Temperature Dependence of Carbon Isotope Fractionation in CAM Plants

The carbon isotope fractionation associated with nocturnal malic acid synthesis in Kalanchoë daigremontiana and Bryophyllum tubiflorum was calculated from the isotopic composition of carbon-4 of malic acid, after appropriate corrections. In the lowest temperature treatment (17°C nights, 23°C days), the isotope fractionation for both plants is −4‰ (that is, malate is enriched in ¹³C relative to the atmosphere). For K. daigremontiana, the isotope fractionation decreases with increasing temperature, becoming approximately 0‰ at 27°C/33°C. Detailed analysis of temperature effects on the isotope fractionation indicates that stomatal aperture decreases with increasing temperature and carboxylation capacity increases. For B. tubiflorum, the temperature dependence of the isotope fractionation is smaller and is principally attributed to the normal temperature dependences of the rates of diffusion and carboxylation steps. The small change in the isotopic composition of remaining malic acid in both species which is observed during deacidification indicates that malate release, rather than decarboxylation, is rate limiting in the deacidification process.     

Effects of Relative Humidity on Carbon Isotope Fractionation in Plants

Four C₃ plants and a C₄ plant were grown from seeds at four levels (30, 45, 60, and 75 %) of relative humidity. All plants were subjected to a 16 h day, at 500 μE/m².s^?1 photon flux density. Mature leaves were analyzed for their carbon isotopic composition. Isotope fractionation decreased by up to 3 ‰ with decreasing relative humidity in all C₃ plants, while the opposite trend was observed in the C₄ plant. The observed shifts in both C₃ and C₄ plants are attributed to decreased stomatal conductance at low relative humidity, resulting in a smaller P_i.     

Dark Fixation of CO(2) by Crassulacean Plants: Evidence for a Single Carboxylation Step

Malic acid isolated from Bryophyllum pinnatum (Lamk.) Oken (B. calycinum Salisb.), Bryophyllum tubiflorum Harv., Kalanchoë diagremontiana Hamet et Perrier and Sedum guatamalense Hemsl. after dark ¹⁴CO₂ fixation was degraded by an in vitro NADP-malic enzyme technique. In the short term (5 to 30 seconds) the malic acid was almost exclusively labeled in the C-4 carboxyl carbon (greater than 90%). The percentage of ¹⁴C in the C-4 carboxyl of malic acid declined slowly with time, reaching 70% in B. tubiflorum and 54% in B. pinnatum after 14 hours of exposure to ¹⁴CO₂. It was found that malic acid-adapted Lactobacillus arabinosus may seriously underestimate the C-4 carboxyl component of label in malic acid-¹⁴C. The amount of substrate which the bacteria can completely metabolize was easily exceeded; there was a significant level of randomization of label even when β-decarboxylation proceeded to completion, and in extended incubation periods, more than 25% of label was removed from malic acid-U-¹⁴C. The significance of these findings in relation to pathways of carbohydrate metabolism and malic acid synthesis in Crassulacean acid metabolism is discussed.     

Carbon Isotope Fractionation of 11 Acetogenic Strains Grown on H2 and CO2

Acetogenic bacteria are able to grow autotrophically on hydrogen and carbon dioxide by using the acetyl coenzyme A (acetyl-CoA) pathway. Acetate is the end product of this reaction. In contrast to the fermentative route of acetate production, which shows almost no fractionation of carbon isotopes, the acetyl-CoA pathway has been reported to exhibit a preference for light carbon. In Acetobacterium woodii the isotope fractionation factor (ε) for ¹³C and ¹²C has previously been reported to be ε = −58.6‰. To investigate whether such a strong fractionation is a general feature of acetogenic bacteria, we measured the stable carbon isotope fractionation factor of 10 acetogenic strains grown on H₂ and CO₂. The average fractionation factor was ε_TIC = −57.2‰ for utilization of total inorganic carbon and ε_acetate = −54.6‰ for the production of acetate. The strongest fractionation was found for Sporomusa sphaeroides (ε_TIC = −68.3‰), the lowest fractionation for Morella thermoacetica (ε_TIC = −38.2‰). To investigate the reproducibility of our measurements, we determined the fractionation factor of 21 biological replicates of Thermoanaerobacter kivui. In general, our study confirmed the strong fractionation of stable carbon during chemolithotrophic acetate formation in acetogenic bacteria. However, the specific characteristics of the bacterial strain, as well as the cultural conditions, may have a moderate influence on the overall fractionation.     

Dark CO(2) Fixation in Gladiolus Cormels and Its Regulation during the Break of Dormancy

The increase in dark CO₂ fixation during cold storage of Gladiolus x gandavensis van Houtte-type grandiflorus cormels is used to monitor changes in their state of dormancy. Dark fixation is also promoted by benzyladenine, which breaks cormel dormancy, and is inhibited by abscisic acid and gibberellin A₃, which inhibit cormel germination. The rate of dark fixation by nondormant cormels is five times higher than that in dormant ones. Dark fixation is not due to microorganisms. It is temperature-dependent and can be measured stoichiometrically in vivo. The apex and base of the cormels accumulate more label than the central part. Dark fixation of both dormant and nondormant cormels is also promoted by imbibition in water. The fate of the labeled assimilates was followed by ion exchange chromatography.     

Isotope Fractionation in Photosynthetic Bacteria during Carbon Dioxide Assimilation

The δ _PDB¹³C values have been determined for the cellular constituents and metabolic intermediates of autotrophically grown Chromatium vinosum. The isotopic composition of the HCO₃^- in the medium and the carbon isotopic composition of the bacterial cells change with the growth of the culture. The δ _PDB¹³C value of the HCO₃^- in the media changes from an initial value of −6.6‰ to +8.1‰ after 10 days of bacterial growth and the δ _PDB¹³C value of the bacterial cells change from −37.5‰ to −29.2‰ in the same period. The amount of carbon isotope fractionation during the synthesis of hexoses by the photoassimilation of CO₂ has a range of −15.5‰ at time zero to −22.0‰ after 10 days. This range of fractionation compares to the range of carbon isotope fractionation for the synthesis of sugars from CO₂ by ribulose 1,5-diphosphate carboxylase and the Calvin cycle.     

Carbon Isotopic Fractionation Does Not Occur during Dark Respiration in C3 and C4 Plants

The magnitude of possible carbon isotopic fractionation during dark respiration was investigated with isolated mesophyll cells from mature leaves of common bean (Phaseolus vulgaris L.), a C3 plant, and corn (Zea mays L.), a C4 plant. Mesophyll protoplasts were extracted from greenhouse-grown leaves and incubated in culture solutions containing different carbohydrate substrates (fructose, glucose, and sucrose) with known [delta]13C values. The CO2 produced by protoplasts after incubation in the dark was collected, purified, and analyzed for its carbon isotope ratio. From observations of the isotope ratios of the substrate and respired CO2, we calculated the carbon isotope discrimination associated with metabolism of each of these substrates. In eight of the 10 treatment combinations, the carbon isotope ratio discrimination was not significantly different from 0. In the remaining two treatment combinations, the carbon isotope ratio discrimination was 1[per mille (thousand) sign]. From these results, we conclude that there is no significant carbon isotopic discrimination during mitochondrial dark respiration when fructase, glucose, or sucrose are used as respiratory substrates.     

Internal CO(2) Supply during Photosynthesis of Sun and Shade Grown CAM Plants in Relation to Photoinhibition

Leaves of Kalanchoë pinnata were exposed in the dark to air (allowing the fixation of CO₂ into malic acid) or 2% O₂, 0% CO₂ (preventing malic acid accumulation). They were then exposed to bright light in the presence or absence of external CO₂ and light dependent inhibition of photosynthetic properties assessed by changes in 77 K fluorescence from photosystem II (PSII), light response curves and quantum yields of O₂ exchange, rates of electron transport from H₂O through Q_B (secondary electron acceptor from the PSII reaction center) in isolated thylakoids, and numbers of functional PSII centers in intact leaf discs. Sun leaves of K. pinnata experienced greater photoinhibition when exposed to high light in the absence of CO₂ if malic acid accumulation had been prevented during the previous dark period. Shade leaves experienced a high degree of photoinhibition when exposed to high light regardless of whether malic acid had been allowed to accumulate in the previous dark period or not. Quantum yields were depressed to a greater degree than was 77 K fluorescence from PSII following photoinhibition.     

Anapleurotic CO(2) Fixation by Phosphoenolpyruvate Carboxylase in C(3) Plants

The role of phosphoenolpyruvate carboxylase in photosynthesis in the C₃ plant Nicotiana tabacum has been probed by measurement of the ¹³C content of various materials. Whole leaf and purified ribulose bisphosphate carboxylase are within the range expected for C₃ plants. Aspartic acid purified following acid hydrolysis of this ribulose bisphosphate carboxylase is enriched in ¹³C compared to whole protein. Carbons 1-3 of this aspartic acid are in the normal C₃ range, but carbon-4 (obtained by treatment of the aspartic acid with aspartate β-decarboxylase) has an isotopic composition in the range expected for products of C₄ photosynthesis (−5‰), and it appears that more than half of the aspartic acid is synthesized by phosphoenolpyruvate carboxylase using atmospheric CO₂/HCO₃⁻. Thus, a primary role of phosphoenolpyruvate carboxylase in C₃ plants appears to be the anapleurotic synthesis of four-carbon acids.     

Products of Dark CO(2) Fixation in Pea Root Nodules Support Bacteroid Metabolism

Products of the nodule cytosol in vivo dark [¹⁴C]CO₂ fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv “Bodil”) nodules. The distribution of the metabolites of the dark CO₂ fixation products was compared in effective (fix⁺) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix⁻) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The ¹⁴C incorporation from [¹⁴C]CO₂ was about threefold greater in the wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the ¹⁴C label in the cytosol was found in organic acids in both symbioses. Malate comprised about half of the total cytosol organic acid content on a molar basis, and more than 70% of the cytosol radioactivity in the organic acid fraction was detected in malate in both symbioses. Most of the remaining ¹⁴C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the ¹⁴C label found in the amino acids of the cytosol was incorporated in aspartate, which on a molar basis comprised only about 1% of the total amino acid pool in the cytosol. The extensive ¹⁴C labeling of malate and aspartate from nodule dark [¹⁴C]CO₂ fixation is consistent with the role of phosphoenolpyruvate carboxlase in nodule dark CO₂ fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more ¹⁴C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated ¹⁴C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained ¹⁴C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid ¹⁴C label was detected in malate and aspartate than their corresponding proportions of the organic acids and amino acids on a molar basis. The proportion of ¹⁴C label in succinate, 2-oxogultarate, citrate, and fumarate in the bacteroids of the wild type greatly exceeded that of the dicarboxylate uptake mutant. The results indicate a central role for nodule cytosol dark CO₂ fixation in the supply of the bacteroids with dicarboxylic acids.     

Enhanced Dark CO(2) Fixation by Preilluminated Chlorella pyrenoidosa and Anacystis nidulans

The products of short time photosynthesis and of enhanced dark ¹⁴CO₂ fixation (illumination in helium prior to addition of ¹⁴CO₂ in dark) by Chlorella pyrenoidosa and Anacystis nidulans were compared. Glycerate 3-phosphate, phosphoenolpyruvate, alanine, and aspartate accounted for the bulk of the ¹⁴C assimilated during enhanced dark fixation while hexose and pentose phosphates accounted for the largest fraction of isotope assimilated during photosynthesis. During the enhanced dark fixation period, glycerate 3-phosphate is carboxyl labeled and glucose 6-phosphate is predominantly labeled in carbon atom 4 with lesser amounts in the upper half of the C₆ chain and traces in carbon atoms 5 and 6. Tracer spread throughout all the carbon atoms of photosynthetically synthesized glycerate 3-phosphate and glucose 6-phosphate. During the enhanced dark fixation period, there was a slow formation of sugar phosphates which subsequently continued at 5 times the initial rate long after the cessation of ¹⁴CO₂ uptake. To explain the kinetics of changes in the labelling patterns and in the limited formation of the sugar phosphates during enhanced dark CO₂ fixation, the suggestion is made that most of the reductant mediating these effects did not have its origin in the preillumination phase.

It is concluded that a complete photosynthetic carbon reduction cycle operates to a limited extent, if at all, in the dark period subsequent to preillumination.

     

Dark CO(2) Fixation and its Role in the Growth of Plant Tissue

Experiments were designed to determine the significance of dark CO₂ fixation in excised maize roots, carrot slices and excised tomato roots grown in tissue culture. Bicarbonate-¹⁴C was used to determine the pathway and amounts of CO₂ fixation, while leucine-¹⁴C was used to estimate protein synthesis in tissues aerated with various levels of CO₂.

Organic acids were labeled from bicarbonate-¹⁴C, with malate being the major labeled acid. Only glutamate and aspartate were labeled in the amino acid fraction and these 2 amino acids comprised over 90% of the ¹⁴C label in the ethanol-water insoluble residue.

Studies with leucine-¹⁴C as an indicator of protein synthesis in carrot slices and tomato roots showed that those tissues aerated with air incorporated 33% more leucine-¹⁴C into protein than those aerated with CO₂-free air. Growth of excised tomato roots aerated with air was 50% more than growth of tissue aerated with CO₂-free air. These studies are consistent with the suggestion that dark fixation of CO₂ is involved in the growth of plant tissues.

     

Contribution of Photorespiration to Changes of Carbon Isotope Characteristics in Plants Affected by Stress Factors

Experimental data available in literature on changes in the carbon isotopic composition of biochemical fractions and metabolites isolated from plant biomass (Clusia minor) and photosynthesizing algae (Chlorella stigmatophora) under the action of environmental stress factors are reviewed and analyzed. Within the framework of previously suggested mechanism of carbon isotope fractionation in photosynthesis, all studied fractions and metabolites obtained from plants and photosynthesizing algae can be divided into two groups according to their carbon isotope composition. The first group includes the fractions and metabolite pools that contain carbon stored by cell during the carboxylase phase of Rubisco functioning. The second group consists of those formed primarily by the photorespiratory carbon flow, generated during the oxygenase phase of Rubisco functioning. The first group represents the assimilatory branch of photosynthesis and is enriched in ¹²C relative to carbon of biomass, whereas the second group represents the photorespiratory branch and is enriched in ¹³C. Under the action of environmental stress factors, such as incident light intensity, moisture availability, and salinity; the isotope composition of metabolites and fractions changes, which reflects variable contributions of the assimilatory and photorespiratory flows to the metabolite synthesis. These isotope shifts were used to study biochemical adaptation of plants to stress conditions and to elucidate the role of photorespiration in this adaptation.     

Carbon Dioxide Fixation in Roots and Nodules of Alnus glutinosa: I. Role of Phosphoenolpyruvate Carboxylase and Carbamyl Phosphate Synthetase in Dark CO(2) Fixation, Citrulline Synthesis, and N(2) Fixation

Detached roots and nodules of the N₂-fixing species, Albus glutinosa (European black alder), actively assimilate CO₂. The maximum rates of dark CO₂ fixation observed for detached nodules and roots were 15 and 3 micromoles CO₂ fixed per gram dry weight per hour, respectively. The net incorporation of CO₂ in these tissues was catalyzed by phosphoenolpyruvate carboxylase which produces organic acids, some of which are used in the synthesis of the amino acids, aspartate, glutamate, and citrulline and by carbamyl phosphate synthetase. The latter accounts for approximately 30 to 40% of the CO₂ fixed and provides carbamyl phosphate for the synthesis of citrulline. Results of labeling studies suggest that there are multiple pools of malate present in nodules. The major pool is apparently metabolically inactive and of unknown function while the smaller pool is rapidly utilized in the synthesis of amino acids. Dark CO₂ fixation and N₂ fixation in nodules decreased after treatment of nodulated plants with nitrate while the percentage of the total ¹⁴C incorporated into organic acids increased. Phosphoenolpyruvate carboxylase and carbamyl phosphate synthetase play key roles in the synthesis of amino acids including citrulline and in the metabolism of N₂-fixing nodules and roots of alder.     

Effect of Fusicoccin on Dark CO(2) Fixation by Vicia faba Guard Cell Protoplasts

When Vicia faba guard cell protoplasts were treated with fusicoccin, dark ¹⁴CO₂ fixation rates increased by as much as 8-fold. Rate increase was saturated with less than 1 micromolar fusicoccin. Even after 6 minutes of dark ¹⁴CO₂ fixation, more than 95% of the incorporated radioactivity was in stable products derived from carboxylation of phosphoenolpyruvate (about 50% and 30% in malate and aspartate, respectively). The relative distribution of ¹⁴C among products and in the C-4 position of malate (initially more than 90% of [¹⁴C]malate) was independent of fusicoccin concentration. After incubation in the dark, malate content was higher in protoplasts treated with fusicoccin. A positive correlation was observed between the amounts of ¹⁴CO₂ fixed and malate content.

It was concluded that (a) fusicoccin causes an increase in the rate of dark ¹⁴CO₂ fixation without alteration of the relative fluxes through pathways by which it is metabolized, (b) fusicoccin causes an increase in malate synthesis, and (c) dark ¹⁴CO₂ fixation and malate synthesis are mediated by phosphoenolpyruvate carboxylase.