Effects of O(2) Tension and Temperature during Light Uptake of CO(2) on the Dark Release of CO(2) by Excised C(3) and C(4) Leaves

A study was conducted on a C₄ (Panicum maximum) and a C₃ (Panicum bisulcatum) species to determine the nature of the dark release of ¹⁴CO₂ with respect to its responses to changes in temperature and O₂ tension during light CO₂ uptake of ¹⁴CO₂.     

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.

     

Responses of Plant Dark Respiration to Doubled CO2 Concentration

Ten species of plants were grown at ambient (350μmol CO2·mol-1 air) and doubled (700 μmol CO2·mol-1 air) CO2 concentrations at ambient temperature and illumination in order to examine changes of dark respiration of whole seedlings or detached leaves. Effects of CO2 on dark respiration were determined by brief exposure ( ≤ 5 min) to corresponding CO2 concentration and temperatures ( 15,20,25,30 and 35 ℃ ) with infrared CO2 analyzer. The reductions in dark respiration on a weight base for leaves of East-Liaoning oak (Quercus liaotungensis Koidz. ) at 15,20 and 25 ℃ and of soybean ( Glycine max L. ) at 20,25,30 and 35 ℃ and for whole seedlings of three- tcoloured amaranth (Amaranthus tricolor L. ) at 15 and 20 ℃ and cucumber ( Cucumis sativus L. ) at 15 cE measured at elevated concentration relative to the ambient CO2 concentration were observed. No significant difference in respiration responded was observed to elevated or ambient CO2 concentrations at 15 ℃ in maize (Zea mays L. ) seedlings and alfalfa (Medicago sativa L. ) leaves, at 35 ℃ in East-Liaoning oak leaves and at 20,25 and 30 ℃ in three-coloured amaranth seedlings. However CO2 efflux in leaves of weeping willow (Salix babylonica L. ), simon poplar (Populus simonii Carr. ) and eucommia (Eucommia ulmoides Oliv. ) at 15,20,25,30 and 35 ℃, alfalfa at 20,25,30 and 35 ℃, East-Liaoning oak at 30 ℃, maize at 15 ℃, seedlings of common buckwheat (Fagotrytum esculentum Moench) at 15,20,25,30 and 35 ℃, cucumber and maize at 20,25,30 and 35 ℃ and three-coloured amaranth at 35 ℃ showed an increase at elevated in contrast to ambient CO2 concentration. In general, at lower temperatures (i. e. 15, 20 ℃ ) there was no significant difference between elevated and ambient CO2 concentration for dark respiration, while at higher temperatures (i. e. 30,35 ℃ ) elevated CO2 concentration positively stimulate clark respiretion. It has not yet been described that double CO2 concentration could enhance plant dark respiration at 30 and 35 ℃. Impacts of the characteristics in dark respiration on the future changes of vegetation and its mechanism were discussed.     

Photoinhibition of CO(2)-Dependent O(2) Evolution by Intact Chloroplasts Isolated from Spinach Leaves

Intact spinach (Spinacia oleracea L.) chloroplasts, when pre-illuminated at 4 millimoles quanta per square meter per second for 8 minutes in a CO₂-free buffer at 21% O₂, showed a decrease (30-70%) in CO₂-dependent O₂ evolution and ¹⁴CO₂ uptake. This photoinhibition was observed only when the O₂ concentration and the quantum fluence rate were higher than 4% and 1 millimole per square meter per second, respectively. There was only a small decrease in the extent of photoinhibition when the CO₂ concentration was increased from 0 to 25 micromolar during the treatment, but photoinhibition was abolished when the CO₂ concentration was increased to 30 micromolar. Addition of small quantities of P-glycerate (40-200 micromolar) or glycerate (160 micromolar) was found to prevent photoinhibition. Other intermediates of the Calvin cycle (fructose-6-P, fructose-1,6-P, ribose-5-P, ribulose-5-P) also prevented photoinhibition to various extents. Oxaloacetate was not effective in preventing photoinhibition in these chloroplasts. The amount of O₂ evolved during treatments with 3-P-glycerate or glycerate was no more than 65% of that measured in the presence of low CO₂ concentrations (9-12 micromolar) which did not prevent photoinhibition. In all cases, the extent to which photoinhibition was prevented by these metabolites was not correlated to the amount of O₂ evolved during the photoinhibitory treatment. It is concluded that in these chloroplasts the prevention of the O₂-dependent photoinhibition of light saturated CO₂ fixation capacity is not linked to the dissipation of excitation energy via the photosynthetic electron transport nor to ATP utilization. The requirement of O₂ for photoinhibition of CO₂ fixation capacity in isolated chloroplasts may be explained by an effect of O₂ in allowing metabolic depletion of Calvin cycle intermediates.     

Low CO(2) Prevents Nitrate Reduction in Leaves

The correlation between CO₂ assimilation and nitrate reduction in detached spinach (Spinacia oleracea L.) leaves was examined by measuring light-dependent changes in leaf nitrate levels in response to mild water stress and to artificially imposed CO₂ deficiency. The level of extractable nitrate reductase (NR) activity was also measured. The results are: (a) In the light, detached turgid spinach leaves reduced nitrate stored in the vacuoles of mesophyll cells at rates between 3 and 10 micromoles per milligram of chlorophyll per hour. Nitrate fed through the petiole was reduced at similar rates as storage nitrate. Nitrate reduction was accompanied by malate accumulation. (b) Under mild water stress which caused stomatal closure, nitrate reduction was prevented. The inhibition of nitrate reduction observed in water stressed leaves was reversed by external CO₂ concentrations (10-15%) high enough to overcome stomatal resistance. (c) Nitrate reduction was also inhibited when turgid leaves were kept in CO₂-free air or at the CO₂-compensation point or in nitrogen. (d) When leaves were illuminated in CO₂-free air, activity of NR decreased rapidly. It increased again, when CO₂ was added back to the system. The half-time for a 50% change in activity was about 30 min. It thus appears that there is a rapid inactivation/activation mechanism of NR in leaves which couples nitrate reductase to net photosynthesis.     

CO(2) Inhibits Respiration in Leaves of Rumex crispus L

Curly dock (Rumex crispus L.) was grown from seed in a glasshouse at an ambient CO₂ partial pressure of about 35 pascals. Apparent respiration rate (CO₂ efflux in the dark) of expanded leaves was then measured at ambient CO₂ partial pressure of 5 to 95 pascals. Calculated intercellular CO₂ partial pressure was proportional to ambient CO₂ partial pressure in these short-term experiments. The CO₂ level strongly affected apparent respiration rate: a doubling of the partial pressure of CO₂ typically inhibited respiration by 25 to 30%, whereas a decrease in CO₂ elicited a corresponding increase in respiration. These responses were readily reversible. A flexible, sensitive regulatory interaction between CO₂ (a byproduct of respiration) and some component(s) of heterotrophic metabolism is indicated.     

Carbon Dioxide Exchanges in Leaves. I. Discrimination Between CO(2) and CO(2) in Photosynthesis

In order to measure CO₂ exchange reactions by leaves using isotopes of CO₂, it is necessary to know precisely the discrimination against ¹⁴CO₂ by leaves. Earlier determinations of discrimination are at variance, and may be inaccurate because of assumptions made about the rate of photorespiration. Maize leaves evolve little or no CO₂ in light, and so provide suitable material for this measurement. Discrimination against ¹⁴CO₂ in photosynthesis by maize leaves is almost precisely the same as in CO₂ absorption by NaOH solution, amounting to 2.1 and 2.0% respectively. The agreement between these values and their close approximation to the relative rates of diffusion of ¹²CO₂ and ¹⁴CO₂, calculated from Graham's law, shows that diffusion into the leaf is primarily responsible for discrimination against ¹⁴CO₂ in photosynthesis.     

Ethylene Contamination of CO(2) Cylinders: Effects on Plant Growth in CO(2) Enrichment Studies

The mechanical extensibilities of stage IVb Phycomyces were measured before and after a humidified wind stimulus. We find that when the humidity of the wind is greater than that of the ambient air, there is an increase in the mechanical extensibility of the cell wall. We also find that a step decrease in wind humidity results in a decrease in the mechanical extensibility of the cell wall.     

Stomatal Behavior and CO(2) Exchange Characteristics in Amphistomatous Leaves

The possibility that differences in stomatal conductance between upper and lower surfaces of amphistomatous leaves are adaptations to differences in CO₂ exchange characteristics for the two surfaces was investigated. The ratio of upper to lower stomatal conductance was found to change little in response to light and humidity for well-watered sunflower (Helianthus annuus L.) plants. Stressing the plants (ψ = −17 bars) and rewatering 1 day before gas exchange measurements reduced upper conductance more severely than lower in both indoor- and outdoor-grown plants, and caused small changes in conductance ratio with light and humidity. A similar pattern was found using outdoor grown sunflower and cocklebur (Xanthium strumarium L.) plants. Calculated intercellular CO₂ concentrations for upper and lower surfaces were always close to identical for a particular set of environmental conditions for both sunflower and cocklebur, indicating that no differences in CO₂ exchange characteristics exist between the two surfaces. By artificially creating a CO₂ gradient across the leaf, the resistance to CO₂ diffusion through the mesophyll was estimated and found to be so low that despite possible nonhomogeneity of the mesophyll, differences in CO₂ exchange characteristics for the two surfaces are unlikely. It is concluded that differences in conductance between upper and lower stomates are not adaptations to differences in CO₂ exchange characteristics.     

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.

     

Role of Glycine and Glyoxylate Decarboxylation in Photorespiratory CO(2) Release

Mechanically isolated soybean leaf cells metabolized added glycolate by two mechanisms, the direct oxidation of glyoxylate and the decarboxylation of glycine. The rate of glyoxylate oxidation was dependent on the cellular glyoxylate concentration and was linear between 0.58 and 2.66 micromoles glyoxylate per milligram chlorophyll. The rate extrapolated to zero at a concentration of zero. The concentration and, therefore, the rate of oxidation of glyoxylate could be decreased by adding glutamate or serine to the cells. These substrates were amino donors for the transamination of glyoxylate to glycine. In the presence of these amino acids more CO₂ was released from added glycolate via the glycine decarboxylation reaction and less by the direct oxidation of glyoxylate.     

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.     

Quantitation of the O(2)-Dependent, CO(2)-Reversible Component of the Postillumination CO(2) Exchange Transient in Tobacco and Maize Leaves

The postillumination transient of CO₂ exchange and its relation to photorespiration has been examined in leaf discs from tobacco (Nicotiana tabacum) and maize (Zea mays). Studies of the transients observed by infrared gas analysis at 1, 21, and 43% O₂ in an open system were extended using the nonsteady state model described previously (Peterson and Ferrandino 1984 Plant Physiol 76: 976-978). Cumulative CO₂ exchange equivalents (i.e. nanomoles CO₂) versus time were derived from the analyzer responses of individual transients. In tobacco (C₃), subtraction of the time course of cumulative CO₂ exchange under photorespiratory conditions (21 or 43% O₂) from that obtained under nonphotorespiratory conditions (1% O₂) revealed the presence of an O₂-dependent and CO₂-reversible component within the first 60 seconds following darkening. This component was absent in maize (C₄) and at low external O₂:CO₂ ratios (i.e. <100) in tobacco. The size of the component in tobacco increased with net photosynthesis as irradiance was increased and was positively associated with inhibition of net photosynthesis by O₂. This relatively simple and rapid method of analysis of the transient is introduced to eliminate some uncertainties associated with estimation of photorespiration based on the maximal rate of postillumination CO₂ evolution. This method also provides a useful and complementary tool for detecting variation in photorespiration.     

Biphasic Activation of Ribulose Bisphosphate Carboxylase in Spinach Leaves as Determined from Nonsteady-State CO(2) Exchange

The activation kinetics of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) following an increase in photon flux density (PFD) were studied by analyzing CO₂ assimilation time courses in spinach leaves (Spinacia oleracea). When leaves were exposed to 45 minutes of darkness before illumination at 690 micromoles per square meter per second, Rubisco activation followed apparent first-order kinetics with a relaxation time of about 3.8 minutes. But when leaves were illuminated for 45 minutes at 160 micromoles per square meter per second prior to illumination at 690 micromoles per square meter per second the relaxation time for Rubisco activation was only 2.1 minutes. The kinetics of this change in relaxation times were investigated by exposing dark-adapted leaves to 160 micromoles per square meter per second for different periods before increasing the PFD to 690 micromoles per square meter per second. It was found that the apparent relaxation time for Rubisco activation changed from 3.8 to 2.1 minutes slowly, requiring at least 8 minutes for completion. This result indicates that at least two sequential, slow processes are involved in light-mediated activation of Rubisco in spinach leaves and that the relaxation times characterizing these two processes are about 4 and 2 minutes, respectively. The kinetics of the first process in the reverse direction and the dependence of the relaxation time for the second process on the magnitude of the increase in PFD were also determined. Evidence that the first slow process is activation of the enzyme Rubisco activase and that the second slow process is the catalytic activation of Rubisco by activase is discussed.