Carbon-13 Nuclear Magnetic Resonance Analysis of Metabolism in Soybean Labeled by CO(2)

Fourier transform ¹³C nuclear magnetic resonance spectra have been obtained of intact, fresh soybean ovules (Glycine max L. cv. Dare) harvested from pods subtended by a trifoliolate exposed to ¹³CO₂ 1 to 3 days earlier. The high resolution spectra are interpreted in terms of the labeled sugars and lipids in the ovule. Comparison of the spectra taken over the 3-day period permits qualitative estimates of sugar metabolism and rates of lipid synthesis. The spectra also contain information about the distribution of labels within the lipid chains. This information leads to a method of estimating the extent to which glucose degradation in the synthesizing soybean ovule is involved in the reactions of the phosphogluconate pathway.     

Short-Term and Long-Term Responses of Crassulacean Acid Metabolism Plants to Elevated CO(2)

For the leaf succulent Agave deserti and the stem succulent Ferocactus acanthodes, increasing the ambient CO₂ level from 350 microliters per liter to 650 microliters per liter immediately increased daytime net CO₂ uptake about 30% while leaving nighttime net CO₂ uptake of these Crassulacean acid metabolism (CAM) plants approximately unchanged. A similar enhancement of about 30% was found in dry weight gain over 1 year when the plants were grown at 650 microliters CO₂ per liter compared with 350 microliters per liter. Based on these results plus those at 500 microliters per liter, net CO₂ uptake over 24-hour periods and dry weight productivity of these two CAM succulents is predicted to increase an average of about 1% for each 10 microliters per liter rise in ambient CO₂ level up to 650 microliters per liter.     

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.     

CO(2) Metabolism in Corn Roots. III. Inhibition of P-enolpyruvate Carboxylase by l-malate

Phosphoenolpyruvate carboxylase was purified from corn root tips about 80-fold by centrifugation, ammonium sulfate fractionation, and anion exchange and gel filtration chromatography. The resulting preparation was essentially free from malate dehydrogenase, isocitrate dehydrogenase, malate enzyme, NADH oxidase, and pyruvate kinase activity. Kinetic analysis indicated that l-malate was a noncompetitive inhibitor of P-enolpyruvate carboxylase with respect to P-enolpyruvate (K_I = 0.8 mm). d-Malate, aspartate, and glutamate inhibited to a lesser extent; succinate, fumarate, and pyruvate did not inhibit. Oxaloacetate was also a noncompetitive inhibitor of P-enolpyruvate carboxylase with an apparent K_I of 0.4 mm. A comparison of oxaloacetate and l-malate inhibition suggested that the mechanisms of inhibition were different. These data indicated that l-malate may regulate CO₂ fixation in corn root tips by a feedback or end product type of inhibition.     

Production of Specifically Labeled Compounds by Methanobacterium espanolae Grown on H(2)-CO(2) plus [C]Acetate

Methanobacterium espanolae, an acidiphilic methanogen, required acetate for maximal growth on H(2)-CO(2). In the presence of 5 to 15 mM acetate, at a growth pH of 5.5, the mu(max) was 0.05 h. M. espanolae consumed 12.3 mM acetate during 96 h of incubation at 35 degrees C with shaking at 100 rpm. At initial acetate levels of 2.5 to 10.0 mM, the amount of biomass produced was dependent on the amount of acetate in the medium. C nuclear magnetic resonance spectra of protein hydrolysates obtained from cultures grown on [1-C]- or [2-C]acetate indicated that an incomplete tricarboxylic acid pathway, operating in the reductive direction, was functional in this methanogen. The amino acids were labeled with a very high degree of specificity and at greater than 90% enrichment levels. Less than 2% label randomization occurred between positions primarily labeled from either the carboxyl or methyl group of acetate, and very little label was transferred to positions primarily labeled from CO(2). The labeling pattern of carbohydrates was typical for glucogenesis from pyruvate. This methanogen, by virtue of the properties described above and its ability to incorporate all of the available acetate (10 mM or lower) from the growth medium, has advantages over other microorganisms for use in the production of specifically labeled compounds.     

Photorespiration in Green Plants During Photosynthesis Estimated by Use of Isotopic CO(2)

Photosynthetic re-absorption of photorespired CO₂ causes underestimation in measured photorespiration and turnover rate of the substrate for photorespiration. Actual values of photorespiration exceed the measured by a factor greater than 1 + R′_w/r_p + [C_L]_x/(r_p·L_x). (R′_w and r_p are the partial resistances to CO₂ uptake between atmosphere, mesophyll evaporating surface, and photosynthetic sink, respectively; L_x is the measured flux of photorespired CO₂ and [C_L]_x is the ambient conc of photorespired CO₂). In 8 species, 1 + R′_w/r_p alone amounted to a correction ranging between 148% and 233%.     

Studies on Effect of Certain Quinones: I. Electron Transport, Photophosphorylation, and CO(2) Fixation in Isolated Chloroplasts

The effect of quinone herbicides and fungicides on photosynthetic reactions in isolated spinach (Spinacia oleracea) chloroplasts was investigated. 2,3-Dichloro-1,4-naphthoquinone (dichlone), 2-amino-3-chloro-1,4-naphthoquinone (06K-quinone), and 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) inhibited ferricyanide reduction as well as ATP formation. Benzoquinone had little or no effect on these reactions. The two reactions showed a differential sensitivity to these inhibitors. Dichlone was a strong inhibitor of both photosystems I and II; photosystem I was more sensitive to 06K-quinone than was photosystem II, whereas the reverse was true of chloranil. Chloranil and 06K-quinone inhibited ferricyanide reduction and the coupled photophosphorylation to the same extent, whereas dichlone affected photophosphorylation to a greater extent than the ferricyanide reduction.     

Limitation of Photosynthesis by Carbon Metabolism : II. O(2)-Insensitive CO(2) Uptake Results from Limitation Of Triose Phosphate Utilization

The occurrence of O₂-insensitive photosynthesis at high quantum flux and moderate temperature in Spinacia oleracea was characterized by analytical gas exchange measurements on intact leaves. In addition photosynthetic metabolite pools were measured in leaves which had been rapidly frozen under defined gas conditions. Upon switching to low O₂ in O₂-insensitive conditions the ATP/ADP ratio fell dramatically within one minute. The P-glycerate pool increased over the same time. Ribulose bisphosphate initially declined, then increased and exceeded the pool size measured in air. The pools of hexose monophosphates and UDPglucose were higher at a partial pressure of O₂ of 21 millibars than at 210 millibars. These results are consistent with the hypothesis that the rate of sucrose synthesis limited the overall rate of assimilation under O₂-insensitive conditions.     

Ecophysiological Significance of CO(2)-Recycling via Crassulacean Acid Metabolism in Talinum calycinum Engelm. (Portulacaceae)

High levels of variability in gas exchange characteristics and degree of CAM-cycling were found in the same and different individuals of Talinum calycinum Engelm. collected from rock outcrops in Missouri. Differences in CO₂ assimilation were mostly correlated with differences in shoot conductance to CO₂ not shoot internal CO₂ concentration. As found previously, CAM acid fluctuations were evident in well-watered plants exhibiting C₃ gas exchange patterns (CAM-cycling) and also in drought-stressed plants with stomata closed, or nearly so, day and night (CAM-idling). Drought stress also resulted in rapid stomatal closure, conserving water during droughts. Maximal CO₂ uptake rates occurred below 35°C; higher temperatures induced decreases in CO₂ assimilation and conductance while shoot internal CO₂ concentrations remained similar. Plant water-use-efficiency was severely curtailed at temperatures above 30°C. Tissue acid fluctuations were the result of changes in malic acid concentrations. Calculations of the amount of water potentially conserved by CAM-cycling yielded values of approximately 5 to 44% of daytime water loss. Thus, CAM-cycling may be an important adaptation minimizing water loss by perennial succulents growing in shallow soil on rock outcrops.     

Use of Carbon Isotopes to Estimate Incorporation of Added CO(2) by Greenhouse-Grown Tomato Plants

A method is presented which uses the ¹³C and ¹⁴C isotope abundance in CO₂-enriched greenhouse crops to determine the percentage of plant organic carbon derived from artificially added CO₂. In a greenhouse experiment with CO₂ concentrations elevated to 1100 ± 100 microliters per liter during part of the daylight hours and maintained at normal atmospheric concentrations (340 microliters per liter) during the rest of the time, it was shown by ¹⁴C analysis that between 41% and 42% of the carbon in tomato plants (Lycopersicon esculentum var 4884) came from the artificially added CO₂. Similar results were obtained from ¹³C analyses when the CO₂ pressure-dependent isotope separation was taken into account.     

Leaf Photosynthesis and Respiration of High CO(2)-Grown Tobacco Plants Selected for Survival under CO(2) Compensation Point Conditions

Four self-pollinated, doubled-haploid tobacco, (Nicotiana tabacum L.) lines (SP422, SP432, SP435, and SP451), selected as haploids by survival in a low CO₂ atmosphere, and the parental cv Wisconsin-38 were grown from seed in a growth room kept at high CO₂ levels (600-700 parts per million). The selected plants were much larger (especially SP422, SP432, and SP451) than Wisconsin-38 nine weeks after planting. The specific leaf dry weight and the carbon (but not nitrogen and sulfur) content per unit area were also higher in the selected plants. However, the chlorophyll, carotenoid, and alkaloid contents and the chlorophyll a/b ratio varied little. The net CO₂ assimilation rate per unit area measured in the growth room at high CO₂ was not higher in the selected plants. The CO₂ assimilation rate versus intercellular CO₂ curve and the CO₂ compensation point showed no substantial differences among the different lines, even though these plants were selected for survival under CO₂ compensation point conditions. Adult leaf respiration rates were similar when expressed per unit area but were lower in the selected lines when expressed per unit dry weight. Leaf respiration rates were negatively correlated with specific leaf dry weight and with the carbon content per unit area and were positively correlated with nitrogen and sulfur content of the dry matter. The alternative pathway was not involved in respiration in the dark in these leaves. The better carbon economy of tobacco lines selected for low CO₂ survival was not apparently related to an improvement of photosynthesis rate but could be related, at least partially, to a significantly reduced respiration (mainly cytochrome pathway) rate per unit carbon.     

The Effect of Temperature on the Occurrence of O(2) and CO(2) Insensitive Photosynthesis in Field Grown Plants

The sensitivity of photosynthesis to O₂ and CO₂ was measured in leaves from field grown plants of six species (Phaseolus vulgaris, Capsicum annuum, Lycopersicon esculentum, Scrophularia desertorum, Cardaria draba, and Populus fremontii) from 5°C to 35°C using gas-exchange techniques. In all species but Phaseolus, photosynthesis was insensitive to O₂ in normal air below a species dependent temperature. CO₂ insensitivity occurred under the same conditions that resulted in O₂ insensitivity. A complete loss of O₂ sensitivity occurred up to 22°C in Lycopersicon but only up to 6°C in Scrophularia. In Lycopersicon and Populus, O₂ and CO₂ insensitivity occurred under conditions regularly encountered during the cooler portions of the day. Because O₂ insensitivity is an indicator of feedback limited photosynthesis, these results indicate that feedback limitations can play a role in determining the diurnal carbon gain in the field. At higher partial pressures of CO₂ the temperature at which O₂ insensitivity occurred was higher, indicating that feedback limitations in the field will become more important as the CO₂ concentration in the atmosphere increases.     

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.     

Method for Simultaneous Measurement of Radioactive and Inactive CO(2) Evolved from Soil Samples During Incubation with Labeled Substrates

An apparatus is described which allows the simultaneous, continuous, and highly sensitive analysis of inactive and radioactive CO₂ evolved from ¹⁴C-supplemented soils or other materials. The apparatus consists of a control unit, a commercially available conductometric CO₂ analyzer, and fraction collector. A number of model experiments were conducted to demonstrate the potentials of the apparatus. These included analysis of the time course of priming action, when ¹⁴C-glucose was added to soil, separation of CO₂ respiration peaks caused by simultaneous degradation of radioactive and inactive soil supplements, and study of the effects of a fungicide, Benomyl, on degradation of ¹⁴C-labeled glucose. In the last experiment, partial degradation of the fungicide could also be followed.     

Rates of Photosynthesis in Characean Cells: I. PHOTOSYNTHETIC 14CO2 FIXATION BY NITELLA TRANSLUCENS

A study has been made of photosynthetic ¹⁴CO₂ fixation by isolated‘mature’ internodes of Nitella translucens. Experimentalconditions were similar to those used in studies of the ionicrelations of these cells. Maximum rates of photosynthesis were33–40µµmoles CO₂, fixed per cm² of surfacearea per second (equivalent to 12–15 /xmoles fixed permg chlorophyll per hour). ^l4CO₂ fixation was inhibited to thedark level by 3(3,4,dichlorophenyl)-1, 1-dimethylurea (at 0-6µM or 10µM) and by the uncoupler carbonyl cyanide-m-chlorophenylhydrazone(SµM). The presence of imidazole or ammonium sulphate(both of which uncouple ATP production in vitro) did not resultin an inhibition of 14CO2 fixation. These results are discussedin relation to published work on solute uptake by Nitella translucens.During photosynthesis there was rapid movement of ¹⁴C-labelledorganic compounds out of the chloroplasts. ¹⁴C-labelled sucrose,ammo-acids, and sugar phosphates were found in samples of vacuolarsap.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N(2) Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO(3)-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a "conventional" Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Effect of a Brief CO(2) Exposure on Ethylene Production

Ethylene production and respiration by Granny Smith apples were inhibited by treatment with 20% CO₂ for 2 hours. A similar effect was observed in tissue slices when treated at either 0 or 25°C.

The inhibition continued even after an extended aeration period. There is also an inhibition of ethylene emission in tissue slices incubated with exogenous 1-aminocyclopropane-1-carboxylic acid (ACC).

In general, CO₂ treatment increased the ACC content of the tissue. These observations are consistent with the idea the action of CO₂ is directed toward the enzyme system responsible for the conversion of ACC into ethylene.