Characterization and Partial Purification of Aldose-6-phosphate Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves

The Pereskia are morphologically primitive, leafed members of the Cactaceae. Gas exchange characteristics using a dual isotope porometer to monitor ¹⁴CO₂ and tritiated water uptake, diurnal malic acid fluctuations, phosphoenolpyruvate carboxylase, and malate dehydrogenase activities were examined in two species of the genus Pereskia, Pereskia grandifolia and Pereskia aculeata. Investigations were done on well watered (control) and water-stressed plants. Nonstressed plants showed a CO₂ uptake pattern indicating C₃ carbon metabolism. However, diurnal fluctuations in titratable acidity were observed similar to Crassulacean acid metabolism. Plants exposed to 10 days of water stress exhibited stomatal opening only during an early morning period. Titratable acidity, phosphoenolpyruvate carboxylase activity, and malate dehydrogenase activity fluctuations were magnified in the stressed plants, but showed the same diurnal pattern as controls. Water stress causes these cacti to shift to an internal CO₂ recycling (“idling”) that has all attributes of Crassulacean acid metabolism except nocturnal stomata opening and CO₂ uptake. The consequences of this shift, which has been observed in other succulents, are unknown, and some possibilities are suggested.     

Characterization of Early Morning Crassulacean Acid Metabolism in Opuntia erinacea var Columbiana (Griffiths) L. Benson

The nature and sequence of metabolic events during phase II (early morning) Crassulacean acid metabolism in Opuntia erinacea var columbiana (Griffiths) L. Benson were characterized. Gas exchange measurements under 2 and 21% O₂ revealed increased O₂ inhibition of CO₂ fixation with progression of phase II. Malate and titratable acidity patterns indicated continued synthesis of C₄ acids for at least 30 minutes into the light period. Potential activities of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme exhibited little change during phase II, while light activation of NADP-malate dehydrogenase, pyruvate, orthophosphate dikinase, and ribulose-1,5-bisphosphate carboxylase was apparent. Short-term ¹⁴CO₂ fixation experiments showed that the per cent of ¹⁴C incorporated into C₄ acids decreased while incorporation into other metabolites increased with time. PEPC exhibited increased sensitivity to 2 millimolar malate, and the K_i(malate) for PEPC decreased markedly with time. Sensitivity of PEPC to malate inhibition was considerably greater at pH 7.5 than at 8.0. The results indicate that decarboxylation and synthesis of malate occur simultaneously during the early morning period, and that phase II acid metabolism is not limited by CO₂ diffusion through stomata. With progression of phase II, CO₂ fixation by PEPC decreases while fixation by ribulose-1,5-bisphosphate carboxylase increases.     

Seasonal Shifts of Photosynthesis in Portulacaria afra (L.) Jacq

Portulacaria afra (L.) Jacq., a perennial facultative Crassulacean acid metabolism (CAM) species, was studied under natural photoperiods and temperatures in San Diego, California. The plants were irrigated every fourth day throughout the study period. Measurements of ¹⁴CO₂ uptake, stomatal resistance, and titratable acidity were made periodically from July 1981 through May 1982. P. afra maintained C₃ photosynthesis during the winter and the spring. Diurnal acid fluctuations were low and maximal ¹⁴CO₂ uptake occurred during the day. The day/night ratio of carbon uptake varied from 5 to 10 and indicated little nocturnal CO₂ uptake. CAM photosynthesis occurred during the summer and a mixture of both C₃ and CAM during the fall. Large acid fluctuations of 100 to 200 microequivalents per gram fresh weight were observed and maximal ¹⁴CO₂ uptake shifted to the late night and early morning hours. Daytime stomatal closure was evident. A reduction in the day/night ratio of carbon uptake to 2 indicated a significant contribution of nocturnal CO₂ uptake to the overall carbon gain of the plant. The seasonal shift from C₃ to CAM was facilitated by increasing daytime temperature and accompanied by reduced daytime CO₂ uptake despite irrigation.     

Crassulacean acid metabolism (CAM) in Kalanchoë: Changes in intercellular CO2 concentration during a normal CAM cycle and during cycles in continuous light or darkness

In the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana, the internal CO₂ concentrations were measured throughout CAM cycles by gas chromatography. Under normal dark-light cycles, the internal CO₂ concentration was near that of the ambient air and increased up to 0.5% during the phase of maximum malate decarboxylation. A sharp increase in internal CO₂ concentration occurring after the first 12 h of the cycle was exhibited by the plants both when there was a normal day-night cycle and when the night was replaced by illumination, and also when the light period was replaced by darkness. Thus, the increase in internal CO₂ in the morning does not appear to be primarily determined by a light-on signal or by alterations of temperature rather than by inherent factors of the leaves. This view is supported further by a steep increase in ¹⁴CO₂ production from labeted malate occurring during extended darkness at a time when the light period would normally begin. The results are discussed in particular in relation to of how CAM can control stomata movement.Abbreviation CAM Crassulacean acid metabolism     

Quantum Requirement for Photosynthesis in Sedum praealtum during Two Phases of Crassulacean Acid Metabolism

The quantum requirement (QR) for photosynthesis in Sedum praealtum, a Crassulacean acid metabolism plant, was compared with that of wheat, a C₃ plant, and maize, a C₄ plant, at 30 C. During the deacidification phase in S. praealtum, approximately 16 moles quanta were absorbed per mole malate consumed. This is equivalent to 16 moles quanta per mole CO₂ fixed, assuming 1 mole CO₂ is assimilated per mole malate decarboxylated. This QR for Crassulacean acid metabolism is similar to that of the C₃ or C₄ plant under atmospheric conditions, even though there are considerable differences in the biochemistry of photosynthesis. During late-afternoon C₃-like fixation of atmospheric CO₂ in S. praealtum, the QR was relatively high with values of 41 under 21% O₂ and 19 under 2% O₂. During the deacidification phase in S. praealtum, the relatively low QR can be accounted for by the repression of photorespiration and saturation of photosynthesis from the elevated CO₂ concentration in the leaves during malate decarboxylation.     

Influence of Photoperiod and Leaf Age on Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq

The possibility that Crassulacean acid metabolism (CAM) is subject to long day photoperiodic control in Portulacaria afra (L.) Jacq., a facultative CAM plant, was studied. Periodic measurements of ¹⁴CO₂ uptake, stomatal resistance, and titratable acidity were made on plants exposed to long and short day photoperiods. Results indicates that waterstressed P. afra had primarily nocturnal CO₂ uptake, daytime stomatal closure, and a large diurnal acid fluctuation in either photoperiod. Mature leaf tissue from nonstressed plants under long days exhibited a moderate diurnal acid fluctuation and midday stomatal closure. Under short days, there was a reduced diurnal acid fluctuation in mature leaf tissue. Young leaf tissue taken from nonstressed plants did not utilize the CAM pathway under either photoperiod as indicated by daytime CO₂ uptake, lack of diurnal acid fluctuation, and incomplete daytime stomatal closure.

The induction of CAM in P. afra appears to be related to the water status of the plant and the age of the leaf tissue. The photosynthetic metabolism of mature leaves may be partly under the control of water stress and of photoperiod, where CAM is favored under long days.

     

Effects of abscisic acid on CAM in Portulacaria afra

Water stress induces Crassulacean acid metabolism (CAM) in Portulacaria afra as manifested by day stomatal closure, organic acid fluctuation, and night CO₂ uptake. We now have evidence that abscisic acid treatment of leaves causes partial stomatal closure that is accompanied by the induction of CAM in a manner similar to water stress. There appears to be an inverse relationship between exogenous CO₂ uptake and decarboxylation of organic acids in that organic acids remain high during the day providing stomata are open. When stomata close, there is consumption of organic acids by decarboxylation. The hypothesis is that stomatal opening controls CAM in this species.This material is based upon work supported by the Science and Education Administration of the USDA under Competitive Grant No. 5901-0410-8-0018-0.     

Malate decarboxylation in isolated mitochondria from the crassulacean acid metabolism plant Sedum praealtum

Mitochondria isolated from the Crassulacean acid metabolism plant Sedum praealtum were demonstrated to decarboxylate added malate at basal rates of 30–50 μmol mg^?1 original chlorophyll h^?1. The basal rate could be stimulated markedly by the addition of ADP, oxaloacetic acid, an uncoupler of oxidative phosphorylation, or NAD, with maximum rates of 70–100 μmol mg^?1 original chlorophyll h^?1 observed. These observed rates were high enough to account for a large proportion of the estimated rate of malate decarboxylation in vivo. The major products of malate oxidation by the mitochondria in most cases were found to be pyruvate and CO₂, indicating that malate oxidation in these mitochondria proceeds mainly through NAD malic enzyme rather than NAD malate dehydrogenase. Under conditions employed little of the pyruvate formed was further oxidized, suggesting a fate other than oxidation (conversion to starch) for this pyruvate. Malate decarboxylation by mitochondria and by partially purified NAD malic enzyme was markedly inhibited by NaHCO₃. A possible physiological role is suggested for this inhibition as a feedback control on the enzyme.     

The correlation between crassulacean acid metabolism and water uptake in Senecio medley-woodii

The combination of a chamber for CO₂ gas exchange with a potometric measuring arrangement allowed concomitant investigations into CO₂ gas exchange, transpiration and water uptake by the roots of whole plants of Senecio medley-woodii, a species which exhibits Crassulacean acid metabolism. The water-uptake rate showed the same daily pattern as malate concentration and osmotic potential. The accumulation of organic acids resulting from nocturnal CO₂ fixation enhanced the water-uptake rate from dusk to dawn. During the day the water-uptake rates decreased with decreasing organic-acid concentration. With gradually increasing water stress, CO₂ dark fixation of S. medley-woodii was increased as long as water could be taken up by the roots. It was also shown that a reestablished water supply after drought caused a similar increase which in both cases ameliorated the water uptake in order to conserve a positive water balance for as long as possible. This water-uptake pattern shows that Crassulacean acid metabolism is not only a water-saving adaptation but also enhances water uptake and is directly correlated with the amelioration of the plant water status.Abbreviation CAM Crassulacean acid metabolism     

Physiological and isotopic aspects of photosynthesis in peperomia

Physiological and isotopic aspects of several Peperomia species were investigated. All but one species had C₃-like stomatal behavior, in that stomata were open during the day and closed during the night. In these species, most atmospheric CO₂ uptake occurred during the day. Concurrent with this stomatal behavior, there were Crassulacean acid metabolism-like acid fluctuations in most species. Carbon and hydrogen isotope ratios of cellulose nitrate from Peperomia reflect their physiological behavior. The δ¹³C values of cellulose nitrate from Peperomia species were similar to values observed in C₃ plants and consistent with the daytime uptake of exogeneous CO₂ via the C₃ photosynthetic pathway. The δD values of cellulose nitrate from Peperomia species approach those of Crassulacean acid metabolism plants. These elevated δD values are caused by fractionations occurring during biochemical reactions and not as a consequence of water relations.     

Modulation of Rubisco Activity during the Diurnal Phases of the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana

The regulation of Rubisco activity was investigated under high, constant photosynthetic photon flux density during the diurnal phases of Crassulacean acid metabolism in Kalanchoë daigremontiana Hamet et Perr. During phase I, a significant period of nocturnal, C₄-mediated CO₂ fixation was observed, with the generated malic acid being decarboxylated the following day (phase III). Two periods of daytime atmospheric CO₂ fixation occurred at the beginning (phase II, C₄–C₃ carboxylation) and end (phase IV, C₃–C₄ carboxylation) of the day. During the 1st h of the photoperiod, when phosphoenolpyruvate carboxylase was still active, the highest rates of atmospheric CO₂ uptake were observed, coincident with the lowest rates of electron transport and minimal Rubisco activity. Over the next 1 to 2 h of phase II, carbamylation increased rapidly during an initial period of decarboxylation. Maximal carbamylation (70%–80%) was reached 2 h into phase III and was maintained under conditions of elevated CO₂ resulting from malic acid decarboxylation. Initial and total Rubisco activity increased throughout phase III, with maximal activity achieved 9 h into the photoperiod at the beginning of phase IV, as atmospheric CO₂ uptake recommenced. We suggest that the increased enzyme activity supports assimilation under CO₂-limited conditions at the start of phase IV. The data indicate that Rubisco activity is modulated in-line with intracellular CO₂ supply during the daytime phases of Crassulacean acid metabolism.     

Carbon Dioxide and Water Vapor Exchange in the Crassulacean Acid Metabolism Plant Kalanchoë pinnáta during a Prolonged Light Period: METABOLIC AND STOMATAL CONTROL OF CARBON METABOLISM

Net CO₂ and water vapor exchange were studied in the Crassulacean acid metabolism plant Kalanchoë pinnáta during a normal 12-hour light/12-hour dark cycle and during a prolonged light period. Leaf temperature and leaf-air vapor pressure difference were kept constant at 20 C and 9 to 10 millibar. There was a 25% increase in the rate of CO₂ fixation during the first 6 hours prolonged light without change in stomatal conductance. This was associated with a decrease in the intracellular partial pressure of CO₂, a decrease in the stimulation of net CO₂ uptake by 2% O₂, and a decrease in the CO₂ compensation point from 45 to 0 microbar. In the normal light period after deacidification, leaves showed a normal light dependence of CO₂ uptake but, in prolonged light, CO₂ uptake was scarcely light-dependent. The increase in titratable acidity in prolonged light was similar to that in the dark.     

Environmental Influences on Open Stomates of a Crassulacean Acid Metabolism Plant, Agave deserti

The major short term stomatal response of Agave deserti was to temperature; increases in leaf temperature led to decreases in water vapor conductance for stomatal opening during the daytime (C₃ mode) as well as at night (Crassulacean acid metabolism or CAM mode). Hourly changes in the water vapor concentration drop from leaf to air had no significant stomatal effect in either mode. Stomatal responses to external CO₂ levels up to 800 microliters per liter were not significant after 15 minutes and only moderate after a few hours, suggesting that CO₂ effects on open stomates of this succulent were indirect in both CAM and C₃ modes.     

Shifts in the Carbon Metabolism of Xerosicyos danguyi H. Humb. (Cucurbitaceae) Brought About by Water Stress : I. General Characteristics

Xerosicyos danguyi H. Humb. (Cucurbitaceae) is an unusual leaf succulent endemic to Madagascar. Under well-watered conditions the plant exhibited Crassulacean acid metabolism (CAM), as characterized by large diurnal changes in titratable acidity, predominantly nighttime stomatal opening and CO₂ uptake, and high δ¹³C values. When plants were exposed to water stress for a minimum of a month, they shifted to a mode of carbon metabolism previously labeled CAM-idling. Under this mode of metabolism, the plants exhibited reduced stomatal opening, reduced CO₂ uptake, dampened diurnal fluctuations in titratable acidity, and no apparent changes in the δ¹³C values. Additionally, investigations showed that the stress hormones 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor) and abscisic acid increased as much as 6-fold in the water-stressed plants. The results are discussed in relation to physiological significance and evolution of the CAM-idling mode of metabolism.     

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.     

Water Relations and Photosynthesis of a Desert CAM Plant, Agave deserti

The water relations and photosynthesis of Agave deserti Engelm., a plant exhibiting Crassulacean acid metabolism, were measured in the Colorado desert. Although no natural stomatal opening of A. deserti occurred in the summer of 1975, it could be induced by watering. The resistance for water vapor diffusion from a leaf (R_WV) became less than 20 sec cm⁻¹ when the soil water potential at 10 cm became greater than −3 bars, as would occur after a 7-mm rainfall. As a consequence of its shallow root system (mean depth of 8 cm), A. deserti responded rapidly to the infrequent rains, and the succulent nature of its leaves allowed stomatal opening to continue for up to 8 days after the soil became drier than the plant. When the leaf temperature at night was increased from 5 to 20 C, R_WV increased 5-fold, emphasizing the importance of cool nighttime temperatures for gas exchange by this plant. Although most CO₂ uptake occurred at night, a secondary light-dependent rise in CO₂ influx generally occurred after dawn. The transpiration ratio (mass of water transpired/mass of CO₂ fixed) had extremely low values of 18 for a winter day, and approximately 25 for an entire year.     

Postillumination burst of carbon dioxide in crassalacean Acid metabolism plants

Immediately following exposure to light, a postillumination burst of CO₂ has been detected in Crassulacean acid metabolism plants. A detailed study with pineapple (Ananas comosus) leaves indicates that the postillumination burst changes its amplitude and kinetics during the course of a day. In air, the postillumination burst in pineapple leaves generally is exhibited as two peaks. The postillumination burst is sensitive to atmospheric CO₂ and O₂ concentrations as well as to the light intensity under which plants are grown. We propose that the CO₂ released in the first postillumination burst peak is indicative of photorespiration since it is sensitive to either O₂ or CO₂ concentration while the second CO₂ evolution peak is likely due to decarboxylation of organic acids involved in Crassulacean acid metabolism.     

Day-night changes in leaf water relations associated with the rhythm of crassulacean acid metabolism in Kalanchoë daigremontiana

A study was made of the day-night changes under controlled environmental conditions in the bulk-leaf water relations of Kalanchoë daigremontiana, a plant showing Crassulacean acid metabolism. In addition to nocturnal stomatal opening and net CO₂ uptake, the leaves of well-watered plants showed high rates of gas exchange during the whole of the second part of the light period. Measurements with the pressure chamber showed that xylem tension increased during the night and then decreased towards a minimum at about midday; a significant increase in xylem tension was also seen in the late afternoon. Cell-sap osmotic pressure paralleled leaf malate content and was maximum at dawn and minimum at dusk. The relationship between these two variables indicated that the nocturnally synthesized malate was apparently behaving as an ideal osmoticum. To estimate bulk-leaf turgor pressure, values for water potential were derived by correcting the pressurechamber readings for the osmotic pressure of the xylem sap. This itself was found to depend on the malate content of the leaves. Bulk-leaf turgor pressure changed rhythmically during the day-night cycle; turgor was low during the late afternoon and for most of the night, but increased quickly to a maximum of 0.20 MPa around midday. In water-stressed plants, where net CO₂ uptake was restricted to the dark period, there was also an increase in bulk-leaf turgor pressure at the start of the light period, but of reduced magnitude. Such changes in turgor pressure are likely to be of considerable ecological importance for the water economy of crassulacean-acid-metabolism plants growing in their natural habitats.Abbreviation and symbols CAM Crassulacean acid metabolism - P turgor pressure - osmotic pressure - water potential Dedicated to Professor Dr. H. Ziegler on the occasion of his 60th birthday     

Perturbations of malate accumulation and the endogenous rhythms of gas exchange in the Crassulacean acid metabolism plant<Emphasis Type="Italic"> Kalanchoë daigremontiana</Emphasis>: testing the tonoplast-as-oscillator model

In continuous light, leaves of the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier exhibit a circadian rhythm of CO₂ uptake, stomatal conductance and leaf-internal CO₂ pressure. According to a current quantitative model of CAM, the pacemaking mechanism involves periodic turgor-related tension and relaxation of the tonoplast, which determines the direction of the net flux of malate between the vacuole and the cytoplasm. Cytoplasmic malate, in turn, through its inhibitory effect on phosphoenolpyruvate carboxylase, controls the rate of CO₂ uptake. According to this mechanism, when the accumulation of malate is disrupted by removing CO₂ from the ambient air, the induction of a phase delay with respect to an unperturbed control plant is expected. First, using the mathematical model, such phase delays were observed in numerical simulations of three scenarios of CO₂ removal: (i) starting at a trough of CO₂ uptake, lasting for about half a cycle (ca. 12 h in vivo); (ii) with the identical starting phase, but lasting for 1.5 cycles (ca. 36 h); and (iii) starting while CO₂ increases, lasting for half a cycle again. Applying the same protocols to leaves of K. daigremontiana in vivo did not induce the predicted phase shifts, i.e. after the end of the CO₂ removal the perturbed rhythm adopted nearly the same phase as that of the control plant. Second, when leaves were exposed to a nitrogen atmosphere for three nights prior to onset of continuous light to prevent malate accumulation, a small, 4-h phase advance was observed instead of a delay, again contrary to the model-based expectations. Hence, vacuolar malic acid accumulation is ruled out as the central pacemaking process. This observation is in line with our earlier suggestion [T.P. Wyka, U. Lüttge (2003) J Exp Bot 54:1471–1479] that in extended continuous light, CO₂ uptake switches gradually from a CAM-like to a C3-like mechanism, with oscillations of the two CO₂ uptake systems being tightly coordinated. It appears that the circadian rhythm of gas exchange in this CAM plant emerges from one or several devices that are capable of generating temporal information in a robust manner, i.e. they are protected from even severe metabolic perturbations.Abbreviations CAM Crassulacean acid metabolism - c_ia Ratio of mesophyll CO₂ concentration to external CO₂ concentration - J_C Rate of carbon dioxide uptake - J_W Transpiration rate - g_W Stomatal conductance - LL Continuous light conditions - PEPC Phosphoenolpyruvate carboxylase - Rubisco d-Ribulose-1,5-bisphosphatecarboxylase/oxygenase - Effective quantum yield of photosystem II     

Day-Night Variations in Malate Concentration, Osmotic Pressure, and Hydrostatic Pressure in Cereus validus

Malate concentration and stem osmotic pressure concomitantly increase during nighttime CO₂ fixation and then decrease during the daytime in the obligate Crassulacean acid metabolism (CAM) plant, Cereus validus (Cactaceae). Changes in malate osmotic pressure calculated using the Van't Hoff relation match the changes in stem osmotic pressure, indicating that changes in malate level affected the water relations of the succulent stems. In contrast to stem osmotic pressure, stem water potential showed little day-night changes, suggesting that changes in cellular hydrostatic pressure occurred. This was corroborated by direct measurements of hydrostatic pressure using the Jülich pressure probe where a small oil-filled micropipette is inserted directly into chlorenchyma cells, which indicated a 4-fold increase in hydrostatic pressure from dusk to dawn. A transient increase of hydrostatic pressure at the beginning of the dark period was correlated with a short period of stomatal closing between afternoon and nighttime CO₂ fixation, suggesting that the rather complex hydrostatic pressure patterns could be explained by an interplay between the effects of transpiration and malate levels. A second CAM plant, Agave deserti, showed similar day-night changes in hydrostatic pressure in its succulent leaves. It is concluded that, in addition to the inverted stomatal rhythm, the oscillations of malate markedly affect osmotic pressures and hence water relations of CAM plants.