primary CO₂ fixation mechanism (C₃ and CAM). For dark adapted leaves measured either at the end of the dark phase or after dark adaptation of plants taken from the light phase a higher P-level fluorescence, a higher variable fluorescence (P-O) and a larger complementary area were found than for leaves of plants taken directly from the light phase. This indicates the presence of largely oxidized photosystem 2 acceptor pools during darkness. During the light phase the fluorescence decline after the P-level was faster than during the dark phase; from this we conclude that the light adaptation of the photosynthetic apparatus (state 1state 2 transition, pH) during the induction period proceeded faster in plants taken from the light phase than in plants taken from the dark phase.Abbreviations C₃ plant plant with primary CO₂ fixation on ribulose-1,5-bis-phosphate (Calvin-Benson cycle) - CAM Crassulacean Acid Metabolism     

Studies on carbon flow in Crassulacean acid metabolism during the initial light period

In the Crassulacean acid metabolism (CAM) plants Kalanchoë tubiflora and Sedum morganianum a shift in the pathways occurs by which external CO₂ enters the metabolism during the initial light period (phase II of the diurnal CAM cycle). At the beginning of phase II, CO₂ is fixed mainly by the C₄ pathway; during late phase II, however, it is fixed mainly via the C₃ pathway. The C₃ pathway contributes to the phosphoenolpyruvate-carboxylase-mediated CO₂ fixation by the provision of three-carbon skeletons. Since the shift in the carbon-flow pathway is delayed after a CO₂-free night when malic-acid accumulation in the vacuoles is prevented, it is very likely that the amount of malic acid in the vacuole is integrated in the mechanism which controls CAM during the initial light period. A light-on signal at the beginning of phase II is not required to bring about the shifts in the carbon-flow pathways, as is shown by the reaction of plants to a prolonged dark period. A model of carbon flow during phase II is proposed.Abbreviations CAM Crassulacean acid metabolism - PEP-Case phosphoenolpyruvate carboxylase     

Comparison of CAM expression,photochemistry and antioxidant responses in Sedum album and Portulaca oleracea under combined stress

Previous studies of crassulacean acid metabolism (CAM) pathway during stress have been directed at individual drought and salinity stress, here, we studied the effects of a combination of drought and salt on CAM expression, chlorophyll fluorescence and antioxidant parameters in the C₃-CAM facultative Sedum album and C₄-CAM facultative Portulaca oleracea plants. While salinity alone was not able to induce functional CAM expression in P. oleracea leaves, we showed that salinity induced low level of nocturnal acid accumulation in S. album species. After 20 d of exposure to the combination of simultaneous salt and drought stress, P. oleracea plants exhibited more resistance to photoinhibition as compared to S. album plants. The decrease of maximum quantum yield (F_v/F_m) in S. album leaves under combined stress was in parallel with the largest suppression of CAM expression of >50%, probably displaying the withdrawal of functional CAM back to C₃ pathway. However, under drought treatment alone, S. album plants exhibited higher photosynthetic flexibility, which was associated with the up-regulation of antioxidant enzymes activities and maintenance of glutathione (GSH) pool, and consequently higher photochemical functioning. The levels of nitric oxide (NO) correlated well with CAM expression, which was observed only in S. album, suggesting that NO acts in a different way in C₃ and C₄ species during CAM induction. Additionally, in both species, over the course of CAM induction, the changes in CAM expression parameters exhibited a similar pattern to that of antioxidant capacity and photochemical functioning parameters.     

Light as a signal influencing the phosphorylation status of plant proteins

The phosphorylation-status of a number of plant enzymes has been shown to be altered in response to light. Phosphoenolpyruvate carboxylase is phosphorylated (more active) in C₄ plants in the light but CAM phosphoenolpyruvate carboxylase is phosphorylated (more active) in the dark. C₄ plant pyruvate, Pi dikinase is dephosphorylated (activated) in the light and sucrose phosphate synthase is less phosphorylated (more active) in the light. The mitochondrial pyruvate dehydrogenase is inactivated (phosphorylated) in the light. The reversal of these events occurs in the dark or when photosynthesis is inhibited. Phytochrome and blue light receptors also alter the phosphorylation-status of proteins. The evidence is rapidly increasing in support of signal transduction networks in plants that involve light reception.     

Kinetic Properties of Phosphoenolpyruvate Carboxylase in Peperomia camptotricha

Kinetic characteristics of phosphoenolpyruvate carboxylase (PEPC) from the epiphytic C₃ or C₄: CAM intermediate plant, Peperomia camptotricha, were investigated. Few day versus night differences in V_max,K_m(PEP)), or malate inhibition were observed, even in extracts from water-stressed plants which characteristically perform CAM, regardless of efforts to stabilize day/night forms. The PEPC extracted from plants during the light period remained stable, without much of an increase or decrease in activity for at least 22 hours at 0 to 4°C. Extracts from mature, fully developed leaves had slightly greater PEPC activity than from very young, developing leaves. Generally, however, the kinetic properties of PEPC extracted from mature leaves of plants grown under short day (SD), long day (LD), or 1-week water-stress conditions, as well as from young, developing leaves, were similar. The PEPC inhibitor, l-malate, decreased the V_max and increased the K_m(PEP) for all treatments. Under specific conditions, malate did not inhibit PEPC rates in the dark extracts as much as the light. The PEPC activator, glucose-6-phosphate (G-6-P), lowered the K_m(PEP) for all treatments. At saturating PEP concentrations, PEPC activity was independent of pH in the range of 7.5 to 9.0. At subsaturating PEP concentrations, the pH optimum was 7.8. The rates of PEPC activity were lower in the light period extracts than the dark, at pH 7.1, but day/night PEPC was equally active at pH 7.8. At pH 7.5 and a subsaturating PEP concentration, G-6-P significantly activated PEPC. At pH 8, however, only slight activation by G-6-P was observed. The lower pH of 7.5 combined with l-malate addition, greatly inhibited PEPC, particularly in extracts from young, developing leaves which were completely inhibited at an l-malate concentration of 1 millimolar. However, malate did not further inhibit PEPC activity in mature leaves when assayed at pH 7.1. The fairly constant day/night kinetic and regulatory properties of PEPC from P. camptotricha are unlike those of PEPC from CAM or C₄ species studied, and are consistent with the photosynthetic metabolism of this plant.     

The photosynthetic apparatus of C3 and CAM-induced Mesembryanthemum crystallinum L.

Accompanying the CAM induction of Mesembryanthemum crystallinum L. grown in high salinity there are changes in the enzymes of carbon metabolism. However, there are no changes in the electron transport activities, Chla/b ratios or in the distribution of chlorophyll amongst the various pigment-protein complexes of isolated thylakoids. Hence with CAM induction there are no changes in the photochemical apparatus of M. crystallinum thylakoids. Despite comparable amounts of chlorophylla/b-proteins of photosystem II to those found in typical C₃ sun plants, both the C₃ and CAM M. crystallinum chloroplasts have relatively more photosystem II, and, concommitantly, less photosystem I complex. This is consistent with greater fluorescence emission at 685 and 695 nm, and lower emission at 735 nm (measured at 77 K) than typically found for C₃ plants, whether sun or shade species. Photoinhibition of isolated C₃ and CAM thylakoids by white light led to comparable decreases in electron transport capacities and fluorescence emission at 77 K with photosystem II being more affected than PSI. We suggest however, that the presence of more core PSII complexes relative to PSI complexes in this CAM-inducible plant, may provide an additional strategy to mitigate photoinhibition in the short-term.     

Diurnal variations in leaf fluorescence induction kinetics: variable fluorescence in crassulacean Acid metabolism plants

The variable fluorescence of leaves from Kalanchoë daigremontiana and pineapple, Ananas comosus, both CAM plants, was found to change over a 24-hour cycle and to exhibit high temperature-dependent maxima during the night period. The time course of the induced fluorescence was correlated with malic acid accumulation but not with other aspects of CAM such as with the nature of the decarboxylation pathway or with stomatal movements. The variable fluorescences of sunflower (Helianthus annuus L.) and corn (Zea mays L.) leaves were compared with the CAM plants diurnally; both plants also exhibit high fluorescence maxima during the night period. We conclude that the assembly of the photosystems in the light is a primary process in photosynthesis induction and may be influenced by other cellular metabolic processes, specifically in the case of CAM leaves by malic acid accumulation.     

Water relations of individual leaf cells ofMesembryanthemum crystallinum plants grown at low and high salinity

Summary The effects of saline conditions on the water relations of cells in intact leaf tissue of the facultative CAM plantMesembryanthemum crystallinum were studied using the pressure probe technique. During a 12-hr light/dark regime a maximum in turgor pressure was recorded for the mesophyll cells of salttreated (CAM) plants at the beginning of the light period followed 6 hr later by a pressure maximum in the bladder cells of the upper epidermis. In contrast, the turgor pressure in the bladder cells of the lower epidermis remained constant during light/dark regime. Turgor pressure maxima were not observed in untreated (C₃) plants.This finding strongly supports the assumption that water movement during malate accumulation and degradation in salttreated plants occurs predominantly between the mesophyll cells and the bladder cells of the upper epidermis. The necessary calculations take differences in the compartment volumes and in the elastic moduli of the cell walls () of the bladder cells of the lower and upper epidermis into account.Measurements of the kinetics of water transport showed that the half-time of water exchange for the two sorts of bladder cells were nearly identical in CAM plants and in C₃ plants. The absolute values of the half-times increased by about 45% in salttreated plants (about 113 sec) compared to the control plants (78 sec). Simultaneously, the half-time of water exchange of the mesophyll cells increased by about 60% from 14 sec (untreated plants) to 22 sec (salt-exposed plants). The leaves of this plant are apparently able to closely maintain the time of propagation of short-term osmotic pressure changes over a large salinity range.A cumulative plot of the data measured on both C₃ and CAM plants showed that the differences between the values of the elastic moduli of bladder cells from the lower and from the upper epidermis are due to differences in volume and suggested that the intrinsic elastic properties of the differently located bladder cells of C₃ and CAM plants were identical.A cumulative plot of the hydraulic conductivity of the membrane obtained both on mesophyll and on bladder cells of salttreated and of untreated plantsvs. the individual turgor pressure yielded a relationship well-known from giant algal cells and some higher plant cells: The hydraulic conductivity increased at very low pressure, indicating that the water permeability properties of the membrane of the various cell types of C₃ and CAM plants are pressure dependent, but otherwise identical.The results suggest that a few fundamental physical relationships control the adaptation of the tissue cells to salinity.     

A comparative immunocytochemical localization study of phosphoenolpyruvate carboxylase in leaves of higher plants

The intracellular localization of phosphoenolpyruvate (PEP) carboxylase in plants belonging to the C₄, Crassulacean acid metabolism (CAM) and C₃ types was invetigated using an immunocytochemical method with an immune serum raised against the sorghum leaf enzyme. The plants studied were sorghum, maize (C₄ type), kalanchoe (CAM type), french bean, and spinach (C₃ type). In the green leaves of C₄ plants, it was shown that the carboxylase was located in the mesophyll and stomatic cells, being largely cytosolic in the mesophyll cells. Similarly, in CAM plants, the enzyme was found mainly outside the chloroplasts. In contrast, in C₃ plants, the PEP carboxylase appeared to be distributed between the cytosol and the chloroplasts of foliar parenchyma. Examination of sections from etiolated leaves showed fluorescence emission from etioplasts and cytosol for the parenchyma of french bean as well as for the bundle sheath and mesophyll of sorghum leaves. This data indicated that during the greening process photoregulation and evolution of PEP carboxylase is dependent on the tissue and on the metabolic type of the plant considered.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate     

Photosynthetic induction in C4 leaves

Simultaneous measurements of CO₂ uptake, transpiration rate, and chlorophyll a fluorescence in leaf strips of C₄ plants during the induction phase of photosynthesis are described. The timecourse of CO₂ fixation is biphasic with the initial phase occurring within the first 1 to 5 min and the secondary phase consisting of a slow rise to the steady-state rate of photosynthesis. Transpiration rate follows the CO₂-fixation timecourse closely but the intercellular CO₂ concentration never falls below saturation for C₄ plants. Chlorophyll a fluorescence quenching occurs exclusively during the initial fast phase of the CO₂-fixation timecourse. The effect of duration of dark pretreatment of leaves on these parameters and the effects of light intensity and CO₂ concentration are examined. These results are discussed with respect to the C₄ cycle and photochemical and non-photochemical chlorophyll fluorescence quenching.Abbreviations IRGA infra-red gas analyser - NADP-ME, NAD-ME and PEP-CK the three groups of C₄ plants utilising the enzymes NADP-malic enzyme, NAD-malic enzyme and phosphoenolpyruvate carboxykinase, respectively, for C₄-acid decarboxylation - PEP phosphoenolpyruvate - 3-PGA 3-phosphoglyceric acid     

Photochemical activities and organization of photosynthetic apparatus of C3 and C4 plants grown under different light intensities

Changes in the photochemical activities, influenced by variation in the growth light intensity, were followed in typical C₃ (Phaseolus, Ipomoea) and C₄ (Amaranthus, Sorghum) plants. Progressive decrease in the growth light intensity accelerated the O-P fluorescence induction in whole leaves. Such acceleration of the fluorescence kinetics was found to be not due to enhanced photosystem II activity but possibly a result of reduced rate of electron flow between the two photosystems. This is supported by 4 lines of evidence: (1) by the Hill activity determined in the presence of electron acceptors functioning before and after plastoquinone; (2) the photosynthetic unit size determined after flash excitation showing variations that were apparently too small to account for the changes observed fluorescence induction; (3) modification of the kinetics of second-range light-induced absorbance changes at 520 nm; and (4) absence of significant changes in the ratio of P₇₀₀/total chlorophyll ratio. The P₇₀₀/cytochrome f ratio, however, increased from the usual 1–1.5 to 3–4 in plants grown under 9% sunlight. Increase in the P₇₀₀/cytochrome f ratio was found to be due to a decrease in the cytochrome f/chlorophyll ratio, and this was due to perhaps to a simultaneous increase in chlorophyll and decrease in cytochrome content.     

Photochemical activities and organization of photosynthetic apparatus of C3 and C4 plants grown under different light intensities

Changes in the photochemical activities, influenced by variation in the growth light intensity, were followed in typical C₃ (Phaseolus, Ipomoea) and C₄ (Amaranthus, Sorghum) plants. Progressive decrease in the growth light intensity accelerated the O-P fluorescence induction in whole leaves. Such acceleration of the fluorescence kinetics was found to be not due to enhanced photosystem II activity but possibly a result of reduced rate of electron flow between the two photosystems. This is supported by 4 lines of evidence: (1) by the Hill activity determined in the presence of electron acceptors functioning before and after plastoquinone; (2) the photosynthetic unit size determined after flash excitation showing variations that were apparently too small to account for the changes observed fluorescence induction; (3) modification of the kinetics of secondrange light-induced absorbance changes at 520 nm; and (4) absence of significant changes in the ratio of P₇₀₀/total chlorophyll ratio. The P₇₀₀/cytochrome f ratio, however, increased from the usual 1–1.5 to 3–4 in plants grown under 9% sunlight. Increase in the P₇₀₀/cytochrome f ratio was found to be due to a decrease in the cytochrome f/chlorophyll ratio, and this was due to perhaps to a simultaneous increase in chlorophyll and decrease in cytochrome content.     

Temperature determines the occurrence of CAM or C<Subscript>3</Subscript> photosynthesis in pineapple plantlets grown <Emphasis Type="Italic">in vitro</Emphasis>

Summary Ananas comosus (L.) Merr. var. Smooth Cayenne plants when grown in vitro under different temperature regimes developed as CAM or as C₃ plants. The plants used in this study were developed from the lateral buds of the nodal etiolated stem explants cultured on Murashige and Skoog medium for 3 mo. The cultures were maintained under a 16-h photoperiod for different thermoperiods. With 28°C light/15°C dark thermoperiod, as compared with constant 28°C light and dark, pineapple plants had a succulence index two times greater, and also a greater nocturnal titratable acidity and phosphoenolpyruvate carboxylase (PEPCase) activity, indicating CAM-type photosynthesis. The highest abscisic acid (ABA) level occurred during the light period, 8 h prior to maximum PEPCase activity, while the indole-3-acetic acid (IAA) peak was found during the dark period, coinciding with the time of highest PEPCase activity. These plants were also smaller with thicker leaves and fewer roots, but had greater dry weight. Their leaves showed histological characteristics of CAM plants, such as the presence of greater quantities of chlorenchyma and hypoderm. In addition, their vascular system was more conspicuous. In contrast, under constant temperature (28°C light/dark) plants showed little succulence in the leaves. There was no significant acid oscillation and diurnal variation in PEPCase activity in these plants, suggesting the occurrence of C₃ photosynthesis. Also, no diurnal variation in ABA and IAA contents was observed. The results of this study clearly indicate a role for temperature in determining the type of carbon fixation pathway in in vitro grown pineapple. Evidence that ABA and IAA participate in CAM signaling is provided.     

Posttranslational regulation of phosphoenolpyruvate carboxylase in c(4) and crassulacean Acid metabolism plants

Control of C₄ photosynthesis and Crassulacean acid metabolism (CAM) is, in part, mediated by the diel regulation of phosphoenolpyruvate carboxylase (PEPC) activity. The nature of this regulation of PEPC in the leaf cell cytoplasm of C₄ and CAM plants is both metabolite-related and posttranslational. Specificially, the regulatory properties of the enzyme vary in accord with the physiological activity of C₄ photosynthesis and CAM: PEPC is less sensitive to feedback inhibition by l-malate under light (C₄ plants) or at night (CAM plants) than in darkness (C₄) or during the day (CAM). While the view that a light-induced change in the aggregation state of the holoenzyme is a general mechanism for the diel regulation of PEPC activity in CAM plants is currently in dispute, there is no supportive in vivo evidence for such a tetramer/dimer interconversion in C₄ plants. In contrast, a wealth of in vitro and in vivo data has accumulated in support of the view that the reversible phosphorylation of a specific, N-terminal regulatory serine residue in PEPC (e.g. Ser-15 or Ser-8 in the maize or sorghum enzymes, respectively) plays a key, if not cardinal, role in the posttranslational regulation of the carboxylase by light/dark or day/night transitions in both C₄ and CAM plants, respectively.     

Is Sedum acre L. a CAM plant?

Summary Sedum acre L. collected from its natural stands south of Darmstadt (Germany) showed ¹³C values typical for C₃ plants. This suggests that in situ at the natural stand CO₂ was fixed mainly via the C₃ mode of photosynthesis rather than via the CAM mode. However, experimental water stress shifts the CO₂ exchange pattern from the C₃ type to CAM type. Simultaneously, a diurnal rhythm of malic acid oscillation, typical for CAM, and increase of PEP-carboxylase and malic enzyme activities developed. Hence, Sedum acre is obviously to be classified as a facultative CAM plant. Because of the temperature characteristics of CO₂ exchange in Sedum acre, in situ CO₂ should be harvested from the atmosphere mainly during the seasons where water stress situations capable of inducing CAM are unlikely to occur.     

Gas Exchange Characteristics of the Submerged Aquatic Crassulacean Acid Metabolism Plant, Isoetes howellii

The submerged aquatic plant Isoetes howellii Engelmann possesses Crassulacean acid metabolism (CAM) comparable to that known from terrestrial CAM plants. Infrared gas analysis of submerged leaves showed Isoetes was capable of net CO₂ uptake in both light and dark. CO₂ uptake rates were a function of CO₂ levels in the medium. At 2,500 microliters CO₂ per liter (gas phase, equivalent to 1.79 milligrams per liter aqueous phase), Isoetes leaves showed continuous uptake in both the light and dark. At this CO₂ level, photosynthetic rates were light saturated at about 10% full sunlight and were about 3-fold greater than dark CO₂ uptake rates. In the dark, CO₂ uptake rates were also a function of length of time in the night period. Measurements of dark CO₂ uptake showed that, at both 2,500 and 500 microliters CO₂ per liter, rates declined during the night period. At the higher CO₂ level, dark CO₂ uptake rates at 0600 h were 75% less than at 1800 h. At 500 microliters CO₂ per liter, net CO₂ uptake in the dark at 1800 h was replaced by net CO₂ evolution in the dark at 0600 h. At both CO₂ levels, the overnight decline in net CO₂ uptake was marked by periodic bursts of accelerated CO₂ uptake. CO₂ uptake in the light was similar at 1% and 21% O₂, and this held for leaves intact as well as leaves split longitudinally. Estimating the contribution of light versus dark CO₂ uptake to the total carbon gain is complicated by the diurnal flux in CO₂ availability under field conditions.     

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

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

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.     

A comparative study on the regulation of C3 and C4 carboxylation processes in the constitutive crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana and the C3-CAM intermediate Clusia minor

A comparison of carbon metabolism in the constitutive crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perr. and the C₃-CAM intermediate Clusia minor L. was undertaken under controlled environmental conditions where plants experience gradual changes in light intensity, temperature and humidity at the start and end of the photoperiod. The magnitude of CAM activity was manipulated by maintaining plants in ambient air or by enclosing leaves overnight in an atmosphere of N₂ to suppress C₄ carboxylation. Measurements of diel changes in carbonisotope discrimination and organic acid content were used to quantify the activities of C₃ and C₄ carboxylases in vivo and to indicate the extent to which the activities of phosphoenolpyruvate carboxylase (PEPCase), ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and decarboxylation processes overlap at the start and end of the photoperiod. These measurements in vivo were compared with measurements in vitro of changes in the diel sensitivity of PEPCase to malate inhibition. The results demonstrate fundamental differences in the down-regulation of PEPCase during the day in the two species. While PEPCase is inactivated within the first 30 min of the photoperiod in K. daigremontiana, the enzyme is active for 4 h at the start and 3 h at the end of the photoperiod in C. minor. Enclosing leaves in N₂ overnight resulted in a two-to threefold increase in PEPCase-mediated CO₂ uptake during Phase II of CAM in both species. However, futile cycling of CO₂ between malate synthesis and decarboxylation does not occur during Phase II in either species. In terms of overall carbon balance, C₄ carboxylation accounted for ≈ 20% of net daytime assimilation in both species under control conditions, increasing to 30–34% after a night in N₂. Although N₂-treated leaves of K. daigremontiana took up 25% more CO₂ than control leaves during the day this was insufficient to compensate for the loss of CO₂ taken up by CAM the previous night. In contrast, in N₂-treated leaves of C. minor, the twofold increase in daytime PEPCase activity and the increase in net CO₂ uptake by Rubisco during Phase III compensated for the inhibition of C₄ carboxylation at night in terms of diel carbon balance.