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.     

Carbon isotope ratios of crassulacean acid metabolism species in relation to climate and phytosociology

Summary The ¹³C, values of the two principal, widely distributed CAM species in Big Bend National Park, Texas, were examined and related to phytosociologic and macroclimatic parameters. Samples of Agave lecheguilla and Opuntia engelmannii were collected along an elevational gradient covering 1370 m, and values for both total and insoluble fractions were measured. Linear regressions were used to correlate changes in values with changes in community importance and mean annual precipitation for both species to determine whether or not they became increasingly C₃-like in their photosynthetic mode at more mesic, upper elevations. In addition, values were measured for other Agave and Opuntia species from different locations and for different seasons and for A. lecheguilla following a short laboratory experiment. Results show that there are very small changes in values for both species along the pronounced temperature and precipitation gradients associated with elevational change. The ranges in values for O. engelmannii are only 1.5 (n=7) and 1.2 (n=6) for total and insoluble fractions respectively. The ranges in values for A. lecheguilla are 2.6 (n=9) and 3.1 (n=9). The results for other species confirm this low variability. We conclude that the species examined are obligate CAM species and that wide elevational distributions are not due to shifts in photosynthetic mode.     

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.     

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.     

Variation in the carbon isotope composition of a plant with crassulacean Acid metabolism

The content of ¹³C varies in plants with Crassulacean acid metabolism. Differences up to 3.5‰ in the ¹³C/¹²C ratios were observed between leaves of different age in the same plant of Bryophyllum daigremontianum. Soluble and insoluble carbon in the same leaf differed up to 8‰, the largest difference occurring in the leaves with the highest Crassulacean acid metabolism activity. Models to account for the isotope discrimination by C₃, C₄, and Crassulacean acid metabolism plants are proposed.     

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.     

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.

     

Carbon isotope ratios of central Mexican Crassulaceae in natural and greenhouse environments

Summary Measurements of carbon isotope ratios of central Mexican Crassulaceae collected over a broad habitat range show consistent patterns of CAM activity with no indications of substantial flexible photosynthetic pathways between C₃ and CAM. The three genera studied — Echeveria, Pachyphytum, and Graptopetalum — are all closely related to Dudleya in which considerable flexible metabolic response has been demonstrated. Comparative measurements of carbon isotope ratios for field collected and greenhouse reared samples of the same taxa showed a uniform occurrence of slightly more negative ¹³C values, but no indication of substantial flexible metabolic response.     

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.     

Electrophoretic analysis of protoplast, vacuole, and tonoplast vesicle proteins in crassulacean Acid metabolism plants

Protoplasts and vacuoles were isolated and purified in large numbers from the CAM plants Ananas comosus (pineapple) and Sedum telephium for protein characterization. Vacuoles were further fractionated to yield a tonoplast vesicle preparation. Polypeptides of protoplasts, vacuoles, and tonoplast vesicles were compared to whole leaf polypeptides from both plants by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis. Approximately 100 vacuole polypeptides could be resolved of which 25 to 30% were enriched in the tonoplast vesicles. The proteins of protoplasts, vacuoles, and tonoplast vesicles from A. comosus were analyzed further by two-dimensional gel electrophoresis. When one-dimensional electrophoretograms of A. comosus polypeptides were stained with a glycoprotein-specific periodic acid Schiff stain, very few polypeptides appeared to be glycosylated, whereas a large number of glycosylated polypeptides were detected with a silver-based glycoprotein stain particularly in tonoplast vesicles. Analysis of the enzymic content of vacuoles from both plants indicated the presence of a variety of hydrolases, including bromelain as a major constituent of A. comosus. No substrate-specific ATPase, however, could be detected in vacuoles or tonoplast vesicles from either plant.     

Carbon, hydrogen, and oxygen isotope ratios of cellulose from plants having intermediary photosynthetic modes

Carbon and hydrogen isotope ratios of cellulose nitrate and oxygen isotope ratios of cellulose from species of greenhouse plants having different photosynthetic modes were determined. When hydrogen isotope ratios are plotted against carbon isotope ratios, four clusters of points are discernible, each representing different photosynthetic modes: C₃ plants, C₄ plants, CAM plants, and C₃ plants that can shift to CAM or show the phenomenon referred to as CAM-cycling. The combination of oxygen and carbon isotope ratios does not distinguish among the different photosynthetic modes. Analysis of the carbon and hydrogen isotope ratios of cellulose nitrate should prove useful for screening different photosynthetic modes in field specimens that grew near one another. This method will be particularly useful for detection of plants which show CAM-cycling.     

Carbon isotope fractionation in plants

Plants with the C₃, C₄, and crassulacean acid metabolism (CAM) photosynthetic pathways show characteristically different discriminations against ¹³C during photosynthesis. For each photosynthetic type, no more than slight variations are observed within or among species. CAM plants show large variations in isotope fractionation with temperature, but other plants do not. Different plant organs, subcellular fractions and metabolises can show widely varying isotopic compositions. The isotopic composition of respired carbon is often different from that of plant carbon, but it is not currently possible to describe this effect in detail. The principal components which will affect the overall isotope discrimination during photosynthesis are diffusion of CO₂, interconversion of CO₂ and HCO^?₃, incorporation of CO₂ by phosphoenolpyruvate carboxylase or ribulose bisphosphate carboxylase, and respiration. Theisotope fractionations associated with these processes are summarized. Mathematical models are presented which permit prediction of the overall isotope discrimination in terms of these components. These models also permit a correlation of isotope fractionations with internal CO₂ concentrations. Analysis of existing data in terms of these models reveals that CO₂ incorporation in C₃ plants is limited principally by ribulose bisphosphate carboxylase, but CO₂ diffusion also contributes. In C₄ plants, carbon fixation is principally limited by the rate of CO₂ diffusion into the leaf. There is probably a small fractionation in C₄ plants due to ribulose bisphosphate carboxylase.     

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     

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.     

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.     

Leaf carbon isotope ratios of plants from a subtropical monsoon forest

Summary Carbon isotope ratios were used to survey the distribution of photosynthetic pathways among taxa, the relationship between photosynthetic pathway and habitat light levels, and the relationship between intercellular CO₂ levels of C₃ plants and habitat light levels within a subtropical monsoon forest in southern China. Of 128 species, most (94) possessed the C₃ photosynthetic pathway; 33 species possessed the C₄ pathway and all of these were restricted to high light locations. There was one epiphytic CAM species. The C₃ species were classified as occurring in open, intermediate, and closed canopy sites. Among C₃ species, carbon isotope ratios tended to become more negative with decreasing light availability in the habitat.C.I.W.D.P.B. Pub no 931     

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.