Metabolite Pool Sizes Research Articles

Environmental Effects on Photosynthesis, Nitrogen-Use Efficiency, and Metabolite Pools in Leaves of Sun and Shade Plants

Effects of varying light intensity and nitrogen nutrition on photosynthetic physiology and biochemistry were examined in the sun plant Phaseolus vulgaris (common bean) and in the shade plant Alocasia macrorrhiza (Australian rainforest floor species). In both Phaseolus and Alocasia, the differing growth regimes produced large changes in photosynthetic capacity and composition of the photosynthetic apparatus. CO(2)-saturated rates of photosynthesis were linearly related to leaf nitrogen (N) content in both species but photosynthesis per unit leaf N was markedly higher for Phaseolus than for Alocasia. Photosynthetic capacity was also higher in Phaseolus per unit ribulose 1,5-bisphosphate (RuBP) carboxylase (RuBPCase) protein. The leaf content of RuBPCase was linearly dependent on leaf N content in the two species. However, the proportion of leaf N which was RuBPCase was greater in Phaseolus than in Alocasia and was more sensitive to growth conditions, ranging from 6% of leaf N at low light to 20% at high light. In Alocasia, this range was much less, 6 to 11%. However, chlorophyll content was much more sensitive to light intensity in Alocasia. Thus, the RuBPCase/chlorophyll ratio was quite responsive to N availability and light intensity in both species (but for different reasons), ranging from 6 grams per gram for Phaseolus and 2 grams per gram for Alocasia at high leaf N and 1.5 gram per gram for Phaseolus and 0.5 gram per gram for Alocasia at low leaf N. These large changes in the proportions of components of the photosynthetic apparatus had marked effects on the sensitivity of these species to photoinhibition. These environmental effects also caused changes in the absolute levels of metabolites of the photosynthetic carbon reduction cycle. Concentrations of RuBP and P-glycerate were approximately 2-fold higher in high light-grown than low light-grown Phaseolus and Alocasia when expressed on a leaf area basis. However, if metabolite pool sizes are expressed on the basis of the RuBPCase catalytic site concentration, then they were little affected by the marked changes in leaf makeup. There appears to be fundamental differences between these species in the mechanism of sun-shade adaptation and N partitioning in the photosynthetic apparatus that result in significant differences in the N-use efficiency of photosynthesis between Phaseolus and Alocasia but similar RuBPCase:substrate:product ratios despite these differences.

The Effect of Abscisic Acid and Other Inhibitors on Photosynthetic Capacity and the Biochemistry of CO2 Assimilation

Abscisic acid (ABA) was shown to reduce the photosynthetic capacity of a leaf through an apparent inhibition of ribulose 1,5-bisphosphate (RuBP) carboxylase (RuBPCase) activity, in addition to promoting stomatal closure. By comparison with the effects of other inhibitors of photosynthesis (cyanazine, methyl viologen, sodium azide, nigericin, sodium cyanide) on whole leaf photosynthesis, RuBPCase activity and metabolite pool sizes, it was demonstrated that the biochemical basis for the apparent effect of ABA on RuBPCase activity was not the result of reduced substrate availability, decarbamylation of the enzyme, or synthesis of carboxyarabinitol 1-phosphate, the naturally occurring tight-binding inhibitor of the enzyme. An inhibition of photosynthetic capacity showing the same biochemical characteristics as ABA-fed leaves was observed in plants grown under saline conditions. We suggest that the common link between environmental stress and reductions in photosynthetic capacity may be ABA. We hypothesize that ABA may affect plasma membrane function and thus indirectly RuBPCase activity through altered ion fluxes. The results of feeding cyanazine, methyl viologen, and nigericin provide additional evidence that regulation of RuBPCase activity by carbamylation/decarbamylation is related to the extent to which the capacity for ATP formation limits photosynthesis.

Salinity and Nitrogen Effects on Photosynthesis, Ribulose-1,5-Bisphosphate Carboxylase and Metabolite Pool Sizes in Phaseolus vulgaris L.

Salinity (100 millimolar NaCl) was found to reduce photosynthetic capacity independent of stomatal closure in Phaseolus vulgaris. This reduction was shown to be a consequence of a reduction in the efficiency of ribulose-1,5-bisphosphate (RuBP) carboxylase (RuBPCase) rather than a reduction in the leaf content of photosynthetic machinery. In control plants, photosynthesis became RuBP-limited at approximately 1.75 moles RuBP per mole 2-carboxyarabinitol bisphosphate binding sites. Salinization caused the RuBP pool size to reach this limiting value for CO(2) fixation at much lower values of intercellular CO(2). Plants grown at low nitrogen and +/- NaCl became RuBP limited at similar RuBP pool sizes as the high nitrogen-grown plants. At limiting RuBP pool sizes and equal values of intercellular CO(2) photosynthetic capacity of salt-stressed plants was less than control plants. This effect of salinity on RuBPCase activity could not be explained by deactivation of the enzyme or inhibitor synthesis. Thus, salinity reduced photosynthetic capacity by reducing both the RuBP pool size by an effect on RuBP regeneration capacity and RuBPCase activity by an unknown mechanism when RuBP was limiting.

Triosephosphates Modulate Leaf Mitochondrial Phosphorylation by Inhibition and Uncoupling of Electron Transport

The effect of TP (triosephosphates:glyceraldehyde-3 phosphate, GAP, +dihydroxyacetone phosphate, DHAP) on respiration, phosphorylation and matrix ATP/ADP ratios of isolated oat mesophyll mitochondria was investigated. With both malate and NADH, a 50% inhibition of state 3-phosphorylation was induced by about 15 to 20 millimolar GAP and 30 to 40 millimolar DHAP. However, the nature of the inhibition appeared to be different with the two respiratory substrates. In the presence of NADH, TP did not inhibit the rate of state 3 (addition of ADP) O(2) consumption. In fact, depending on concentration, TP gradually increased the rates measured without ADP towards those seen under state 3, acting as uncouplers. When malate was the substrate for respiration, state 3 rates were decreased. The effect was comparable to that of rotenone and could be abolished by the addition of NADH. These observations indicate a dual action of TP: inhibition of electron transport around site I and uncoupling. In any case, the intramitochondrial ATP/ADP ratio decreased upon addition of TP. The effective TP concentrations as well as the changes in mitochondrial ATP/ADP ratios were comparable to results on changes of compartmental pool sizes of adenylates and other metabolites during dark/light transition of oat mesophyll protoplasts (R. Hampp, M. Goller, H. Füllgraf, and I. Eberle 1985 Plant Cell Physiol 24: 99). The possible role of TP in the regulation of mitochondrial respiration in the light, as well as modes of interference, are discussed.

Role of pyruvate carboxylation in the energy-linked regulation of pool sizes of tricarboxylic acid-cycle intermediates in the myocardium.

The increase in the metabolite pool size of the tricarboxylic acid cycle in the isolated perfused rat heart after a decrease in the ATP consumption by KCl-induced arrest was used to study the anaplerotic mechanisms. During net anaplerosis the label incorporation into the tricarboxylic acid-cycle intermediates from [1-14C]pyruvate increased and occurred mainly by pathways not involving prior release of the label to CO2. A method for determination of the specific radioactivity of mitochondrial pyruvate was devised, and the results corroborated the notion that tissue alanine can be used as an indicator of the specific radioactivity of intracellular pyruvate.

Accumulation and disposal of tricarboxylic acid cycle intermediates during propionate oxidation in the isolated perfused rat heart

The role of the metabolite disposal mechanisms in the regulation of the tricarboxylic acid cycle pool size was studied in isolated perfused rat hearts oxidizing 2 mM propionate. Malate and succinate accumulated during the propionate metabolism. A further 118% increase in the malate concentration and 600% increase in the succinate concentration and a slight inhibition of the propionate uptake were observed during a subsequent KCl-induced arrest of the heart metabolizing propionate. When the mechanical activity of the heart was restored, the malate and succinate concentrations returned to the same levels as before the arrest of the heart, but the propionate uptake did not rise significantly. The mean disposal rates of the tricarboxylic acid cycle metabolites during the cardiac arrest and subsequent restoration of the activity were 1.4 and 2.4 μmol/min per g dry weight, respectively. During cardiac arrest the malate carbon disposed was almost totally recovered as C 3 compounds, whereas after the increase in the ATP-consumption most of it was oxidized. The results show that propionate is oxidized by heart muscle at an appreciable rate but the disposal rate of the tricarboxylic acid cycle intermediates is not tightly regulated by the cellular energy state. Although the metabolite pool size of the tricarboxylic acid cycle responds to change in the ATP consumption, the energy state appears to have a greater effect on the fate of the C 3 compounds formed than on the actual rate of C 4 compound disposition.

Growth Regulator Effects, Metabolite Changes, and Respiration during Shoot Initiation in Cultured Cotyledon Explants of Pinus radiata

Adventitious shoot initiation in cotyledon explants from 6-7-day-old germinated seed of Pinus radiata requires exogenous cytokinin. This requirement was examined in terms of both concentration and duration. Treatments resulting in an insufficient cytokinin supply, particularly during the first 3 days in culture, dramatically reduced organogenesis. Other phytohormones and some other growth-active substances also reduced this cytokinin-induced shoot formation and promoted callus formation. Changes in metabolite pool sizes and in respiratory activity were monitored over the first 21 days in culture, during which meristematic tissue formation occurred along the entire length of the cotyledon. Lipids and free sugars were depleted ca. 10-fold and sixfold, respectively, and there was a steady decline in free amino-N levels. However, the protein-N pool remained relatively large. While there was a continued increase in cotyledon fresh weight, respiration decreased initially and then increased slightly during later...

Inhibition of C4 photosynthesis by (benzamidooxy)acetic acid

(Benzamidooxy)acetic acid (common name benzadox) which has herbicidal properties was evaluated as a potential inhibitor of photosynthesis in C4 plants. Among enzymes of the C4 pathway, it was a relatively strong inhibitor of alanine aminotransferase in in vitro experiments at concentrations of 5mM. In benzadox treated leaves of Panicum miliaceum, a NAD-malic enzyme type C4 species, there was strong inhibition of both alanine and aspartate aminotransferase and of photosynthetic O2 evolution within one hour. Consistent with the inhibition of these enzymes of the C4 cycle, the pool sizes of metabolites of the cycle was altered: the aspartate level was increased two fold, while the levels of other metabolites such as pyruvate, alanine, oxalacetate and malate were decreased. Kinetic studies with partially purified alanine aminotransferase showed that benzadox is a competitive inhibitor with respect to alanine and a noncompetitive inhibitor with respect to 2-oxoglutarate. Comparisons between the structures and inhibitory actions of benzadox and (aminooxy)acetic acid, the latter a potent inhibitor of alanine and aspartate aminotransferases, suggest that in vivo, benzadox may exert its effect through metabolism to (aminooxy)acetic acid.

Insulin-stimulated glucose uptake, leucine incorporation into protein, and uridine incorporation into RNA in skin fibroblast cultures from patients with diabetes mellitus.

Since intrinsic cellular abnormalities have previously have reported in diabetes mellitus, skin fibroblasts from patients with insulin-dependent diabetes and non-insulin-dependent diabetes, and from age-matched young and old controls, were examined for stimulation by insulin (4--4000 ng/ml) of glucose uptake, leucine incorporation into protein, and uridine incorporation into RNA. No differences in insulin-stimulated glucose uptake were seen between donor types. At 40 and 400 ng/ml, insulin did not stimulate as much leucine incorporation into protein in insulin-dependent diabetics as in young controls (p less than 0.05) and at 4000 ng/ml, less insulin-stimulated leucine incorporation was seen in insulin-dependent diabetics than in young controls or non-insulin dependent diabetics (p less than 0.01). Lower insulin-simulated uridine incorporation into RNA in old controls than in other cell lines appeared to be largely secondary to a two-fold increase in basal incorporation in these old controls. These results provide additional evidence for intrinsic cellular abnormalities in diabetes mellitus. Whether the differences in basal or insulin-stimulated response between fibroblasts of different donor types are attributable to alterations in protein or RNA synthesis, metabolite pool size or turnover have yet to be determined.

The synthesis of models to describe metabolism and its integration.

The wide range of metabolic, nutritional and physiological information which must be integrated in analyses of animal metabolism and function indicates that modelling techniques can be utilized and contribute to the research process. Advantages gained through the use of modelling techniques in support of research include evaluations of available concepts and results for adequacy as quantitative and dynamic explanations of the behaviour of complex (metabolic, physiological) systems, evaluation of alternate hypotheses for probable adequacy, interpretation or extension or both of available information, and identification of critical experiments. Achievement of these objectives requires a close interaction of modelling and experimental analyses. Additional requirements imposed by these objectives are that the elements of research models represent specific biological entities (model must be causal), that the basic models be firmly based on established principles and contain no hypothetical elements, and that numerical inputs to the model derive directly from experimental measurements. Emphasis in developing this presentation was placed upon illustration of these principles and objectives of research modelling. Examples were drawn from three ongoing modelling analyses; animal metabolism, animal growth, and tissue metabolism. Models of whole animal metabolism The development of early models of animal metabolism required extensive searches of the literature and summarization and integration of a wide range of information. Results utilized included estimates of organ weights and energy expenditures, extracellular and intracellular pool sizes of metabolites, nutrient absorption, turnover and utilization rates, and oxidation rates of metabolites (Smith, 1971; Smith et al. 1975). Mathematical representations of tissue metabolism require consideration of energy requirements for basal metabolism and for variable biosynthetic and work functions, affinities of tissues for metabolites, biosynthetic and oxidative capacities of tissues and specific regulatory mechanisms. A generalized equation set for a typical tissue expressed in a modified FORTRAN format follows: DADP=ATPUSE- METOX:SATP,

Kinetic analysis of 25-hydroxyvitamin D3 metabolism in strontium-induced rickets in the chick.

Kinetic data analysis was used to derive a six-compartment computer model which describes the in vivo [3H]25-hydroxyvitamin D3 ([3H]25-OHD3) metabolism in control and strontium rachitic chicks. Plasma concentrations of 25-OHD3 (13 pmol/ml) and 25, 25-dihydroxyvitamin D3 (0.9 pmol/ml) were 18 and 125% greater than controls, respectively, whereas the corresponding level for 1alpha,25-dihydroxyvitamin D3 (0.3 pmol/ml) was only 30% of control. Plasma disappearance of 25-HOD3 was fitted using a two-compartment model in which the metabolite extrapolated half-life was nearly twice as large for strontium rachitic chicks (71 compared to 41 h). Intestinal sequestration of 1alpha,25-dihydroxyvitamin D3 was assumed to be irreversible and was fitted by a single exponential term in which metabolite uptake rate and tissue concentration in strontium rickets was suppressed to 20 and 10% of control, respectively. In contrast, uptake of 25-OHD3 by the intestine was observed to occur by a reversible process in which metabolite concentration was 45% greater in the strontium rachitic compared to control group. The developed compartment model accepts time-dependent control or perturbed metabolite data for the plasma and (or) intestinal pools and provides quantitative values for metabolite pool size, flux rate, and turnover time.

Steady-State Photosynthesis in Alfalfa Leaflets

When the CO(2) concentration to which Medicago sativa L. var. El Unico leaflets were exposed was increased from half-saturation to saturation (doubled rate of photosynthesis), glycolate and glycine production apparently decreased due to inhibition of a portion of the glycolate pathway. Serine and glycerate production was not inhibited. We conclude that serine and glycerate were made from 3-phosphoglycerate and not from glycolate and that the conversion of glycine to serine may not be the major source of photorespiratory CO(2) in alfalfa. In investigations of glycolate and photorespiratory metabolism, separate labeling data should be obtained for glycine and serine as those two amino acids may be produced from different precursors and respond differently to environmental perturbations. The increased photosynthetic rate (at saturating CO(2)) resulted in greater labeling of both soluble and insoluble products. Sucrose labeling increased sharply, but there was no major shift of tracer carbon flow into sucrose relative to other metabolites. The flow of carbon from the reductive pentose phosphate cycle into the production of tricarboxylic acid cycle intermediates and amino acids increased. Only small absolute increases occurred in steady-state pool sizes of metabolites of the reductive pentose phosphate cycle at elevated CO(2), providing further evidence that the cycle is well regulated.

Increasing Photosynthesis by Inhibiting Photorespiration with Glyoxylate

Glyoxylate treatment doubles net photosynthetic carbon dioxide fixation by tobacco leaf disks because inhibition of glycolate synthesis by glyoxylate results in decreased photorespiration. These observations show that photorespiration can be metabolically regulated and suggest that genetic or chemical alteration of pool sizes of certain metabolites can produce plants with increased photosynthesis.

Optimizing the sensitivity of enzyme systems to perturbations from equilibrium

Algebraic derivations and numerical examples illustrate how metabolite pool sizes and enzyme rate constants influence the rate at which a multireactant enzyme system, initially poised in a near-equilibrium steady state, responds to small perturbations in the concentrations of the reactants. Certain enzymes, such as those employing the ordered bi bi catalytic mechanism, become relatively insensitive to perturbations when the reactants are all present at high concentrations. Other enzymes, such as those employing the ping-pong bi bi mechanism, are most sensitive to perturbations at high reactant concentrations. The ratio of the reactant concentrations to one another significantly alters sensitivity to perturbations; equations are presented for calculation of the reactant concentrations yielding maximal sensitivity to perturbations. Natural selection could choose metabolite pool sizes and enzyme rate constants which would optimize the performance of these systems, but changing metabolic loads (naturally or experimentally imposed) constantly alter the sensitivity of these systems to perturbations, changing the relative strengths of various connections in metabolic control networks.

Dynamics of metabolism of normal and virus-transformed chick cells in culture.

Application of steady-state tracer technique to normal and transformed cells in tissue culture allows quantitation of intracellular pool sizes of many metabolites and determination of rate of carbon flow along diverse paths. Using a unique apparatus to control the environmental conditions, we show that the glucose carbon flow into tricarboxylic acid cycle intermediates and amino acids is unchanged upon transformation. The increased glycogen formation and glycolysis varies with the glucose concentration in the medium, correlates with the faster glucose transport of transformed cells, and cannot be explained by a difference in growth rate alone.