Radioassay of orotic acid phosphoribosyltransferase and orotidylate decarboxylase utilizing a high-voltage paper electrophoresis technique or an improved 14CO2- release method.

Orotic acid phosphoribosyltransferase (EC 2.4.2.10) and orotidylate decarboxylase (EC 4.1.1.23) can be assayed independently of one another by the high voltage paper electrophoresis method described here, which separates orotic acid, OMP, and UMP, the substrates and/or products of these enzymes, from each other. The relative migration of other compounds, mainly other nucleotides, their bases, or other intermediates of the UMP biosynthetic pathway, has also been recorded. The method has allowed us to observe that OMP is not released to any significant degree from the enzyme complex of these two enzymes that occurs in Ehrlich ascites cells; rather orotic acid is converted stoichiometrically by the enzyme complex to UMP. For purification of the enzyme complex, we have found the release of ¹⁴CO₂ from [¹⁴C]carboxyl-labeled orotic acid (when phosphoribosyl pyrophosphate and Mg²⁺ are present) preferable to the HVPE method as a routine assay procedure. The most economical CO₂-absorbant for the assay of the enzyme complex or for orotidylate decarboxylase (and possibly for other enzymes which release CO₂) is an NaOH-soaked paper strip. As detailed here, its use allows one to repeatedly reuse the scintillation vials and fluid.     

Products of photosynthetic 14CO2 fixation and related enzyme activities in fruiting structures of chickpea

Fruiting structures of a number of legumes including chickpea are known to carry out photosynthetic CO₂ assimilation, but the pathway of CO₂ fixation and particularly the role of phosphoenolpyruvate carboxylase (EC 4.1.1.31) in these tissues is not clear. Activities of some key enzymes of the Calvin cycle and C₄ metabolism, rates of ¹⁴CO₂ fixation in light and dark, and initial products of photosynthetic ¹⁴CO₂ fixation were determined in podwall and seedcoat (fruiting structures) and their subtending leaf in chickpea (Cicer arietinum L.). Compared to activities of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and other Calvin cycle enzyme, viz. NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13), NAD⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and ribulose-5-phosphate kinase (EC 2.7.1.19), the levels of phosphoenolpyruvate carboxylase and other enzymes of C₄ metabolism viz. NADP⁺-malate dehydrogenase (EC 1.1.1.82), NAD⁺-malate dehydrogenase (EC 1.1.1.37), NADP⁺ malic enzyme (EC 1.1.1.40), NAD⁺-malic enzyme (EC 1.1.1.39), glutamate oxaloacetate transaminase (EC 2.6.1.1) and glutamate pyruvate transaminase (EC 2.6.1.2), were generally much higher in podwall and seedcoat than in the leaf. Podwall and seedcoat fixed ¹⁴CO₂ in light and dark at much higher rates than the leaf. Short-term assimilation of ¹⁴CO₂ by illuminated fruiting structures produced malate as the major labelled product with less labelling in 3-phosphoglycerate, whereas the leaf showed a major incorporation into 3-phosphoglycerate. It seems likely that the fruiting structures of chickpea utilize phosphoenolpyruvate carboxylase for recapturing the respired carbon dioxide.     

Agarose-DTNB column for binding sulfhydryl-containing proteins and peptides.

The counting rate of [1-¹⁴C]trichloroacetic acid (TCA) was not stable in a standard toluene/Triton X-100 liquid scintillation solution because this compound becomes partially degraded to ¹⁴CO₂ and CHCl₃. Both toluene and Triton were contributing factors in causing this degradation. The NCS solubilizer added to the toluene/Triton scintillation solution trapped ¹⁴CO₂ and stabilized counting rates of [1-¹⁴C]TCA. When [2-¹⁴C]TCA was used, ¹⁴CHCl₃ remained in the scintillation solution resulting in stable counting rates without the addition of NCS.     

Carbon and nitrogen metabolism in a barley (Hordeum vulgare L.) mutant with impaired chloroplast dicarboxylate transport

A mutant line, RPr79/2, of barley (Hordeum vulgare L. cv. Maris Mink) has been isolated that has an apparent defect in photorespiratory nitrogen metabolism. The metabolism of ¹⁴C-labelled glutamine, glutamate and 2-oxoglutarate indicates that the mutant has a greatly reduced ability to synthesise glutamate, especially in air, although in-vitro enzyme analysis indicates the presence of wild-type activities of glutamine synthetase (EC 6.3.1.2) glutamate synthase (EC 1.4.7.1 and EC 1.4.1.14) and glutamate dehydrogenase (EC 1.4.1.2). Several characteristics of RPr79/2 are very similar to those described for glutamate-synthase-deficient barley and Arabidopsis thaliana mutants, including the pattern of labelling following fixation of ¹⁴CO₂, and the rapid rise in glutamine content and fall in glutamate in leaves on transfer to air. The CO₂-fixation rate in RPr79/2 declines much more slowly on transfer from 1% O₂ to air than do the rates in glutamate-synthase-deficient plants, and RPr79/2 plants do not die in air unless the temperature and irradiance are high. Analysis of (glutamine+NH₃+2-oxoglutarate)-dependent O₂ evolution by isolated chloroplasts shows that chloroplasts from RPr79/2 require a fivefold greater concentration of 2-oxoglutarate than does the wild-type for maximum activity. The levels of 2-oxoglutarate in illuminated leaves of RPr79/2 in air are sevenfold higher than in Maris Mink. It is suggested that RPr79/2 is defective in chloroplast dicarboxylate transport.     

Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase

Five mutant lines of barley (Hordeum vulgare L.), which are only able to grow at elevated levels of CO₂, contain less than 5% of the wild-type activity of ferredoxin-dependent glutamate synthase (EC 1.4.7.1). Two of these lines (RPr 82/1 and RPr 82/9) have been studied in detail. Leaves and roots of both lines contain normal activities of NADH-dependent glutamate synthase (EC 1.4.1.14) and the other enzymes of ammonia assimilation. Under conditions that minimise photorespiration, both mutants fix CO₂ at normal rates; on transfer to air, the rates drop rapidly to 15% of the wild-type. Incorporation of ¹⁴CO₂ into sugar phosphates and glycollate is increased under such conditions, whilst incorporation of radioactivity into serine, glycine, glycerate and sucrose is decreased; continuous exposure to air leads to an accumulation of ¹⁴C in malate. The concentrations of malate, glutamine, asparagine and ammonia are all high in air, whilst aspartate, alanine, glutamate, glycine and serine are low, by comparison with the wild-type parent line (cv. Maris Mink), under the same conditions. The metabolism of [¹⁴C]glutamate and [¹⁴C]glutamine by leaves of the mutants indicates a very much reduced ability to convert glutamine to glutamate. Genetic analysis has shown that the mutation in RPr 82/9 segregates as a single recessive nuclear gene.Abbreviations GDH glutamate dehydrogenase (EC 1.4.1.2) - GS glutamine synthetase (EC 6.3.1.2) - RuBP ribulose 1,5-bisphosphate     

Anaerobic degradation of toluene by pure cultures of denitrifying bacteria

Several denitrifying Pseudomonas spp., isolated with various aromatic compounds, were tested for the ability to degrade toluene in the absence of molecular oxygen. Four out of seven strains were able to degrade toluene in the presence of N₂O. More than 50% of the ¹⁴C from ring-labelled toluene was released as CO₂, and up to 37% was assimilated into cell material. Furthermore it was demonstrated for two strains that they were able to grow on toluene as the sole carbon and energy source in the presence of N₂O. Suspensions of cells pre-grown on toluene degraded toluene, benzaldehyde or benzoate without a lag phase and without accumulation of intermediates. p-Cresol, p-hydroxybenzylalcohol, p-hydroxybenzaldehyde or p-hydroxybenzoate was degraded much slower or only after distinct lag times. In the presence of fluoroacetate [¹⁴C]toluene was transformed to [¹⁴C]benzoate, which suggests that anaerobic toluene degradation proceeds through oxidation of the methyl side chain to benzoate.     

The effect of Cd2+ on photosynthetic reactions of mesophyll protoplasts

Photosynthetic CO₂-fixation of mesophyll protoplasts of lambs lettuce [Valerianella locusta (L.) Betcke] was inhibited by short time exposure to Cd⁺. Inhibition was due to uptake of the metal ion into the protoplasts and increased with increasing Cd²⁺ concentrations and the time of preincubation. A 10 min pretreatment at 2 mM Cd²⁺ reduced CO₂-fixation by 40–60%. Inhibition of photosynthesis was independent of the light intensity to which the protoplasts were exposed. Measurement of the lightinduced electrochromic pigment absorption change at 518nm and chlorophyll fluorescence studies revealed that primary photochemical reactions associated with the thylakoid membranes were not affected by the metal ion. Also, light activation of the ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) was not inhibited by Cd²⁺. Under rate-limiting CO₂ concentrations, inhibition of CO₂-fixation was smaller than at V_max of CO₂ reduction indicating that the carboxylation reaction of the Calvin cycle is not susceptible to Cd²⁺. Cd²⁺ treatment of protoplasts significantly extended the lagphase of CO₂-supported O₂-evolution and partly inhibited light activation of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and the ribulose-5-phosphate kinase (EC 2.7.1.19). Measurement of relative concentrations of [¹⁴C]-labeled Calvin cycle intermediates showed that Cd²⁺ caused a decrease in the 3-phosphoglycerate/triose phosphate ratio and an increase in the triose phosphate/ribulose-1,5-bisphosphate ratio. It is concluded that in protoplasts Cd²⁺ affects photosynthesis mainly at the level of dark reactions and that the site of inhibition may be localized in the regenerative phase of the Calvin cycle.     

Incorporation of 14CO2 into C4 acids by leaves of C3-C4 intermediate and C3 species of Moricandia and Panicum at the CO2 compensation concentration

Comparative ¹⁴CO₂ pulse-¹²CO₂ chase studies performed at CO₂ compensation ()-versus air-concentrations of CO₂ demonstrated a four-to eightfold increase in assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of the C₃-C₄ intermediate species Panicum milioides Nees ex Trin., P. decipiens Nees ex Trin., Moricandia arvensis (L.) DC., and M. spinosa Pomel at . Specifically, the distribution of ¹⁴C in malate and aspartate following a 10-s pulse with ¹⁴CO₂ increases from 2% to 17% (P. milioides) and 4% to 16% (M. arvensis) when leaves are illuminated at the CO₂ compensation concentration (20 l CO₂/l, 21% O₂) versus air (340 l CO₂/l, 21% O₂). Chasing recently incorporated ¹⁴C for up to 5 min with ¹²CO₂ failed to show any substantial turnover of label in the C₄ acids or in carbon-4 of malate. The C₄-acid labeling patterns of leaves of the closely related C₃ species, P. laxum Sw. and M. moricandioides (Boiss.) Heywood, were found to be relatively unresponsive to changes in pCO₂ from air to . These data demonstrate that the C₃-C₄ intermediate species of Panicum and Moricandia possess an inherently greater capacity for CO₂ assimilation via phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) at the CO₂ compensation concentration than closely related C₃ species. However, even at , CO₂ fixation by PEP carboxylase is minor compared to that via ribulosebisphosphate carboxylase (EC 4.1.1.39) and the C₃ cycle, and it is, therefore, unlikely to contribute in a major way to the mechanism(s) facilitating reduced photorespiration in the C₃-C₄ intermediate species of Panicum and Moricandia.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - PEP phosphoenolpyruvate - CO₂ compensation concentration - 3PGA 3-phosphoglycerate - SuP sugar monophosphates - SuP₂ sugar bisphosphates Published as Paper No. 8249, Journal Series, Nebraska Agricultural Research Division     

Carbon assimilation by different developmental stages of Laminaria saccharina

Various stages of the life cycle of the marine brown alga Laminaria saccharina (L.) Lamour. (Laminariales, Phaeophyta) including male and female gametophytes, female gametes, zygotes and young sporophytes of different age were investigated for their potentials of carbon dioxide (¹⁴CO₂) fixation. Rates of photosynthesis attain the same order of magnitude in all stages. Photosynthetic ¹⁴CO₂-fixation is accompanied by a substantial light independent carbon assimilation. This is confirmed by rate determinations of the equivalent carboxylating enzymes present in the plants, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxokinase (EC 4.1.1.32) as well as by chromatographic analyses of the appropriate [¹⁴C]-assimilate patterns.Abbreviations RuBP-C ribulose-1,5-bisphosphate carboxylase - PEP-CK phosphoenolpyruvate carboxykinase - PEP phosphoenolpyruvate - PS photosynthesis - DF dark fixation     

Photosynthetic Assimilation of Sun versus Shade Norway Spruce [Picea abies (L.) Karst] Needles Under the Long-Term Impact of Elevated CO2 Concentration

The long-term impact of elevated concentration of CO₂ on assimilation activity of sun-exposed (E) versus shaded (S) foliage was investigated in a Norway spruce stand [Picea abies (L.) Karst, age 14 years] after three years of cultivation in two domes with adjustable windows (DAW). One DAW was supplied with ambient air [AC, ca. 350 µmol(CO₂) mol^–1) and the second with elevated CO₂ concentration [EC = AC plus 350 µmol(CO₂) mol^–1]. The pronounced vertical profile of the photosynthetic photon flux density (PPFD) led to the typical differentiation of the photosynthetic apparatus between the shaded and sun needles. Namely, photon-saturated values of maximal net photosynthetic rate (P _Nmax) and apparent quantum yield () were significantly higher/lower for E-needles as compared with the S-ones. The prolonged exposure to EC was responsible for the apparent assimilatory activity stimulation observed mainly in deeply shaded needles. The degree of this stimulation decreases in the order: S-needles dense part > S-needles sparse part > E-needles dense part > E-needles sparse part. In exposed needles some signals on a manifestation of the acclimation depression of the photosynthetic activity were found. The long-term effect of EC was responsible for the decrease of nitrogen content of needles and for its smoother gradient between E- and S-needles. The obtained results indicate that the E- and S-foliage respond differently to the long-term impact of EC.     

Characterization of carbon metabolism in Opuntia ficus-indica Mill. exhibiting the idling mode of Crassulacean acid metabolism

Upon transfer from well-watered conditions to total drought, long-day-grown cladodes of Opuntia ficus-indica Mill. shift from full Crassulacean acid metabolism (CAM) to CAM-idling. Experiments using ¹⁴C-tracers were conducted in order to characterize the carbon-flow pattern in cladodes under both physiological situations. Tracer was applied by ¹⁴CO₂ fumigations and NaH¹⁴CO₃ injections during the day-night cycle. The results showed that behind the closed stomata, mesophyll cells of CAM-idling plants retained their full capacity to metabolize CO₂ in light and in darkness. Upon the induction of CAM-idling the level of the capacity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) was maintained. By contrast, malate pools decreased, displaying finally only a small or no day-night oscillation. The capacity of NADP-malic enzyme (EC 1.1.1.40) decreased in parallel with the reduction in malate pools. Differences in the labelling patterns, as influenced by the mode of tracer application, are discussed.Abbreviations CAM Crassulacean acid metabolism - PEP-Case phosphoenolpyruvate carboxylase     

Preparation of [U-14C]-labelled glycogen, maltosaccharides, maltose, and D-glucose by photoassimilation of 14CO2 in Anacystis nidulans and selective enzymic degradation.

A procedure is described for the isolation from the phototrophic procaryole Anacystis nidulans of [U-¹⁴C]-labelled glycogen, with high specific radioactivity,formed when NaH¹⁴CO₃ was added to non-dividing cells that continued to photoassimilate CO₂. [U-¹⁴C]-Labelled glycogen was then treated with isoamylase (EC 3.2.1.68), isoamylase plus beta-amylase (EC 3.2.1.2), or glucoamylase (EC 3.2.1.3) to give [U-¹⁴C]-labelled maltosaccharides, maltose-U-¹⁴C, or d-glucose-U-¹⁴C, respectively.     

Isolation and characterization of a bacterium that mineralizes toluene in the absence of molecular oxygen

A bacterium tentatively identified as a Pseudomonas sp. was isolated from a laboratory aquifer column in which toluene was degraded under denitrifying conditions. The organism mineralized toluene in pure culture in the absence of molecular oxygen. In carbon balance studies using [ring-UL-¹⁴C]toluene, more than 50% of the radioactivity was recovered as ¹⁴CO₂. Nitrate and nitrous oxide served as electron acceptors for toluene mineralization. The organism was also able to degrade m-xylene, benzoate, benzaldehyde, p-cresol, p-hydroxybenzaldehyde, p-hydroxybenzoate and cyclohexanecarboxylic acid in the absence of molecular oxygen.     

Stimulating the anaerobic degradation of aromatic hydrocarbons in contaminated sediments by providing an electrode as the electron acceptor

The possibility that electrodes might serve as an electron acceptor to simulate the degradation of aromatic hydrocarbons in anaerobic contaminated sediments was investigated. Initial studies with Geobacter metallireducens demonstrated that although toluene was rapidly adsorbed onto the graphite electrodes it was rapidly oxidized to carbon dioxide with the electrode serving as the sole electron acceptor. Providing graphite electrodes as an electron acceptor in hydrocarbon‐contaminated sediments significantly stimulated the removal of added toluene and benzene. Rates of toluene and benzene removal accelerated with continued additions of toluene and benzene. [¹⁴C]‐Toluene and [¹⁴C]‐benzene were quantitatively recovered as [¹⁴C]‐CO₂, demonstrating that even though the graphite adsorbed toluene and benzene they were degraded. Introducing an electrode as an electron acceptor also accelerated the loss of added naphthalene and [¹⁴C]‐naphthalene was converted to [¹⁴C]‐CO₂. The results suggest that graphite electrodes can serve as an electron acceptor for the degradation of aromatic hydrocarbon contaminants in sediments, co‐localizing the contaminants, the degradative organisms and the electron acceptor. Once in position, they provide a permanent, low‐maintenance source of electron acceptor. Thus, graphite electrodes may offer an attractive alternative for enhancing contaminant degradation in anoxic environments.     

Nitrogenase activity and dark CO2 fixation in the lichen Peltigera aphthosa Willd

The lichen Peltigera aphthosa consists of a fungus and green alga (Coccomyxa) in the main thallus and of a Nostoc located in superficial packets, intermixed with fungus, called cephalodia. Dark nitrogenase activity (acetylene reduction) of lichen discs (of alga, fungus and Nostoc) and of excised cephalodia was sustained at higher rates and for longer than was the dark nitrogenase activity of the isolated Nostoc growing exponentially. Dark nitrogenase activity of the symbiotic Nostoc was supported by the catabolism of polyglucose accumulated in the ligh and which in darkness served to supply ATP and reductant. The decrease in glucose content of the cephalodia paralleled the decline in dark nitrogenase activity in the presence of CO₂; in the absence of CO₂ dark nitrogenase activity declined faster although the rate of glucose loss was similar in the presence and absence of CO₂. Dark CO₂ fixation, which after 30 min in darkness represented 17 and 20% of the light rates of discs and cephalodia, respectively, also facilitated dark nitrogenase activity. The isolated Nostoc, the Coccomyxa and the excised fungus all fixed CO₂ in the dark; in the lichen most dark CO₂ fixation was probably due to the fungus. Kinetic studies using discs or cephalodia showed highest initial incorporation of ¹⁴CO₂ in the dark in to oxaloacetate, aspartate, malate and fumarate; incorporation in to alanine and citrulline was low; incorporation in to sugar phosphates, phosphoglyceric acid and sugar alcohols was not significant. Substantial activities of the enzymes phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and carbamoyl-phosphate synthase (EC 2.7.2.5 and 2.7.2.9) were detected but the activities of PEP carboxykinase (EC 4.1.1.49) and PEP carboxyphosphotransferase (EC 4.1.1.38) were negligible. In the dark nitrogenase activity by the cephalodia, but not by the free-living Nostoc, declined more rapidly in the absence than in the presence of CO₂ in the gas phase. Exogenous NH ₄ ⁺ inhibited nitrogenase activity by cephalodia in the dark especially in the absence of CO₂ but had no effect in the light. The overall data suggest that in the lichen dark CO₂ fixation by the fungus may provide carbon skeletons which accept NH ₄ ⁺ released by the cyanobacterium and that in the absence of CO₂, NH ₄ ⁺ directly, or indirectly via a mechanism which involves glutamine synthetase, inhibits nitrogenase activity.Abbreviations CP carbamoyl phosphate - EDTA ethylenedi-amine tetraacetic acid - PEP phosphoenolpyruvate - RuBP ribulose 1,5 bisphosphate     

METABOLITES AND ENZYMES OF THE PENTOSE PHOSPHATE PATHWAY IN ISOLATED NERVE ENDINGS1

Abstract— The activities of each enzyme associated with the pentose phosphate pathway as well as the non-enzymatic intermediates in this pathway were measured in synaptosomes isolated from rat cerebral cortex. The specific activities of transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) were significantly lower in synaptosomes than cerebral cortex; however, the specific activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribosephosphate isomerase (EC 5.3.1.6) and ribulosephosphate epimerase (EC 5.1.3.1.) were comparable in homogenates of synaptosomal fractions and cerebral cortex. Concentrations of most intermediates of the pentose pathway were also similar in extracts of synaptosomes and brain homogenates. Six hours after treatment of rats with the nicotinamide analog, 6-aminonicotinamide (6-AN), 6-phosphogluconate levels in synaptosomes were increased 5-fold; however, glucose-6-phosphate levels remained unchanged. During a 30 min in uitro incubation 6-phosphogluconate levels increased approx 2-fold in synaptosomes obtained from 6-AN treated rats but did not change in synaptosomes from untreated rats. During the same period glucose-6-phosphate levels decreased in synaptosomes from both control and 6-AN treated rats. The conversion of both [1-¹⁴C]glucose and [6-¹⁴C]glucose to ¹⁴CO₂ was depressed in synaptosomes from 6-AN treated rats; however, the ratio of the two isotopes converted to ¹⁴CO₂ was essentially the same. It is concluded that the pentose phosphate pathway is active in nerve endings both in vivo and in vitro.     

Carboxylating enzymes and pathway of photosynthetic carbon assmilation in different marine algae—Evidence for the C4-pathway?

Experiments on short-term photosynthesis in H¹⁴CO₃ ^- (2–5 s) using various species of different algal classes resulted in predominant ¹⁴C-labelling (>90% of total ¹⁴C-incorporation) of phosphorylated compounds. The percentage of malate and aspartate usually accounts for distinctly less than 10% of the total ¹⁴C-labelling. These findings are consistent with data from enzymatic analyses, since 97–100% of the carboxylation capacity is due to ribulose-1.5-biphosphate carboxylase (EC 4.1.1.39) in Rhodophyceae and Chlorophyceae. Phaeophyceae are generally characterized by considerable activity of phosphoenolpyruvate carboxykinase (EC 4.1.1.32): at least 10% of carboxylation is confined to this enzyme. Similar ratios are obtained when rates of photosynthesis and of light-independent CO₂-fixation are compared. Activity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) could not be detected in the species investigated. The results are discussed with emphasis on the pathway of photosynthetic carbon assimilation in marine algae.Abbreviations PEP-CK phosphoenolpyruvate carboxykinase (EC 4.1.1.32) - PEP-C phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RubP-C ribulose-1.5-biphosphate carboxylase (EC 4.1.1.39) Dedicated to Professor H. Fischer, Bonn, on his 65th birthday     

Photosynthesis in Plants of Four Tropical Species Growing under Elevated CO2

We studied the responses of leaf gas exchange and growth to an increase in atmospheric CO₂ concentration in four tropical deciduous species differing in carbon fixation metabolism: Alternanthera crucis, C3-C4; Ipomoea carnea, C3; Jatropha gossypifolia, C3; and Talinum triangulare, inducible-CAM. In the first stage, plants were grown in one open-top chamber at a CO₂ concentration of 560±40 mol mol^-1 (EC), one ambient CO₂ concentration chamber (AC), and one unenclosed plot (U). In the second stage, plants were grown in five EC chambers (CO₂ concentration = 680±30 mol mol^-1), five AC chambers, and five unenclosed plots. During the first weeks under EC in the first stage, plants of all the species had a very marked increase in their maximal net photosynthetic rates (P _max) of 3.5 times on average; this stimulatory effect was maintained for 11-15 weeks, rates dampening afterward to values still higher than controls for 37 weeks. After a suspension of CO₂ enrichment for 6 weeks, an increase in P _max of EC plants over the controls was found in plants of all the species until week 82 of the experiment. Stomatal conductance (g) showed no response to EC. Carboxylation efficiency decreased in all the species under EC     

Photosynthetic carbon metabolism during leaf ontogeny in Zea mays L.: Enzyme studies

The activities of several enzymes, including ribulose-1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured as a function of leaf age in Z. mays. Mature leaf tissue had a RuDP-carboxylase activity of 296.7 mol CO₂ g^-1 fresh weight h^-1 and a PEP-carboxylase activity of 660.6 mol CO₂ g^-1 fresh weight h^-1. In young corn leaves the activity of the two enzymes was 11 and 29%, respectively, of the mature leaves. In senescent leaf tissue, RuDP carboxylase activity declined more rapidly than that of any of the other enzymes assayed. On a relative basis the activities of NADP malic enzyme (EC 1.1.1.40), aspartate (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2), and NAD malate dehydrogenase (EC 1.1.1.37) exceeded those of both PEP and RuDP carboxylase in young and senescent leaf tissue. Pulse-chase labeling experiments with mature and senescent leaf tissue show that the predominant C₄ acid differs between the two leaf ages. Labeling of alanine in senescent tissue never exceeded 4% of the total ¹⁴C remaining during the chase period, while in mature leaf tissue alanine accounted for 20% of the total after 60 s in ¹²CO₂. The activity of RuDP carboxylase during leaf ontogeny in Z. mays parallels the development of the activity of this enzyme in C₃ plants.Abbreviations RuDP ribulose-1,5-diphosphate - PEP phosphoenol pyruvate - PGA 3-phosphoglycerate     

The Effect of CO2 Enrichment on the Growth of Nodulated and Non-Nodulated Isogenic Types of Soybean Raised Under Two Nitrogen Concentrations

To find the effects of CO₂ enrichment on plant development and photosynthetic capacity of nodulated (line A62-1) and non-nodulated (line A62-2) isogenic lines of soybean (Glycine max Merr.), we examined the interactions among two CO₂ treatments (36±3 Pa = AC and 70±5 Pa = EC), and two nitrogen concentrations [0 g(N) m⁻²(land area) = 0N; 30 g(N) m⁻²(land area) = 30N]. Nodules were found in both CO₂ treatments in 0N of A62-1 where the number and dry mass of nodules increased from AC to EC. While the allocation of dry mass to root and shoot and the amount of N in each organ did not differ between the growth CO₂ concentrations, there was larger N allocation to roots in 0N than in 30N for A62-2. The CO₂-dependence of net photosynthetic rate (P _N) for A62-1 was unaffected by both CO₂ and N treatments. In contrast, the CO₂-dependence of P _N was lower in 0N than in 30N for A62-2, but it was independent of CO₂ treatment. P _N per unit N content was unaffected by CO₂ concentrations. The leaf area of both soybean lines grown in 30N increased in EC. But in 0N, only the nodulated A62-1 showed an increase in leaf area in EC. Nitrogen use efficiency of plants, NUE [(total dry mass of the plant)/(amount of N accumulated in the plant)] in 30N was unaffected by CO₂ treatments. In 0N, NUE in EC was lower than in AC in A62-1, and was higher than that at AC in A62-2. Hence, the larger amount and/or rate of N fixation with the increase of the sink-size of symbiotic microorganisms supplied adequate N to the plant under EC. In EC, N deficiency caused the down-regulation of the soybean plant. This revised version was published online in June 2006 with corrections to the Cover Date.