CO(2) Fixation in Opuntia Roots

Nonautotrophic CO₂ metabolism in Opuntia echinocarpa roots was studied with techniques of manometry and radiometry. The roots were grown in a one-quarter strength nutrient solution for several days; the distal 2 cm was used for physiological studies. The roots assimilated significant quantities of ¹⁴CO₂ and appeared to show a crassulacean-type acid metabolism with respect to quality and quantity. Most of the ¹⁴C activity was associated with the distal portion of the elongating root indicating correlation with metabolic activity. The ¹⁴CO₂ assimilation was comparable to a crassulacean leaf succulent, but 3 times greater than that found for stem tissue of the same Opuntia species.

The rates of O₂ and CO₂ exchange and estimated CO₂ fixation were 180, 123, and 57 μl/g per hour. A respiratory quotient of 0.66 was found.

The products of ¹⁴CO₂ fixation were similar in most respects to reported experiments with leaf succulents. Equilibration of the predominant malic acid with isocitric, succinic, and fumaric acids was not evident. The latter observation was interpreted as metabolic isolation of the fixation products rather than poor citric acid cycle activity.

A rapid turnover of the fixed ¹⁴CO₂ was measured by following decarboxlyation kinetics and by product analysis after a postincubation period. The first order rate constant for the steady state release was 4.4 × 10⁻³ min⁻¹ with a half-time of 157.5 minutes. Amino acids decayed at a more rapid rate than organic acids.

     

CO(2) Exchange and Dinitrogen Fixation of Subterranean Clover in Response to Light Level

Small swards of nodulated subterranean clover plants were grown in pots to a common dry weight under controlled conditions. The rooting medium was a porous calcined clay. All mineral nutrients except nitrogen were supplied daily in solution. Pots then were placed in an assimilation chamber for 3 days for the measurement of net CO₂ exchange at light levels ranging from 0.1 to 2.0 millieinsteins per square meter per second. N₂-fixation (acetylene reduction) of each pot was measured subsequently. H₂-evolution and N₂-fixation were measured for similar treatments in separate experiments using smaller pots.     

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.     

Green Light Drives CO2 Fixation Deep within Leaves

Maximal ^l4CO₂-fixation in spinach occurs in the middle of thepalisade mesophyll [Nishio et al. (1993) Plant Cell 5: 953],however, ninety percent of the blue and red light is attenuatedin the upper twenty percent of a spinach leaf [Cui et al. (1991)Plant Cell Environ. 14: 493]. In this report, we showed thatgreen light drives ¹⁴CO₂-fixation deep within spinach leavescompared to red and blue light. Blue light caused fixation mainlyin the palisade mesophyll of the leaf, whereas red light drovefixation slightly deeper into the leaf than did blue light.¹⁴CO₂-fixation measured under green light resulted in less fixationin the upper epidermal layer (guard cells) and upper most palisademesophyll compared to red and blue light, but led to more fixationdeeper in the leaf than that caused by either red or blue light.Saturating white, red, or green light resulted in similar maximal¹⁴CO₂-fixation rates, whereas under the highest irradiance ofblue light given, carbon fixation was not saturated, but itasymptotically approached the maximal ¹⁴CO₂-fixation rates attainedunder the other types of light. The importance of green lightin photosynthesis is discussed. ¹Supported in part by grants from Competitive Research GrantsOffice, U.S. Department of Agriculture (Nos. 91-37100-6672 and93-37100-8855).     

Regulation of Soybean Net Photosynthetic CO(2) Fixation by the Interaction of CO(2), O(2), and Ribulose 1,5-Diphosphate Carboxylase

Kinetic properties of soybean net photosynthetic CO₂ fixation and of the carboxylase and oxygenase activities of purified soybean (Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO₂ concentration, and O₂ concentration. With leaves, O₂ inhibition of net photosynthetic CO₂ fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO₂ fixation at higher temperatures was caused by a reduced affinity of the leaf for CO₂ and an increased affinity of the leaf for O₂. With purified ribulose 1,5-diphosphate carboxylase, O₂ inhibition of CO₂ incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O₂ sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO₂ and a slightly increased affinity of the enzyme for O₂. The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO₂ and O₂ provides further evidence that the carboxylase regulates the O₂ response of photosynthetic CO₂ fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO₂ and O₂ fixation in vivo is proposed.     

Photosynthetic CO(2) Fixation at Air Levels of CO(2) by Isolated Spinach Chloroplasts

A system has been developed for the study of photosynthetic CO₂ fixation by isolated spinach chloroplasts at air levels of CO₂. Rates of CO₂ fixation were typically 20 to 60 micromoles/milligrams chlorophyll per hour. The rate of fixation was linear for 10 minutes but then declined to less than 10% of the initial value by 40 minutes. Ribulose 1,5-bisphosphate (RuBP) levels remained unchanged during this period, indicating that they were not the cause for the decline. The initial activity of the RuBP carboxylase in the chloroplast was high for 8 to 10 minutes and then declined similar to the rate of CO₂ fixation, suggesting that the decline in CO₂ fixation may have been caused by deactivation of the enzyme.     

Metabolite Control Overrides Circadian Regulation of Phosphoenolpyruvate Carboxylase Kinase and CO(2) Fixation in Crassulacean Acid Metabolism

Phosphoenolpyruvate carboxylase (PEPc) catalyzes the primary fixation of CO₂ in Crassulacean acid metabolism plants. Flux through the enzyme is regulated by reversible phosphorylation. PEPc kinase is controlled by changes in the level of its translatable mRNA in response to a circadian rhythm. The physiological significance of changes in the levels of PEPc-kinase-translatable mRNA and the involvement of metabolites in control of the kinase was investigated by subjecting Kalanchoë daigremontiana leaves to anaerobic conditions at night to modulate the magnitude of malate accumulation, or to a rise in temperature at night to increase the efflux of malate from vacuole to cytosol. Changes in CO₂ fixation and PEPc kinase activity reflected those in kinase mRNA. The highest rates of CO₂ fixation and levels of kinase mRNA were observed in leaves subjected to anaerobic treatment for the first half of the night and then transferred to ambient air. In leaves subjected to anaerobic treatment overnight and transferred to ambient air at the start of the day, PEPc-kinase-translatable mRNA and activity, the phosphorylation state of PEPc, and fixation of atmospheric CO₂ were significantly higher than those for control leaves for the first 3 h of the light period. A nighttime temperature increase from 19°C to 27°C led to a rapid reduction in kinase mRNA and activity; however, this was not observed in leaves in which malate accumulation had been prevented by anaerobic treatment. These data are consistent with the hypothesis that a high concentration of malate reduces both kinase mRNA and the accumulation of the kinase itself.     

Effects of Powdery Mildew Infection on the Efficiency of CO(2) Fixation and Light Utilization by Sugar Beet Leaves

Sugar beet leaves (Beta vulgaris L.) infected with powdery mildew (Erysiphe polygoni D.C.) show declining rates of net photosynthesis as the disease develops; relative to healthy controls, reductions of 35, 70, and 75% were observed at 9, 16, and 22 days after inoculation, respectively. A leaf gas exchange procedure in which an air stream flowed through the leaf showed that mesophyll conductance declined in parallel with photosynthesis in mildew-infected leaves. Viscous flow conductance of diseased leaves also declined over the same period suggesting that stomatal aperture was reduced. From the magnitude and time course of disease effects on stomatal aperture and mesophyll conductance, it appears that the effects at the mesophyll level were primarily responsible for mediating the decline in net photosynthesis. Changes in mesophyll conductance were closely correlated with reduced activity of ribulose-1,5-bisphosphate carboxylase on a leaf area basis. This decrease could be attributed to a reduction in the concentration of the enzyme, a reduction which was greater than the reduction in total soluble protein. The quantum efficiency of light use was also decreased by the disease. Mildew-infected leaves had quantum yields that were reduced, relative to healthy leaves, by 17 and 22% at 14 and 18 days after inoculation, respectively.     

Reversible pH Changes in Cells of Chlamydomonas reinhardi Resulting from CO(2) Fixation in the Light and Its Evolution in the Dark

Illumination of a suspension of Chlamydomonas reinhardi causes an increase in the pH of the medium which is reversed in the dark. This pH change is a manifestation of CO₂ uptake in the light and its evolution in the dark. Simultaneous measurements of pH changes and oxygen evolution reveal that the photosynthetic coefficient approaches one.     

Chloroplast Integrity and ATP-Dependent CO(2) Fixation in Spinacia oleracea

Washed whole chloroplasts of Spinacia oleracea isolated and assayed in a tris (hydroxymethyl aminomethane)-HCl buffered sucrose solution exhibited low dark CO₂ fixing activity, whereas washed whole chloroplasts isolated in the same buffer but assayed in that buffer without sucrose exhibited much greater dark CO₂ fixing activity. The lowered activity could be attributed to the impermeability of the chloroplast membrane to ribose-5-phosphate or adenosine triphosphate. The preservation of the integrity of the chloroplast membrane, as reflected by its impermeability to either or both of the abovementioned compounds, was measured by the fixation of ¹⁴CO₂ into acid-stable products in the presence of ribose-5-phosphate and adenosine triphosphate by the whole chloroplast as compared with fixation by the chloroplast extract. An effect (i.e., apparent resistance to the passage of ribose-5-phosphate or adenosine-5-triphosphate into the chloroplast) similar to, but less pronounced than, that produced by the presence of sucrose in the isolation medium was observed upon the addition of MnCl₂ or CaCl₂ to the buffered sucrose isolation medium. The addition of KCl enhanced slightly the effect produced by addition of sucrose alone to the isolation medium. The presence of MgCl₂ in the isolation medium, however, either caused the chloroplasts to become leaky or more fragile since more of the activity of the carboxylative phase enzymes appeared in the cytoplasm. When a mixture of all of the metal ions was added to the buffered sucrose suspending medium, the chloroplasts exhibited the same response observed with MgCl₂ alone. The addition of ethylene diaminetetraacetate or dithiothreitol appeared to alter the permeability of the chloroplast membrane nonspecifically when the assay was conducted in the absence of sucrose. Specific activities (μmoles CO₂ fixed/mg chlorophyll × hr) as high as 329.6 have been observed for dark fixation by chloroplasts. The phosphoenolpyruvate carboxylase activity in the chloroplasts was only one-seventh that of ribulose diphosphate carboxylase. The phosphoenolpyruvate carboxylase activity in the cytoplasm was 5 times that of the chloroplasts.     

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.     

Localized Tetrazolium Reduction in Relation to N(2) Fixation, CO(2) Fixation, and H(2) Uptake in Aquatic Filamentous Cyanobacteria

The aquatic filamentous cyanobacteria Anabaena oscillarioides and Trichodesmium sp. reveal specific cellular regions of tetrazolium salt reduction. The effects of localized reduction of five tetrazolium salts on N(2) fixation (acetylene reduction), CO(2) fixation, and H(2) utilization were examined. During short-term (within 30 min) exposures in A. oscillarioides, salt reduction in heterocysts occurred simultaneously with inhibition of acetylene reduction. Conversely, when salts failed to either penetrate or be reduced in heterocysts, no inhibition of acetylene reduction occurred. When salts were rapidly reduced in vegetative cells, CO(2) fixation and H(2) utilization rates decreased, whereas salts exclusively reduced in heterocysts were not linked to blockage of these processes. In the nonheterocystous genus Trichodesmium, the deposition of reduced 2,3,5-triphenyl-2-tetrazolium chloride (TTC) in the internal cores of trichomes occurs simultaneously with a lowering of acetylene reduction rates. Since TTC deposition in heterocysts of A. oscillarioides occurs contemporaneously with inhibition of acetylene reduction, we conclude that the cellular reduction of this salt is of use in locating potential N(2)-fixing sites in cyanobacteria. The possible applications and problems associated with interpreting localized reduction of tetrazolium salts in cyanobacteria are presented.     

Cellular Localization of CO(2) Fixation and Translocation of Metabolites

Leaves of maize (Zea mays L.) and sugar beet (Beta vulgaris L.) were enclosed in an illuminated chamber in air for 30 min after which time ¹⁴CO₂ was released into the chamber. Two min after the ¹⁴CO₂ was released, the leaves were removed from the chamber, and small sections were cut from them. The sections were put in small wire baskets and frozen in isopentane cooled by liquid nitrogen. Approximately 1.5 min elapsed from the removal of the leaf from the illuminated chamber until the tissue was frozen. The tissue was freeze-dried, embedded in paraffin and the cellular location of the isotopic activity was determined by radiography of leaf cross sections. Isotopic activity in maize leaves was localized in bundle sheath parenchyma. In contrast, the label in sugar beet leaves was generally distributed in the mesophyll cells. The bundle sheath cells in maize contain specialized chloroplasts which appear to have a unique capacity to incorporate CO₂. Translocation from leaves of maize was 3-fold as rapid as from sugar beet leaves in the same environment. Low light intensity did not alter the distribution pattern of fixed CO₂.     

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.