Phosphoenolpyruvate carboxykinase in plants exhibiting crassulacean Acid metabolism

Phosphoenolpyruvate carboxykinase has been found in significant activities in a number of plants exhibiting Crassulacean acid metabolism. Thirty-five species were surveyed for phosphoenolpyruvate carboxykinase, phosphoenolpyruvate carboxylase, ribulose diphosphate carboxylase, malic enzyme, and malate dehydrogenase (NAD). Plants which showed high activities of malic enzyme contained no detectable phosphoenolpyruvate carboxykinase, while plants with high activities of the latter enzyme contained little malic enzyme. It is proposed that phosphoenolpyruvate carboxykinase acts as a decarboxylase during the light period, furnishing CO₂ for the pentose cycle and phosphoenolpyruvate for gluconeogenesis.     

C/C ratio changes in crassulacean Acid metabolism plants

¹³C/¹²C ratios have been found in totally combusted leaves of Crassulacean acid metabolism plants to range from −14 to −33 δ ¹³C‰ compared with a limestone standard. Crassulacean acid metabolism plants apparently utilize both ribulose-1, 5-diphosphate carboxylase and phosphoenolpyruvate carboxylase to assimilate atmospheric CO₂ and, depending on environmental conditions, have ¹³C/¹²C ratios indicative of either carboxylase or to any intermediate value. The degree of discrimination against ¹³C and the resultant ¹³C/¹²C ratio from the photosynthetically fixed CO₂ is influenced by environmental conditions and is not a specific and fixed characteristic of a Crassulacean acid metabolism plant. Certain Crassulacean acid metabolism plants may shift their ratios as much as 17 δ ¹³C‰ in specific environments.     

Hydrogen, oxygen, and carbon isotope ratios of cellulose from submerged aquatic crassulacean Acid metabolism and non-crassulacean Acid metabolism plants

Isotope ratios of cellulose and cellulose nitrate from aquatic Crassulacean acid metabolism (CAM) and non-CAM plants were determined. Cellulose oxygen istope ratios for all plants that grew together were virtually identical, whereas large differences were observed for hydrogen isotope ratios of cellulose nitrate between CAM and non-CAM plants. Carbon isotope ratios of cellulose nitrate did not differentiate CAM from non-CAM plants.     

The effect of rishitin on potato tonoplast vesicle and vacuole proton transport

Rishitin, a known potato phytoalexin, was tested for its effects on proton transport. Like the pterocarpan phytoalexins, glyceollin and phaseollin, rishitin was found to inhibit proton transport. At 100 μM rishitin, proton transport in potato tonoplast vesicles was inhibited by >95%. This inhibition appears to be due to an increase in proton conductance and not to inhibition of the tonoplast ATPase. Potato vacuoles were also shown to have increased proton leakage in the presence of rishitin.     

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.

     

The rapid isolation of vacuoles from leaves of crassulacean Acid metabolism plants

A technique is presented for the isolation of vacuoles from Sedum telephium L. leaves. Leaf material is digested enzymically to produce protoplasts rapidly which are partially lysed by gentle osmotic shock and the inclusion of 5 millimolar ethyleneglycol-bis (β-aminoethyl ether)N,N′-tetraacetic acid in the wash medium. Vacuoles are isolated from the partially lysed protoplasts by brief centrifugation on a three-step Ficoll-400 gradient consisting of 5, 10, and 15% (w/v) Ficoll-400. A majority of the vacuoles accumulate at the 5 to 10% Ficoll interface, whereas a smaller proportion sediments at the 10 to 15% Ficoll-400 interface. The total time required for vacuole isolation is 2 to 2.5 hours, beginning from leaf harvest.     

Carbon isotope ratios in crassulacean Acid metabolism plants: seasonal patterns from plants in natural stands

A year round study of photosynthesis and carbon isotope fractionation was conducted with plants of Opuntia phaeacantha Engelm. and Yucca baccata Torr. occurring in natural stands at elevations of 525, 970, 1450 and 1900 m. Plant water potentials and the daytime pattern of ¹⁴CO₂ photosynthesis were similar for all cacti along the elevational gradient, despite significant differences in temperature regime and soil water status. Carbon isotope ratios of total tissue and soluble extract fractions were relatively constant throughtout the entire year. Additionally, the σ¹³C values were similar in all plants of the same species along the elevational gradient, i.e. −12.5 ± 0.86 ‰ for O. phaeacantha and −15.7 ± 0.95 ‰ for Y. baccata. The results of this study indicate Crassulacean acid metabolism predominates as the major carbon pathway of these plants, which do not facultatively utilize the reductive pentose phosphate cycle of photosynthesis as the primary carboxylation reaction.     

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.     

Fructose 2,6-bisphosphate, carbohydrate partitioning, and crassulacean Acid metabolism

To initiate structural studies of the ADPglucose pyrophosphorylase from spinach an improved purification procedure was devised. The modified purification scheme allowed the isolation of 20 to 30 milligrams pure enzyme from 10 kilogram of spinach leaves. Electrophoresis of the purified enzyme confirmed an earlier study which showed that the enzyme was putatively composed of two subunits (Copeland L, J Preiss 1981 Plant Physiol 68: 996-1001). The two subunits migrate as 51 and 54 kilodalton proteins upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both proteins can be detected on Western blots of leaf homogenates prepared under denaturing conditions suggesting that both subunits exist in vivo. Anion-exchange chromatography in the presence of urea allowed resolution of the 51 and 54 kilodalton proteins. They possess different N-terminal amino acid sequences and tryptic peptide maps. Western blot analysis reveals that the 51 and 54 kilodalton proteins are antigenically dissimilar. The 51 but not the 54 kilodalton protein is immunologically related to the ADPglucose pyrophosphorylase from maize endosperm and potato tuber.     

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.     

Reappearance of hydrolytic activities and tonoplast proteins in the regenerated vacuole of evacuolated protoplasts

Mesophyll protoplasts of tobacco (Nicotiana tabacum L. cv. Xanthi) were evacuolated by centrifugation in a density gradient. Evacuolation resulted in the quantitative loss of vacuolar hydrolytic activities. The evacuolated miniprotoplasts were cultivated under different conditions, and the regeneration of the central vacuole was investigated by light and electron microscopy as well as by the determination of activities of vacuolar marker enzymes. Vacuoles and hydrolytic activities, as well as cell wall material reappeared faster when the cells were cultivated at low osmotic strength. A newly synthesized tonoplast polypeptide could be detected using a polyspecific serum raised against tonoplast proteins of barley (Hordeum vulgare L.). Both vacuolar proton pumps, the ATPase as well as the pyrophosphatase appear to be newly synthesized during the regeneration of the vacuole.Abbreviations GAP-DH NADP-dependent glyceraldehyde 3-phosphate dehydrogenase - PEP phosphoenolpyruvate - PPi pyrophosphate - PPase pyrophosphatase We thank Dr. Ernst Wehrli, Labor für Elektronenmikroskopie I, ETH Zürich, for taking micrographs. Esther Vogt assisted in the determination of the hydrolases. Bafilomycin was kindly provided by Professor Altendorf, Osnabrück FRG. This work was supported by the Swiss National Foundation grant No. 31-25196.88.     

Temperature features of enzymes affecting crassulacean Acid metabolism

Enzymes involved in malic acid production via a pathway with 2 carboxylation reactions and in malic acid conversion via total oxidation have been demonstrated in mitochondria of Bryophyllum tubiflorum Harv. Activation of the mitochondria by Tween 40 was necessary to reveal part of the enzyme activities. The temperature behavior of the enzymes has been investigated, revealing optimal activity of acid-producing enzymes at 35°. Even at 53° the optimum for acid-converting enzymes was not yet reached. From the simultaneous action of acid-producing and acid-converting enzyme systems the overall result at different temperatures was established. Up to 15° the net result was a malic acid production. Moderate temperatures brought about a decrease in this accumulation, which was partly accompanied by a shift to isocitrate production, while at higher temperatures total oxidation of the acids exceeded the production.     

Daily free sulphate fluctuations in tropical plants with crassulacean acid metabolism

A difference in the dawn and dusk concentrations of free sulphatein leaf-cell sap, similar to the typical oscillation of titratableacidity in CAM plants, was observed in Fourcroya humboldtiana(Agavaceae) sampled in the succession of a cloud forest in Venezuela.Similar results were found in Clusia rosea and Clusia sp. (Clusiaceae),Tillandsia flexuosa (Bromeliaceae) and Subpilocereus ottonis(Cactaceae) growing in a natural oil spill. The concentrationof free sulphate was related to the pH of the cell sap. Theleaf-cell sap of F. humboldtiana dusk samples was titrated withmalic acid to obtain the low pH usually present in the morning,and the sulphate concentrations were compared. An increase inthe initial value of sulphate was observed, as found in thefield in the dawn samples where the acidity is high. The presenceof K₂SO₄ stored mainly in the vacuole and its dissociation atlow pH almost completely explains the daily oscillation of sulphatein F. humboldtiana. The salts MgSO₄ and CaSO₄ can also be dissociated,but the daily oscillations of soluble Mg and Ca are much lowerthan that of soluble K. In C. rosea, Clusia sp. and S. ottonisthe fluctuation of sulphate is higher than the oscillation ofcations and in C. rosea the oscillation of Ca is twice as muchas that of K. Key words: Sulphate, CAM, Fourcroya humboldtiana, Clusiaceae     

A malic Acid permease in isolated vacuoles of a crassulacean Acid metabolism plant

Pine (Pinus silvestris L.) trees subjected to relatively low concentration of SO₂ in the field emit H₂S from the needles, as demonstrated by gas chromatographic analysis after preconcentration on a molecular sieve. H₂S is the only reduced sulfurous compound emitted from SO₂ fumigated leaves. The emission is light and SO₂ concentration dependent. Pine trees in the field and in laboratory experiments continue to emit H₂S several hours after the termination of prolonged SO₂ fumigation. The maximum emission rates observed from pine trees in the field and in laboratory experiments, 14 and 20 nanomoles per milligram chlorophyll per hour respectively, are about the activity expected for the sulfur assimilation pathway in the chloroplasts.     

Seasonal diurnal acid rhythms in two aquatic crassulacean acid metabolism plants

Summary The diurnal change in titrable acidity in two aquatic CAM plants, Littorella uniflora var. americana (Fern.) Gl. and Isoetes macrospora Duriev., growing at two sites, was monitored at biweekly intervals for two years during the ice-free period. Both plants exhibited the classic pattern of CAM acitivity, with deacidification 60 to 90% complete by noon. The maximum diurnal acid rhythms observed were 169±7, and 154±20 eq·g^-1 fresh weight for the two Littorella populations, and 182±9 and 133±16 eq·g^-1 fresh weight for the two Isoetes populations. The seasonal pattern of the diurnal acid rhythm was correlated with temperature and light. The maximum activity occurred in midsummer, with negligible activity under ice cover. This pattern was similar to that of terrestrial CAM plants from non-arid environments. Comparison of CAM activity for populations of the same species indicates that the magnitude of CAM activity is closely related to plant productivity, and appears to be related to light and perhaps CO₂ availability. In these plants, CAM extends the diel period of carbon accumulation and contributes 40 to 50% of the annual carbon gain. The prolonged period of carbon acquisition and effective conservation of respired CO₂ via CAM is of paramount importance in the growth and productivity of these plants in oligotrophic environments.     

Recent developments in mesophyll conductance in C3, C4, and crassulacean acid metabolism plants

The conductance of carbon dioxide (CO₂) from the substomatal cavities to the initial sites of CO₂ fixation (g_m) can significantly reduce the availability of CO₂ for photosynthesis. There have been many recent reviews on: (i) the importance of g_m for accurately modelling net rates of CO₂ assimilation, (ii) on how leaf biochemical and anatomical factors influence g_m, (iii) the technical limitation of estimating g_m, which cannot be directly measured, and (iv) how g_m responds to long‐ and short‐term changes in growth and measurement environmental conditions. Therefore, this review will highlight these previous publications but will attempt not to repeat what has already been published. We will instead initially focus on the recent developments on the two‐resistance model of g_m that describe the potential of photorespiratory and respiratory CO₂ released within the mitochondria to diffuse directly into both the chloroplast and the cytosol. Subsequently, we summarize recent developments in the three‐dimensional (3‐D) reaction‐diffusion models and 3‐D image analysis that are providing new insights into how the complex structure and organization of the leaf influences g_m. Finally, because most of the reviews and literature on g_m have traditionally focused on C₃ plants we review in the final sections some of the recent developments, current understanding and measurement techniques of g_m in C₄ and crassulacean acid metabolism (CAM) plants. These plants have both specialized leaf anatomy and either a spatially or temporally separated CO₂ concentrating mechanisms (C₄ and CAM, respectively) that influence how we interpret and estimate g_m compared with a C₃ plants.     

Physiological potential for survival of propagules of crassulacean Acid metabolism species

Terminal stem joints from three opuntias were detached and maintained for 160 days under natural climatic conditions in the winter and summer. Neither Crassulacean acid metabolism (CAM) nor CAM-idling, as evidenced by a diurnal malate flux, was maintained throughout the two periods; ceasing earlier in the summer period. A 13 to 20% fresh weight loss occurred over the winter period, as opposed to a 30 to 40% loss over the summer period, although tissue water potentials remained above −1.5 megapascals. Chlorophyll and protein contents remained essentially constant in the winter but decreased in the summer. Starch content decreased slightly over the winter but more significantly over the summer. Mucilage content increased slightly in winter and declined slightly in summer. The initiation of rooting was found to be inversely related to spine density and dependent upon orientation and season. Comparison of these data suggest rooting coincided with the cessation of CAM-idling in both climatic periods and was uncoupled from the occurrence of precipitation. The physiological limit for survival of these propagules after detachment was lower than anticipated being of only a few months' duration.     

Stomatal regulation and water economy in crassulacean acid metabolism plants: an optimization model

It has been found that the stomatal behaviour of C₃ and C₄ plants can be predicted from the assumption that they increase transpiration whenever the increase δW in daily water loss is offset by a gain of at least λδW in carbon assimilation, where the minimum acceptable marginal conversion efficiency λ is determined by long term ecological factors, and is essentially constant within the course of a day. This paradigm is here extended to plants with Crassulacean acid metabolism (CAM). During daytime assimilation the results are the same as for C₃ and C₄ metabolisms. However night-time assimilation can be inhibited by the accumulation of malate in the cell vacuoles. In this event our model predicts that in a constant environment the stomatal conductance remains proportional to the square root of the mesophyll conductance as the latter declines. This is intermediate between keeping a constant stomatal conductance and keeping a constant intercellular CO₂ concentration (as tends to occur in the C₃/C₄ model when illumination varies), and results in an increasing intercellular CO₂ concentration towards the end of the night. This prediction accords with the Agave data of Nobel & Hartsock 1979. The model also predicts the allocation of time between C₃ and CAM for different degrees of water stress. The results agree qualitatively with observation, even though physiological changes (other than stomatal conductance) are not included in the model.