Crassulacean acid metabolism in Kalanchoë species collected in various climatic zones of Madagascar: a survey by δ13C analysis

Summary The carbon isotope compositions of samples of Kalanchoë species collected at the natural stands in Madagascar were determined. The results suggest that all species of the genus Kalanchoë are capable of crassulacean acid metabolism. The observed ¹³C values cover the whole range from –10 to –30. This high diversity of the ¹³C values was found among the species of the genus as well as, in certain cases, within a single species. This suggest that the CAM patterns in Kalanchoë are generally very flexible. The ¹³C values show a clear correlation with the climate of the habitats from where the samples derived. Values indicative of CO₂ fixation taking place exclusively during the night were found in the dry regions of Madagascar, whereas ¹³C values indicative of mixed CO₂ fixation during night and day or of CO₂ fixation entirely during the day are distributed in the humid zones.     

Leaf δ13C in Pinus resinosa trees and understory plants: variation associated with light and CO2 gradients

 Our objective was to evaluate the relative importance of gradients in light intensity and the isotopic composition of atmospheric CO₂ for variation in leaf carbon isotope ratios within a Pinus resinosa forest. In addition, we measured photosynthetic gas exchange and leaf carbon isotope ratios on four understory species (Dryopteris carthusiana, Epipactus helleborine, Hieracium floribundum, Rhamnus frangula), in order to estimate the consequence of the variation in the understory light microclimate for carbon gain in these plants. During midday, CO₂ concentration was relatively constant at vertical positions ranging from 15 m to 3 m above ground. Only at positions below 3 m was CO₂ concentration significantly elevated above that measured at 15 m. Based on the strong linear relationship between changes in CO₂ concentration and δ¹³C values for air samples collected during a diurnal cycle, we calculated the expected vertical profile for the carbon isotope ratio of atmospheric CO₂ within the forest. These calculations indicated that leaves at 3 m height and above were exposed to CO₂ of approximately the same isotopic composition during daylight periods. There was no significant difference between the daily mean δ¹³C values at 15 m (–7.77‰) and 3 m (–7.89‰), but atmospheric CO₂ was significantly depleted in ¹³C closer to the ground surface, with daily average δ¹³C values of –8.85‰ at 5 cm above ground. The light intensity gradient in the forest was substantial, with average photosynthetically active radiation (PAR) on the forest floor approximately 6% of that received at the top of the canopy. In contrast, there were only minor changes in air temperature, and so it is likely that the leaf-air vapour pressure difference was relatively constant from the top of the canopy to the forest floor. For red pine and elm tree samples, there was a significant correlation between leaf δ¹³C value and the height at which the leaf sample was collected. Leaf tissue sampled near the forest floor, on average, had lower δ¹³C values than samples collected near the top of the canopy. We suggest that the average light intensity gradient through the canopy was the major factor influencing vertical changes in tree leaf δ¹³C values. In addition, there was a wide range of variation (greater than 4‰) among the four understory plant species for average leaf δ¹³C values. Measurements of leaf gas exchange, under natural light conditions and with supplemental light, were used to estimate the influence of the light microclimate on the observed variation in leaf carbon isotope ratios in the understory plants. Our data suggest that one species, Epipactus helleborine, gained a substantial fraction of carbon during sunflecks. Received: 21 March 1996 / Accepted: 13 August 1996     

Vertical canopy gradients in δ13C correspond with leaf nitrogen content in a mixed-species conifer forest

Stable carbon isotope composition varies markedly between sun and shade leaves, with sun leaves being invariably more enriched (i.e., they contain more¹³C). Several hypotheses have emerged to explain this pattern, but controversy remains as to which mechanism is most general. We measured vertical gradients in stable carbon isotope composition (δ¹³C) in more than 200 trees of nine conifer species growing in mixed-species forests in the Northern Rocky Mountains, USA. For all species except western larch, δ¹³C decreased from top to bottom of the canopy. We found that δ¹³C was strongly correlated with nitrogen per unit leaf area (N _area), which is a measure of photosynthetic capacity. Usually weaker correlations were found between δ¹³C and leaf mass per area, nitrogen per unit leaf mass, height from the ground, or depth in the canopy, and these correlations were more variable between trees than for N _area. Gradients of δ¹³C (per meter canopy depth) were steeper in small trees than in tall trees, indicating that a recent explanation of δ¹³C gradients in terms of drought stress of upper canopy leaves is unlikely to apply in our study area. The strong relationship between N _area and δ¹³C here reported is consistent with the general finding that leaves or species with higher photosynthetic capacity tend to maintain lower CO₂ concentrations inside leaves. We conclude that photosynthetic capacity is a strong determinant of δ¹³C in vertical canopy profiles, and must be accounted for when interpreting δ¹³C values in conifer forests.     

Comparisons of δ13C values in leaves of aquatic macrophytes from different habitats in Britain and Finland; some implications for photosynthetic processes in aquatic plants

Summary The ¹³C values of submerged aquatic plants from contrasting but relatively defined habitats, and the ¹³C values of emergent, floating and submerged leaves of dimorphic aquatic plants, were measured. In many instances the ¹³C values of dissolved inorganic carbon in the water were also measured. Plant ¹³C values in the vicinity of-40 to-50 were found in rapidly flowing spring waters with carbonate ¹³C values of-16 to-21, consistent with the notion that species such as Fontinalis antipyretica almost exclusively assimilate free CO₂ via RuP₂ carboxylase. Plant ¹³C values in the vicinity of-10 to-15 in sluggish water with carbonate ¹³C values of about-5 were observed, consistent with the notion that boundary layer diffusion and/or HCO₃ ^- uptake may determine the ¹³C value of submerged aquatic plants in these circumstances. Comparisons of ¹³C values of the same or related species growing in waters of similar carbonate ¹³C value but different flow rates confirmed this view; more negative ¹³C values were frequently associated with plants in fast moving water. In Britain, but not in Finland, the ¹³C values of submerged leaves of dimorphic plants were almost invariably more negative than in aerial leaves. The ¹³C value of carbonate from chalk streams and in acid springs indicate substantial inputs of respiratory CO₂, as opposed to atmospheric carbon. The contributions of these variations in ¹³C of the carbon source, and of isotope fractionation in diffusion, to the ¹³C value of submerged parts of dimorphic plants is discussed.     

Are polyamines involved in the induction and regulation of the Crassulacean acid metabolism?

Leaves of plants with Crassulacean acid metabolism (CAM) were analyzed for variation in the content of polyamines in connection with the metabolism of malic acid in the dark and in the light, and with the induction of full-CAM activity. Under conditions (long days) resulting in extremely low CAM activity, young leaves of K. blossfeldiana have very low content in the polyamine-precursor arginine and in putrescine. The content in these two substances was increased dramatically by full-CAM induction with short days. During the course of the night/day cycle two peaks of putrescine content were observed in leaves of Kalanchoe blossfeldiana Poelln. Tom Thumb performing full-CAM operation: a large increase occurs toward the end of the day and the first half of the night, and its kinetics corresponds to the increase in the rate of malic acid synthesis; another peak, very sharp, appears during the first hours of the day, concomitant with the time of release of malic acid from the vacuole into the cytoplasm. In the case of Bryophyllum daigremontianum Berger similar variations were observed for the content in spermidine. These results support the hypothesis that polyamines could be involved in countering the tendency toward acidification of the cytoplasm at those moments of CAM operation at which the local concentration of malic acid is increased (i.e., during active synthesis in the dark and during the efflux from the vacuole in the light).Abbreviation CAM Crassulacean acid metabolism     

Climatic implications of δ13C and δ18O ratios from C3 and C4 plants growing in a tropical montane habitat in southern India

The stable carbon and oxygen isotope ratios in cellulose of C3 and C4 plants growing on the surface of a montane peat bog in the Nilgiri hills, southern India, were measured. The mean monthly δ¹³C values in cellulose of both C3 and C4 plants are found to be significantly related to rainfall, while the δ¹⁸O values are sensitive to changes in maximum temperature and relative humidity of the region. Further, higher δ¹⁸O values were observed in C4 plants compared to C3 plants, suggesting that C4 plants are probably less sensitive to relative humidity as compared to C3 plants and are able to photosynthesize even during drier conditions. The plant isotope-climate correlations thus established can be used for reconstructing the past temperature and rainfall conditions of the tropics from the isotopic ratios of peat deposits, derived from a mixture of C3 and C4 plants in the region.     

Contribution of C3 carboxylation to the circadian rhythm of carbon dioxide uptake in a Crassulacean acid metabolism plant Kalanchoë daigremontiana

During the endogenous circadian rhythm of carbon dioxide uptake in continuous light by a Crassula cean acid metabolism plant, Kalancho? daigremontiana, the two carboxylating enzymes, phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco), are active simultaneously, although, until now, only the role of PEPC in generating the rhythm has been acknowledged. According to the established model, the rhythm is primarily regulated at the PEPC activity level, modulated by periodic compartmentation of its inhibitor, malate, in the vacuole and controlled by tension/relaxation of the tonoplast. However, the circadian accumulation of malic acid (the main indicator of PEPC activity) dampened significantly within the first few periods without affecting the rhythm's amplitude. Moreover, the amount of malate accumulated during a free-running oscillation was several-fold lower than the amount expected if PEPC were the key carboxylating enzyme, based on a 1:1 stoichiometry of CO(2) and malate. Together with the observation that rates of CO(2) uptake under continuous light were higher than in darkness, the evidence shows that C(3) carboxylation greatly contributes to the generation of rhythmic CO(2) uptake in continuous light in this 'obligate' CAM plant. Because the shift from predominantly CAM to predominantly C(3) carboxylation is smooth and does not distort the trajectory of the rhythm, its control probably arises from a robust network of oscillators, perhaps also involving stomata.     

Fractionation and turnover of stable carbon isotopes in animal tissues: Implications for δ13C analysis of diet

The use of stable carbon isotopes as a means of studying energy flow is increasing in ecology and paleoecology. However, secondary fractionation and turnover of stable isotopes in animals are poorly understood processes. This study shows that tissues of the gerbil (Meriones unguienlatus) have different δ¹³C values when equilibrated on corn (C₄) or wheat (C₃) diets with constant ¹³C/¹²C contents. Lipids were depleted 3.0‰ and hair was enriched 1.0‰ relative to the C₄ diet. Tissue δ¹³C values were ranked hair>brain>muscle>liver>fat. After changing the gerbils to a wheat (C₃) diet, isotope ratios of the tissues shifted in the direction of the δ¹³C value of the new diet. The rate at which carbon derived from the corn diet was replaced by carbon derived from the wheat diet was adequately described by a negative exponential decay model for all tissues examined. More metabolically active tissues such as liver and fat had more rapid turnover rates than less metabolically active tissues such as hair. The half-life for carbon ranged from 6.4 days in liver to 47.5 days in hair. The results of this study have important implications for the use of δ¹³C values as indicators of animal diet. Both fractionation and turnover of stable carbon isotopes in animal tissues may obscure the relative contributions of isotopically distinct dietary components (such as C₃ vs. C₄, or marine vs. terrestrial) if an animal's diet varies through time. These complications deserve attention in any study using stable isotope ratios of animal tissue as dietary indicators and might be minimized by analysis of several tissues or products covering a range of turnover times.     

Effect of tranexamic acid and δ-aminovaleric acid on lipoprotein(a) metabolism in transgenic mice

The assembly of lipoprotein(a) (Lp(a)) is a two-step process which involves the interaction of kringle-4 (K-IV) domains in apolipoprotein(a) (apo(a)) with Lys groups in apoB-100. Lys analogues such as tranexamic acid (TXA) or δ-aminovaleric acid (δ-AVA) proved to prevent the Lp(a) assembly in vitro. In order to study the in vivo effect of Lys analogues, transgenic apo(a) or Lp(a) mice were treated with TXA or δ-AVA and plasma levels of free and low density lipoprotein bound apo(a) were measured. In parallel experiments, McA-RH 7777 cells, stably transfected with apo(a), were also treated with these substances and apo(a) secretion was followed. Treatment of transgenic mice with Lys analogues caused a doubling of plasma Lp(a) levels, while the ratio of free:apoB-100 bound apo(a) remained unchanged. In transgenic apo(a) mice a 1.5-fold increase in plasma apo(a) levels was noticed. TXA significantly increased Lp(a) half-life from 6 h to 8 h. Incubation of McA-RH 7777 cells with Lys analogues resulted in an up to 1.4-fold increase in apo(a) in the medium. The amount of intracellular low molecular weight apo(a) precursor remained unchanged. We hypothesize that Lys analogues increase plasma Lp(a) levels by increasing the dissociation of cell bound apo(a) in combination with reducing Lp(a) catabolism.     

Patterns of δ^13C and δ^15N in wolverine Gulo gulo tissues from the Brooks Range,Alaska

Knowledge of carnivore diets is essential to understand how carnivore populations respond demographically to variations in prey abundance. Analysis of stable isotopes is a useful complement to traditional methods of analyzing carnivore diets. We used data on δ^13C and δ^15N in wolverine tissues to investigate patterns of seasonal and annual diet variation in a wolverine Gulo gulo population in the western Brooks Range, Alaska, USA. The stable isotope ratios in wolverine tissues generally reflected that of terrestrial carnivores, corroborating previous diet studies on wolverines. We also found variation in δ^13C and δ^15N both between muscle samples collected over several years and between tissues with different assimilation rates, even after correcting for isotopic fractionation. This suggests both annual and seasonal diet variation. Our results indicate that data on δ^13C and δ^15N holds promise for qualitative assessments of wolverine diet changes over time. Such temporal variation may be important indicators of ecological responses to environmental perturbations, and we suggest that more refined studies of stable isotopes may be an important tool when studying temporal change in diets of wolverines and similar carnivores [ Current Zoology 55 （3）： 188- 192, 2009].     

Differences in δ15N and δ13C between embryonic and maternal tissues of the ovoviviparous bluntnose sixgill shark Hexanchus griseus

Stable nitrogen (δ¹⁵N) and carbon (δ¹³C) isotope ratios from muscle, liver and yolk were analysed from the mother and embryos of an ovoviviparous shark, Hexanchus griseus. Embryonic liver and muscle had similar δ¹⁵N and δ¹³C ratios or were depleted in heavy isotopes, compared to the same maternal somatic and reproductive yolk tissues, but no relationship existed between δ¹⁵N or δ¹³C and embryo length, as expected, because a switch to placental nourishment is lacking in this species. This study expands the understanding of maternal nourishment and embryonic stable isotope differences in ovoviviparous sharks.     

Uptake of organic matter by the roots of sweet potato: Analysis of the δ14C value of plants

Summary To determine whether sweet potato (Ipomoea batatas (L.) Poir.) takes up organic matter through the roots from the medium, the concentrations of natural¹⁴C (¹⁴C) in plant organic matter, atmospheric CO₂ and compost applied to media were examined under soil and sand culture conditions. In these experiments, three kinds of composts of different ¹⁴C were used. CO₂ derived from the mineralization of compost was continuously pumped out from the pots and its direct uptake by the leaves was prevented.¹⁴C of plant parts harvested after the 43 days experimental period were affected by the ¹⁴C of the compost in the treatments where the compost of rice straw was applied, and which suggested that a significant amount of plant carbon was derived from the compost.     

Cadmium induced oxidative stress in soybean plants also by the accumulation of δ-aminolevulinic acid

Cadmium toxicity has been extensively studied in plants, however its biochemical mechanism of action has not yet been well established. To fulfil this objective, four-weeks-old soybean nodulated plants were treated with 200 μM Cd²⁺ for 48 h. δ-aminolevulinic acid dehydratase (ALA-D, E.C. 4.2.1.24) activity and protein expression, as well as δ-aminolevulinic acid (ALA) and porphobilinogen (PBG) concentrations were determined in nodules, roots and leaves. In vitro experiments carried out in leaves were performed using leaf discs to evaluate the oxidant and antioxidant properties of ALA and S-adenosyl-l-methinone (SAM), respectively. Oxidative stress parameters such as thiobarbituric acid reactive substances (TBARS) and GSH levels as well as superoxide dismutase (SOD, E.C. 1.15.1.1), and guaiacol peroxidase (GPOX, E.C. 1.11.1.7) were also determined. Cadmium treatment caused 100% inhibition of ALA-D activity in roots and leaves, and 72% inhibition in nodules whereas protein expression remained unaltered in the three studied tissues. Plants accumulated ALA in nodules (46%), roots (2.5-fold) and leaves (104%), respect to controls. From in vitro experiments using leaf discs, exposed to ALA or Cd²⁺, it was found that TBARS levels were enhanced, while GSH content and SOD and GPOX activities and expressions were diminished. The protective role of SAM against oxidative stress generated by Cd²⁺ and ALA was also demonstrated. Data presented in this paper let us to suggest that accumulation of ALA in nodules, roots and leaves of soybean plants due to treatment with Cd²⁺ is highly responsible for oxidative stress generation in these tissues.     

Stratification of δ13C values of leaves in Amazonian rain forests

Summary The contribution of soil respiration to the photosynthesis of the shade flora in the Amazon forest was evaluated by measuring the ¹³C values of leaves collected at different levels in two forest communities. Canopy leaves have an average ¹³C of-30.5 in the podsol forest and-28.7 in the laterite forest. Leaves from plants in the lower forest strata have a significantly lower value of-35.2 in the podsol forest and-34.3 in the laterite forest.Mailing address of the first author: Before May 31, 1980: Department of Biological Sciences, Stanford University, Stanford, California 94305 USA. After May 31: Centro de Ecologia, IVIC Aptdo. 1827. Caracas, Venezuela     

Biosynthesis of δ-aminolevulinic acid in Rhodopseudomonas sphaeroides

The biosynthesis of δ-aminolevulinic acid was investigated in three strains of Rhodopseudomonas sphaeroides. A wild-type strain (NCIB 8253) possessed both δ-aminolevulinic acid synthetase and γ,δ-dioxovaleric acid transaminase in the cytoplasmic and membrane cell fractions. δ-Aminolevulinic acid synthetase activities were not detected in extracts of mutant strains H5 and H5D. However, γ,δ-dioxovaleric acid transaminase was found in the cytoplasmic and membrane fractions of these latter two strains. Strain H5 required exogenously added δ-aminolevulinic acid for growth and bacteriochlorophyll synthesis. Strain H5D did not require this compound for growth and bacteriochlorophyll synthesis. γ,δ-Dioxovaleric acid added in the growth medium did not support the growth of H5, although it was actively transported into the cells. Addition of γ,δ-dioxovaleric acid to the growth medium did not enhance the growth of either the wild-type or H5D strains. These results indicate that ALA synthetase is not required for growth and bacteriochlorophyll synthesis in H5D and that γ,δ-dioxovaleric acid is probably not an intermediate in the formation of δ-aminolevulinic acid in the strains of Rhodopseudomonas sphaeroides studied. In strain H5D another pathway may function in the formation of δ-aminolevulinic acid other than that catalyzed by δ-aminolevulinic acid synthetase or γ,δ-dioxovaleric acid transaminase.     

Environment or development? Lifetime net CO2 exchange and control of the expression of Crassulacean acid metabolism in Mesembryanthemum crystallinum

The relative influence of plant age and environmental stress signals in triggering a shift from C(3) photosynthesis to Crassulacean acid metabolism (CAM) in the annual halophytic C(3)-CAM species Mesembryanthemum crystallinum was explored by continuously monitoring net CO(2) exchange of whole shoots from the seedling stage until seed set. Plants exposed to high salinity (400 mm NaCl) in hydroponic culture solution or grown in saline-droughted soil acquired between 11% and 24% of their carbon via net dark CO(2) uptake involving CAM. In contrast, plants grown under nonsaline, well-watered conditions were capable of completing their life cycle by operating in the C(3) mode without ever exhibiting net CO(2) uptake at night. These observations are not consistent with the widely expressed view that the induction of CAM by high salinity in M. crystallinum represents an acceleration of preprogrammed developmental processes. Rather, our study demonstrates that the induction of the CAM pathway for carbon acquisition in M. crystallinum is under environmental control.     

How closely do the delta(13)C values of Crassulacean Acid metabolism plants reflect the proportion of CO(2) fixed during day and night?

The extent to which Crassulacean acid metabolism (CAM) plant delta(13)C values provide an index of the proportions of CO(2) fixed during daytime and nighttime was assessed. Shoots of seven CAM species (Aloe vera, Hylocereus monocanthus, Kalanchoe beharensis, Kalanchoe daigremontiana, Kalanchoe pinnata, Vanilla pauciflora, and Xerosicyos danguyi) and two C(3) species (teak [Tectona grandis] and Clusia sp.) were grown in a cuvette, and net CO(2) exchange was monitored for up to 51 d. In species exhibiting net dark CO(2) fixation, between 14% and 73.3% of the carbon gain occurred in the dark. delta(13)C values of tissues formed inside the cuvette ranged between -28.7 per thousand and -11.6 per thousand, and correlated linearly with the percentages of carbon gained in the light and in the dark. The delta(13)C values for new biomass obtained solely during the dark and light were estimated as -8.7 per thousand and -26.9 per thousand, respectively. For each 10% contribution of dark CO(2) fixation integrated over the entire experiment, the delta(13)C content of the tissue was, thus, approximately 1.8 per thousand less negative. Extrapolation of the observations to plants previously surveyed under natural conditions suggests that the most commonly expressed version of CAM in the field, "the typical CAM plant," involves plants that gain about 71% to 77% of their carbon by dark fixation, and that the isotopic signals of plants that obtain one-third or less of their carbon in the dark may be confused with C(3) plants when identified on the basis of carbon isotope content alone.     

Variation of bone collagen amino acid δ13C values in archaeological humans and fauna with different dietary regimes: developing frameworks of dietary discrimination

We present bone collagen amino acid (AA) δ(13)C values for a range of archaeological samples representing four "benchmark" human diet groups (high marine protein consumers, high freshwater protein consumers, terrestrial C(3) consumers, and terrestrial C(4) consumers), a human population with an "unknown" diet, and ruminants. The aim is to establish an interpretive palaeodietary framework for bone collagen AA δ(13)C values, and to assess the extent to which AA δ(13)C values can provide additional dietary information to bulk collagen stable isotope analysis. Results are analyzed to determine the ability of those AAs for which we have a complete set, to discriminate between the diet groups. We show that very strong statistical discrimination is obtained for all interdiet group comparisons. This is often obvious from suitably chosen bivariate plots using δ(113)C values that have been normalized to compensate for interdiet group differences in bulk δ(13)C values. Bi-plots of non-normalized phenylalanine and valine δ(13)C values are useful for distinguishing aquatic diets (marine and freshwater) from terrestrial diets. Our interpretive framework uses multivariate statistics (e.g., discriminant analysis) to optimize the separation of the AA δ(13)C values of the "benchmark"' diet groups, and is capable of accurately assigning external samples to their expected diet groups. With a growing body of AA δ(13)C values, this method is likely to enhance palaeodietary research by allowing the "unknown" diets of populations under investigation to be statistically defined relative to the well-characterized or "known" diets of previously investigated populations.     

Reliability of δ13C and δ15N in faeces for reconstructing savanna herbivore diet

We tested the reliability of herbivore faecal δ¹³C and δ¹⁵N values for reconstructing diet through review of an extensive database derived from a 3-year study of ungulates in South Africa's Kruger National Park. Faeces are a useful material for stable isotope studies of diet because they record dietary turnover at very short time scales, and because sampling is non-invasive. However, the validity of faecal isotope proxies may be questioned because they represent only undigested food remains. Results from Kruger Park confirm that free-ranging browsers have faecal δ¹³C consistent with C₃ feeding, grazer faeces are C₄, and mixed-feeder faeces intermediate. Although the respective ranges do not overlap, there is significant variation in faecal δ¹³C of browsers and grazers (～2.0–4.0‰) across space and through time. We demonstrate that most (～70%) of this variation can be ascribed to corresponding patterns of variation in the δ¹³C of C₃ and C₄ plants, respectively, re-enforcing the fidelity of faecal isotope proxies for diet but highlighting a need for mixing models that control for variations in plant δ¹³C in order to achieve accurate diet reconstructions. Predictions for the effects of climate (rainfall) and ecophysiology on ¹⁵N-abundance variations in mammals do not persist in faeces. Rather, faecal δ¹⁵N tracks changes in plant δ¹⁵N, with further fractionation occurring primarily due to variations in dietary protein (reflected by %N). Controlling for these effects, we show that a dual-isotope multiple source mixing model (Isosource) can extend diet reconstructions for African savanna herbivores beyond simplified C₃/C₄ distinctions, although further understanding of variations in mammal δ¹⁵N are needed for greater confidence in this approach.