Malate synthesis by dark carbon dioxide fixation in leaves

The rates of dark CO₂ fixation and the label distribution in malate following dark ¹⁴CO₂ fixation in a C-4 plant (maize), a C-3 plant (sunflower), and two Crassulacean acid metabolism plants (Bryophyllum calycinum and Kalanchoë diagremontianum leaves and plantlets) are compared. Within the first 30 minutes of dark ¹⁴CO₂ fixation, leaves of maize, B. calycinum, and sunflower, and K. diagremontianum plantlets fix CO₂ at rates of 1.4, 3.4, 0.23, and 1.0 μmoles of CO₂/mg of chlorophyll· hour, respectively. Net CO₂ fixation stops within 3 hours in maize and sunflower, but Crassulaceans continue fixing CO₂ for the duration of the 23-hour experiment.

A bacterial procedure using Lactobacillus plantarum ATCC No. 8014 and one using malic enzyme to remove the β-carboxyl (C₄) from malate are compared. It is reported that highly purified malic enzyme and the bacterial method provide equivalent results. Less purified malic enzyme may overestimate the label in C₄ as much as 15 to 20%.

The contribution of carbon atom 1 of malate is between 18 and 21% of the total carboxyl label after 1 minute of dark CO₂ fixation. Isotopic labeling in the two carboxyls approached unity with time. The rate of increase is greatest in sunflower leaves and Kalanchoë plantlets. In addition, Kalanchoë leaves fix ¹⁴CO₂ more rapidly than Kalanchoë plantlets and the equilibration of the malate carboxyls occurs more slowly. The rates of fixation and the randomization are tissue-specific. The rate of fixation does not correlate with the rate of randomization of isotope in the malate carboxyls.

     

Malate Metabolism in the Dark After CO(2) Fixation in the Crassulacean Plant Kalanchoë tubiflora

The metabolism of [¹³C]malate was studied in the Crassulacean plant Kalanchoë tubiflora following exposure to ¹³CO₂ for 2 hour intervals during a 16 hour dark cycle. Nuclear magnetic resonance spectroscopy of [¹³C]malate extracted from labeled tissue revealed that the transient flux of malate to the mitochondria, estimated by the randomization of [4-¹³C]malate to [1- ¹³C]malate by fumarase, varied substantially during the dark period. At both 15 and 25°C, the extent of malate label randomization in the mitochondria was greatest during the early and late parts of the dark period and was least during the middle of the night, when the rate of ¹³CO₂ uptake was highest. Randomization of labeled malate continued for many hours after malate synthesis had initially occurred. Internally respired ¹²CO₂ also served as a source of carbon for malate formation. At 15°C, 15% of the total malate was formed from respired ¹²CO₂, while at 25°C, 49% of the accumulated malate was derived from respired ¹²CO₂. Some of the malate synthesized from external ¹³CO₂ was also respired during the night. The proportion of the total [¹³C]malate respired during the dark period was similar at 15 and 25°C, and respiration of newly formed [¹³C]malate increased as the night period progressed. These data are discussed with regard to the relative fluxes of malate to the mitochondria and the vacuole during dark CO₂ fixation.     

Diurnal regulation of phosphoenolpyruvate carboxylase from crassula

Phosphoenolpyruvate carboxylase appears to be located in or associated with the chloroplasts of Crassula. As has been found with this enzyme in other CAM plants, a crude extract of leaves gathered during darkness and rapidly assayed for phosphoenolpyruvate carboxylase (PEPc) activity is relatively insensitive to inhibition by malate. After illumination begins, the PEPc activity becomes progressively more sensitive to malate. This enzyme also shows a diurnal change in activation by glucose-6-phosphate, with the enzyme from dark leaves more strongly activated than that from leaves in the light.

When the enzyme is partially purified in the presence of malate, the characteristic sensitivity of the day leaf enzyme is largely retained. Partial purification of the enzyme from dark leaves results in a small increase in sensitivity to malate inhibition.

Partially purified enzyme is found by polyacrylamide gel electrophoresis analysis to have two bands of PEPc activity. In enzymes from dark leaves, the slower moving band predominates, but in the light, the faster moving band is preponderant. Both of these bands are shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be composed of the same subunit of 103,000 daltons.

The enzyme partially purified from night leaves has a pH optimum of 5.6, and is relatively insensitive to malate inhibition over the range from pH 4.5 to 8. The enzyme from day leaves has a pH optimum of 6.6 and is strongly inhibited by malate at pH values below 7, but becomes insensitive at higher pH values.

Gel filtration of partially purified PEPc showed two activity peaks, one corresponding approximately to a dimer of the single subunit, and the other twice as large. The larger protein was relatively insensitive to malate inhibition, the smaller was strongly inhibited by malate.

Kinetic studies showed that malate is a mixed type inhibitor of the sensitive, day, enzyme, increasing K_m for phosphoenolpyruvate and reducing V_max. With the insensitive, night, enzyme, malate is a K type inhibitor, reducing the K_m for phosphoenolpyruvate, but having little effect on V_max. The inhibition of the insensitive enzyme by malate appears to be hysteretic, taking several minutes to be expressed during assay, probably indicating a change in the conformation or aggregation state of the enzyme.

Activation by glucose-6-phosphate is of the mixed type for the day form of the enzyme, causing both a decreased K_m for phosphoenolpyruvate and an increased V_max, but the night, or insensitive, form shows only an increase in V_max in response to glucose-6-phosphate.

     

Effect of carbon dioxide on nitrate accumulation and nitrate reductase induction in corn seedlings

Exposure of the leaf canopy of corn seedlings (Zea mays L.) to atmospheric CO₂ levels ranging from 100 to 800 μl/l decreased nitrate accumulation and nitrate reductase activity. Plants pretreated with CO₂ in the dark and maintained in an atmosphere containing 100 μl/l CO₂ accumulated 7-fold more nitrate and had 2-fold more nitrate reductase activity than plants exposed to 600 μl/l CO₂, after 5 hours of illumination. Induction of nitrate reductase activity in leaves of intact corn seedlings was related to nitrate content. Changes in soluble protein were related to in vitro nitrate reductase activity suggesting that in vitro nitrate reductase activity was a measure of in situ nitrate reduction. In longer experiments, levels of nitrate reductase and accumulation of reduced N supported the concept that less nitrate was being absorbed, translocated, and assimilated when CO₂ was high. Plants exposed to increasing CO₂ levels for 3 to 4 hours in the light had increased concentrations of malate and decreased concentrations of nitrate in the leaf tissue. Malate and nitrate concentrations in the leaf tissue of seven of eight corn genotypes grown under comparable and normal (300 μl/l CO₂) environments, were negatively correlated. Exposure of roots to increasing concentrations of potassium carbonate with or without potassium sulfate caused a progressive increase in malate concentrations in the roots. When these roots were subsequently transferred to a nitrate medium, the accumulation of nitrate was inversely related to the initial malate concentrations. These data suggest that the concentration of malate in the tissue seem to be related to the accumulation of nitrate.     

Effect of Varying CO(2) Partial Pressure on Photosynthesis and on Carbon Isotope Composition of Carbon-4 of Malate from the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana Hamet et Perr

Intact leaves of Kalanchoë daigremontiana were exposed to CO₂ partial pressures of 100, 300, and 1000 microbars. Malic acid was extracted, purified, and degraded in order to obtain isotopic composition of carbon-1 and carbon-4. From these data, it is possible to calculate the carbon isotope composition of newly fixed carbon in malate. In all three treatments, the isotopic composition of newly introduced carbon is the same as that of the CO₂ source and is independent of CO₂ partial pressures over the range tested. Comparison with numerical models described previously (O'Leary 1981 Phytochemistry 20: 553-567) indicates that we would expect carbon 4 of malate to be 4‰ more negative than source CO₂ if diffusion is totally limiting or 7‰ more positive than source CO₂ if carboxylation is totally limiting. Our results demonstrate that stomatal aperture adjusts to changing CO₂ partial pressures and maintains the ratio of diffusion resistance to carboxylation resistance approximately constant. In this study, carboxylation and diffusion resistances balance so that essentially no fractionation occurs during malate synthesis. Gas exchange studies of the same leaves from which malate was extracted show that the extent of malate synthesis over the whole night is nearly independent of CO₂ partial pressure, although there are small variations in CO₂ uptake rate. Both the gas exchange and the isotope studies indicate that the ratio of external to internal CO₂ partial pressure is the same in all three treatments. Inasmuch as a constant ratio will result in constant isotope fractionation, this observation may explain why plants in general have fairly invariable ¹³C contents, despite growing under a variety of environmental conditions.     

Equilibration of Label in Malate during Dark Fixation of CO(2) in Kalanchoë fedtschenkoi

In vitro studies of dark ¹⁴CO₂ fixation with isolated cell aggregates of Kalanchoë fedtschenkoi showed that malate synthesized after 20 sec is predominantly (85 to 92%) labeled at carbon 4, while after 20 min only 65 to 69% of the radioactivity was located in this position. The intramolecular labeling pattern of malate could not be changed by supplementing the cells with carboxylation reaction substrates such as ribulose diphosphate or phosphoenolpyruvate. The kinetic decline of label at carbon 4 of malate occurs independently of CO₂ fixation, since 4-¹⁴C-labeled aspartate fed to the cells gave rise to malate labeled 62% at carbon 4 after 20 min. Furthermore, the cells were capable of converting fed malate to fumarate. It is concluded that synthesis of malate during dark CO₂ fixation is accomplished by a single carboxylation step via phosphoenolpyruvate carboxylase and labeling patterns observed in malate are a consequence of the action of fumarase.     

Light-Stimulated Burst of Carbon Dioxide Uptake following Nocturnal Acidification in the Crassulacean Acid Metabolism Plant Kalanchoë diagremontiana

CO₂ exchange characteristics were studied during the light-stimulated burst of CO₂ uptake (MB) immediately following a period of nocturnal CO₂ fixation in the Crassulacean acid metabolism plant Kalanchoë daigremontiana. During the early parts of the MB, stimulation of net CO₂ uptake by low ambient O₂ concentration (1.5%) was small, and leaves showed the capacity for net CO₂ uptake at low ambient CO₂ partial pressure (30 microbars) and when the MB was interrupted by darkness. During the later phase of the MB, stimulation of net CO₂ uptake by 1.5% O₂ was increased, and net CO₂ loss was recorded both at 30 microbars CO₂ and during dark interruptions. These results suggest that CO₂ fixation during the MB occurs simultaneously via phosphoenolpyruvate carboxylase (predominant during the early phase of the MB) and via ribulose bisphosphate carboxylase (predominant during the later phase of the burst). The magnitude and duration of the MB was increased by a reduction in the length of the dark period and by low (15°C) compared to high (30°C) leaf temperatures.     

Changes in enzyme activities involved in malate metabolism in oak leaves during rhythmic growth

Amide content, ATP level and activities of enzymes linked to malate metabolism were determined in leaves of three successive flushes of common oak during the development of the third flush. In the expanding leaves, all studied enzymes showed a maximum activity around the 7th day after budbreak. Phosphoenolpyruvate carboxylase (PEPc), NAD-malate dehydrogenase (MDH) and NADP-malic enzyme (ME) maintained high activity up to full leaf expansion. In contrast, fumarase (FUM), pyruvate kinase (PK) and NADP-MDH activities sharply decreased to reach, on the 10th day after budbreak, the same low activity levels as those measured in mature leaves. Two patterns were observed in oak leaves during growth. Firstly (7th–10th day after budbreak), PK, FUM and NADP-MDH could contribute to the supply of ATP through glycolysis and Krebs cycle; the ATP profile corroborated those results. Secondly (after the 10th day), the maintenance of an active PEPc pathway led to a respiratory CO₂ refixation and provided carbon skeletons for amino acid synthesis. Furthermore, nitrate reductase (NR) activity was high in young oak leaves. Slight changes in activities of NR as well as NAD(P)-ME, NAD(P)-MDH can be noted on days 7 and 10 after budbreak in the mature leaves. These changes could be necessary in supplying the third flush with amino acids. These data suggest that MDH, ME, PK and PEPc have important functions in the young leaves which are not directly linked to C₃ photosynthesis but rather to nitrate assimilation and malate provision to mitochondria.     

Veränderliche Markierungsmuster bei 14CO2-Fütterung von Bryophyllum tubiflorum zu verschiedenen Zeitpunkten der Hell-Dunkelperiode

Summary The distribution of radioactivity between the products of ¹⁴CO₂ light fixation in phyllodia of Bryophyllum tubiflorum could be influenced experimentally by manipulating the malic acid content of the cells. Accelerating the deacidification of the tissue during the light period by application of higher light intensities accelerated the increase of malate labelling and the decrease of the sucrose labelling after ¹⁴CO₂ light fixation under our standard conditions (10 min preillumination, 15 min ¹⁴CO₂ light fixation, 8000 lux).In other experiments different malate contents of the tissues were induced by treating the phyllodia with different temperatures during the night period. In the morning, phyllodia with low malate content transferred most of the label into malate, and phyllodia with high malate content incorporated most of the ¹⁴C radioactivity into sugars. However, this was true only after preillumination of 1 hour. When the phyllodia fixed ¹⁴CO₂ without preillumination, no differences in the labelling patterns between acidified and non-acidified phyllodia could be observed.In experiments using leaf tissue slices of Bryophyllum daigremontianum we could again observe that malate was labelled more heavily in the deacidified tissue than in the acidified controls, with less radioactivity being transferred into phosphate esters and sugars. The rates of ¹⁴CO₂ light fixation were identical in tissue slices with high and low malate content. However, the rates of CO₂ dark fixation in the acidified samples were clearly lower than those in the deacidified ones. The low rate of CO₂ dark fixation in acidified samples could not be inhibited by an inhibitor of PEP-carboxylase as the high CO₂ dark fixation rate of the deacidified tissue could be inhibited.The results are discussed in relation to the feed back inhibition of PEP-carboxylase in vivo by malate. Compartmentation also seemed to be involved in controlling the flow of carbon during CO₂ light fixation in succulent tissue.     

Photosynthesis at High Temperature in Tuber-Bearing Solanum Species : A Comparison between Accessions of Contrasting Heat Tolerance

Differences in the photosynthetic performance between pairs of heat tolerant (HT) and heat sensitive (HS) accessions of tuber-bearing Solanum species were measured at 40 °C, after treating plants at 40/30 °C. After 1 to 9 days of heat treatment, both HT and HS accessions showed progressive inhibitory effects, primarily decreased rates of CO₂ fixation, and loss of leaf chlorophyll. These effects were most pronounced in the HS accessions. Stomatal conductivity and internal CO₂ concentrations were lower for both accessions at 40 °C especially for the HS accessions, suggesting that at ambient CO₂ concentrations, stomatal conductance was limiting CO₂ availability at the higher temperature. In the HT accessions, stomatal limitations were largely attributed to differences in vapor pressure deficit between 25° and 40 °C, while the HS accessions exhibited significant nonstomatal limitations. The young expanding leaves of the HS accession showed some HT characteristics, while the oldest leaves showed severe senescence symptoms after 9 days at 40/30 °C. The data suggest that differences in heat sensitivity between HT and HS accessions are the result of accelerated senescence, chlorophyll loss, reduced stomatal conductance, and inhibition of dark reactions at high temperature.     

Period and phase control by temperature in the circadian rhythm of carbon dioxide fixation in illuminated leaves of Bryophyllum fedtschenkoi

The rhythm of CO₂ assimilation exhibited by leaves of Bryophyllum fedtschenkoi maintained in light and normal air occurs only at constant ambient temperatures between 10°C and 30°C. Over this range the period increases linearly with increasing temperature from the extremely low value of 15.7 h to 23.3 h, but shows a considerable degree of temperature compensation. Outside the range 10°C–30°C the rhythm is inhibited but re-starts on changing the temperature to 15°C. Prolonged exposure of leaves to high (40°C) and low (2°C) temperature inhibits the rhythm by driving the basic oscillator to fixed phase points in the cycle which differ by 180°, and which have been characterised in terms of the malate status of the leaf cells. At both temperatures loss of the circadian rhythm of CO₂ assimilation is due to the inhibition of phosphoenolpyruvate carboxylase (PEPCase) activity, but the inhibition is apparently achieved in different ways at 40°C and 2°C. High temperature appears to inhibit directly PEPCase activity, but not the activity of the enzymes responsible for the breakdown of malate, with the result that the leaf acquires a low malate status. In contrast, low temperature does not directly inhibit PEPCase activity, but does inhibit enzymes responsible for malate breakdown, so that the malate level in the leaf increases to a high value and PEPCase is eventually allosterically inhibited. The different malate status of leaves held at these two temperatures accounts for the phases of the rhythms being reversed on returning the leaves to 15°C. After exposure to high temperature, CO₂ fixation by PEPCase activity can begin immediately, whereas after exposure to low temperature, the large amount of malate accumulated in the leaves has to be decarboxylated before CO₂ fixation can begin.     

Effects of osmotic gradients on vacuolar malic Acid storage: a basic principle in oscillatory behavior of crassulacean Acid metabolism

Malate synthesis by CO₂ dark fixation and malate accumulation in the vacuoles of leaf slices of Kalanchoë daigremontiana Hamet et Perrier, a plant performing crassulacean acid metabolism, occurs only in external solutions where the osmotic pressure difference between the cells and the medium is low. Conversely, malate loss from the vacuoles depends on a high osmotic pressure difference between the cells and the medium and is observed in media of low osmotic pressure. This suggests that the diurnal oscillations of malate levels in crassulacean acid metabolism leaf cells are regulated by osmotic gradients. These findings support a model which is introduced to explain how the rhythm of crassulacean acid metabolism may function in the intact plant.     

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.     

Diurnal changes in volume and specific tissue weight of crassulacean Acid metabolism plants

The diurnal variations in volume and in specific weight were determined for green stems and leaves of Crassulacen acid metabolism (CAM) plants. Volume changes were measured by a water displacement method. Diurnal variations occurred in the volume of green CAM tissues. Their volume increased early in the light period reaching a maximum about mid-day, then the volume decreased to a minimum near midnight. The maximum volume increase each day was about 2.7% of the total volume. Control leaves of C₃ and C₄ plants exhibited reverse diurnal volume changes of 0.2 to 0.4%. The hypothesis is presented and supported that green CAM tissues should exhibit a diurnal increase in volume due to the increase of internal gas pressure from CO₂ and O₂ when their stomata are closed. Conversely, the volume should decrease when the gas pressure is decreased.

The second hypothesis presented and supported was that the specific weight (milligrams of dry weight per square centimeter of green surface area) of green CAM tissues should increase at night due to the net fixation of CO₂. Green CAM tissues increased their specific weight at night in contrast to control C₃ and C₄ leaves which decreased their specific weight at night. With Kalanchoë daigremontiana leaves, the calculated increase in specific leaf weight at night based on estimates of carbohydrate available for net CO₂ fixation was near 6% and the measured increase in specific leaf weight was 6%.

Diurnal measurements of CAM tissue water content were neither coincident nor reciprocal with their diurnal patterns of either volume or specific weight changes.

     

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.     

Effect of elevated CO2 on growth and crassulacean-acid-metabolism activity of Kalanchoë pinnata under tropical conditions

 Kalancho? pinnata (Lam.) Pers. (Crassulaceae), a succulent-leaved crassulacean-acid-metabolism plant, was grown in open-top chambers at ambient and elevated (two times ambient) CO₂ concentrations under natural conditions at the Smithsonian Tropical Research Institute, Republic of Panama. Nocturnal increase in titratable acidity and nocturnal carbon gain were linearly related, increased with leaf age, and were unaffected by CO₂ treatments. However, under elevated CO₂, dry matter accumulation increased by 42–51%. Thus, the increased growth at elevated CO₂ was attributable entirely to increased net CO₂ uptake during daytime in the light. Malic acid was the major organic acid accumulated overnight. Nocturnal malate accumulation exceeded nocturnal citrate accumulation by six-to eightfold at both CO₂ concentrations. Basal (predawn) starch levels were higher in leaves of plants grown at elevated CO₂ but diurnal fluctuations of starch were of similar magnitude under both ambient and elevated CO₂. In both treatments, nocturnal starch degradation accounted for between 78 and 89% of the nocturnal accumulation of malate and citrate. Glucose, fructose, and sucrose were not found to exhibit marked day-night fluctuations. Received: 4 March 1996 / Accepted: 25 May 1996     

Diurnal modulation of phosphoenolpyruvate carboxylation in pea leaves and roots as related to tissue malate concentrations and to the nitrogen source

Phosphoenolpyruvate (PEP) carboxylation was measured as dark ¹⁴CO₂ fixation in leaves and roots (in vivo) or as PEP carboxylase (PEPCase) activity in desalted leaf and roof extracts (in vitro) from Pisum sativum L. cv. Kleine Rheinländerin. Its relation to the malate content and to the nitrogen source (nitrate or ammonium) was investigated. In tissue from nitrate-grown plants, PEP carboxylation varied diurnally, showing an increase upon illumination and a decrease upon darkening. Diurnal variations in roots were much lower than in leaves. Fixation rates in leaves remained constantly low in continuous darkness or high in continuous light. Dark CO₂ fixation of leaf slices also decreased when leaves were preilluminated for 1 h in CO₂-free air, suggesting that the modulation of dark CO2 fixation was related to assimilate availability in leaves and roots. Phosphoenolpyruvate carboxylase activity was also measured in vitro. However, no difference in maximum enzyme activity was found in extracts from illuminated or darkened leaves, and the response to substrate and effectors (PEP, malate, glucose-6-phosphate, pH) was also identical. The serine/threonine protein kinase inhibitors K252b, H7 and staurosporine, and the protein phosphatase 2A inhibitors okadaic acid and cantharidin, fed through the leaf petiole, did not have the effects on dark CO₂ fixation predicted by a regulatory system in which PEPCase is modulated via reversible protein phosphorylation. Therefore, it is suggested that the diurnal modulation of PEP carboxylation in vivo in leaves and roots of pea is not caused by protein phosphorylation, but rather by direct allosteric effects. Upon transfer of plants to ammonium-N or to an N-free nutrient solution, mean daily malate levels in leaves decreased drastically within 4–5 d. At that time, the diurnal oscillations of PEP carboxylation in vivo disappeared and rates remained at the high light-level. The coincidence of the two events suggests that PEPCase was de-regulated because malate levels became very low. The drastic decrease of leaf malate contents upon transfer of plants from nitrate to ammonium nutrition was apparently not caused by increased amino acid or protein synthesis, but probably by higher decarboxylation rates.Abbreviations CAM crassulacean acid metabolism - PEP Phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PP protein phosphatase - PK protein kinase This work was supported by the Deutsche Forschungsgemeinschaft. B. Baur was a recipient of a doctoral grant, and L. Leport recipient of a post-doctoral grant of the DFG. The skilled technical assistance of Eva Wirth and Maria Lesch is gratefully acknowledged.     

C Nuclear Magnetic Resonance Studies of Crassulacean Acid Metabolism in Intact Leaves of Kalanchoë tubiflora

¹³C nuclear magnetic resonance spectroscopy of intact leaves of Kalanchoë tubiflora was used to observe Crassulacean acid metabolism in vivo. ¹³C signals from C-4 of malate were observed after overnight exposure of leaves to ¹³CO₂. Illumination of the labeled leaves resulted in a gradual decrease in the malate signals. After a period of darkness in normal air, ¹³C signals were detected in all four carbons of malate in the previously labeled leaves. The ¹³C nuclear magnetic resonance spectrum of malate in solution was pH dependent, which allowed an estimation of the vacuolar pH from the whole leaf spectrum. The pH was 4.0 following a 14-hour dark period, but rose to greater than 6.0 after 6 hours of illumination.     

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.     

Carbonic Anhydrase Activity and CO2-Transfer Resistance in Zn-Deficient Rice Leaves

It has been reported that carbonic anhydrase (CA) activity in plant leaves is decreased by Zn deficiency. We examined the effects of Zn deficiency on the activity of CA and on photosynthesis by leaves in rice plants (Oryza sativa L.). Zn deficiency increased the transfer resistance from the stomatal cavity to the site of CO₂ fixation 2.3-fold and, consequently, the value of the transfer resistance relative to the total resistance in the CO₂-assimilation process increased from 10% to 21%. This change led to a reduced CO₂ concentration at the site of CO₂ fixation, resulting in an increased gradient of CO₂ between the stomatal cavity and this site. The present findings support the hypothesis that CA functions to facilitate the supply of CO₂ from the stomatal cavity to the site of CO₂ fixation. We also showed that the level of mRNA for CA decreased to 13% of the control level during Zn deficiency. This decrease resembled the decrease in CA activity, suggesting the possible involvement of the CA mRNA level in the regulation of CA activity.