Steven A. Hill

Fluxes of carbohydrate metabolism in ripening bananas

The major fluxes of carbohydrate metabolism were estimated during starch breakdown by ripening bananas (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21° C. Production of CO2 and the contents of starch, sucrose, glucose and fructose of intact fruit were determined for a period of 10 d that included the climacteric. The detailed distribution of label was determined after supplying the following to cores of pulp from climacteric fruit: [U-14C]-, [1-14C]-, [3,4-14C]-and [6-14C]glucose, [U-14C]glycerol, 14CO2. The data obtained were used to estimate the following fluxes, values given as μmol hexose · (g FW)−1 · h−1 in parenthesis: starch to hexose monophosphates (5.9) and vice versa (0.4); hexose monophosphates to sucrose (7.7); sucrose to hexose (4.7); hexose to hexose monophosphate (3.8); glycolysis (0.5–1.6); triose phosphate to hexose monophosphates (0.14); oxidative pentose-phosphate pathway (0.48); CO2 fixation in the dark (0.005). These estimates are related to our understanding of carbohydrate metabolism during ripening.

The contribution of adenosine 5'-diphosphoglucose pyrophosphorylase to the control of starch synthesis in potato tubers

Abstract. The aim of this work was to investigate the extent to which starch synthesis in potato (Solanum tuberosum L.) tubers is controlled by the activity of ADPglucose pyrophosphorylase (EC 2.7.7.27; AGPase). In order to do this, fluxes of carbohydrate metabolism were measured in tubers that had reduced AGPase activity as a result of the expression of a cDNA encoding the B subunit in the antisense orientation. Reduction in AGPase activity led to a reduction in starch accumulation, and an increase in sucrose accumulation. The control coefficient of AGPase on starch accumulation in intact plants was estimated to be around 0.3. The fluxes of carbohydrate metabolism were measured in tuber discs from wild-type and transgenic plants by investigating the metabolism of [U-14C]glucose. In tuber discs, the control coefficient of AGPase over starch synthesis was estimated as 0.55, while the control coefficient of the enzyme over sucrose synthesis was −0.47. The values obtained suggest that AGPase activity exerts appreciable control over tuber metabolism in potato.

The control of source to sink carbon flux during tuber development in potato

We have used top-down metabolic control analysis to investigate the control of carbon flux through potato (Solanum tuberosum) plants during tuberisation. The metabolism of the potato plant was divided into two blocks of reactions (the source and sink blocks) that communicate through the leaf apoplastic sucrose pool. Flux was measured as the transfer of 14 C from CO2 to the tuber. Flux and apoplastic sucrose concentration were varied either by changing the light intensity or using transgenic manipulations that specifically affect the source or sink blocks, and elasticity coefficients were measured. We have provided evidence in support of our assumption that apoplastic sucrose is the only communicating metabolite between the source and sink blocks. The elasticity coefficients were used to calculate the flux control coefficients of the source and sink blocks, which were 0.8 and 0.2, respectively. This work suggests that the best strategy for the manipulation of tuber yield in potato will involve increases in photosynthetic capacity, rather than sink metabolism.

The effect of hypoxia on the control of carbohydrate metabolism in ripening bananas

The effect of exogenous glucose on the major fluxes of carbohydrate metabolism in cores of climacteric fruit of banana (Musa cavendishii Lamb ex Paxton) was determined with the intention of using the effects in the application of top-down metabolic control analysis. Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21 °C. Cores were removed from climacteric fruit and incubated in 100 or 200 mM glucose for 4 or 6 h. The rates of starch breakdown, sucrose and fructose accumulation and CO2 production were measured. The steady-state contents of hexose monophosphates, adenylates and pyruvate were determined. In addition, the detailed distribution of label was determined after supply of the following: [U-14C]-, [1-14C]-, [3,414C]and [6-14C]glucose, and [U-14C]glycerol. The data were used to estimate the major fluxes of carbohydrate metabolism. Supply of exogenous glucose led to increases in the size of the hexose-monophosphate pools. There was a small stimulation of the rate of sugar synthesis and a major increase in the rate of starch synthesis. Starch breakdown was inhibited. Respiration responded to the demand for ATP by sugar synthesis. The effect of glucose on fluxes and metabolite pools is discussed in relation to our understanding of the control and regulation of carbohydrate metabolism in ripening fruit.

Regulation of primary metabolic pathways in plants

Rubisco: Attempts to Reform a Promiscuous Enzyme M.A.J. Parry, et al. 2. Insights into the Active Site of the Plant Alternative Oxidase and its Relationship to Function C. Affourtit, A.L. Moore. 3. The Many-Faceted Function of Phosphoenolpyruvate Carboxykinase in Plants R.C. Leegood, et al. 4. Folate Synthesis and Compartmentation in Higher Plants F. Rebeille, R. Douce. 5. Structure and Function of Plastid Metabolite Transporters U.-I. Flugge, et al. 6. Integration of Metabolism within Non-Photosynthetic Plastids, and with the Cytosol M.J. Emes, et al. 7. Carbon Flux to Fatty Acids in Plastids S. Rawsthorne, et al. 8. Compartmentation of Metabolites between the Subcellular Compartments of leaves, the Apoplast, the Phloem and the Storage Tissue of Different Crop Plants G. Lohaus, et al. 9. Regulation of Starch Synthesis in Storage Organs A.M. Smith. 10. The Integration of Sucrose and Fructan Metabolism in Temperate Grasses and Cereals C.J. Pollock, et al. 11. Expression of Fructosyltransferase Genes in Transgenic Plants I. Vijn, et al. 12. The Application of Transgenic Technology to the Study of Sink Metabolism in Potato R. Trethewey, L. Willmitzer. 13. Increasing the Flux in a Metabolix Pathway: A Metabolic Control Analysis Perspective D.A. Fell, S. Thomas. 14. Nitrate Acts as a Signal to Control Gene Expression, Metabolism and Biomass Allocation M. Stitt, W.-R. Scheible.

Control of succinate oxidation by cucumber (Cucumis sativus L.) cotyledon mitochondria: The role of the adenine-nucleotide translocator and extra-mitochondrial reactions

The aim of this work was to assess the extent to which mitochondria control the gluconeogenic flux in cucumber (Cucumis sativus L.) cotyledons, by quantifying the distribution of control of succinate oxidation by cotyledon mitochondria. The methods of metabolic control analysis were applied under state 3 and state 4 conditions and in the presence of cell-free extracts in order to simulate in-vivo conditions. Oxygen uptake by isolated cotyledon mitochondria oxidising succinate under state 3 conditions was examined using inhibitor titrations. During lipid mobilisation in light-grown cotyledons (3-4 d post-imbibition), control was shared between the adenine-nucleotide translocator (flux-control coefficient, C = 0.25–0.28) and the dicarboxylate-uptake system (C = 0.69–0.72). The dicarboxylate-uptake system was also important in dark-grown cotyledons at this stage (C = 0.55–0.57). In the photosynthetic phase of development (more than 5 d post-imbibition) control rested with the respiratory chain. Application of an external ATP demand provided either by cell-free extracts of cucumber cotyledons or a glucose/hexokinase ADP-regenerating system showed that the reactions outside the mitochondria exert control (C = 0.45–0.54 and C = 0.24–0.38, for cytosolic extract and glucose/hexokinase, respectively). The adenine-nucleotide translocator was a controlling step of both oxygen uptake (C = 0.11–0.32) and the flux between succinate and hexose phosphates (C = 0.28). Other mitochondrial steps made a significant contribution to control. Control of oxygen uptake was dependent on both the nature of the external load and on the rate of phosphorylation. A potential role for mitochondrial membrane-transport processes, including the adenine-nucleotide translocator, is proposed for the integration of lipid breakdown and gluconeogenesis in vivo.

Mitochondrial Pyruvate Dehydrogenase (Molecular Cloning of the E1[alpha] Subunit and Expression Analysis)

A polymerase chain reaction-based approach was used to isolate cDNA clones encoding the E1[alpha] subunit of the mitochondrial pyruvate dehydrogenase from higher plants. Putative full-length clones were identified on the basis of similarity to E1[alpha] sequences from nonplant sources. Southern blot analysis revealed a small family of genes in potato (Solanum tuberosum L.), whereas in cucumber (Cucumis sativus) there are only one or two genes. Tissue-spefific variation in the relative amounts of E1[alpha] mRNA was observed in northern blot analysis of different potato tissues, with the highest steady-state transcript levels found in floral tissue. Measurement of pyruvate dehydrogenase activity in cucumber cotyledons showed that there is a transient increase to a maximum at 4 to 5 d postimbibition. Western blot analysis revealed that the amount of E1[alpha] protein also peaks at this time. Steady-state transcript levels in germinating cucumber cotyledons also show transient accumulation, peaking 2 d postimbibition. These data are consistent with regulation of E1[alpha] at the level of transcription and/or mRNA stability in postgerminative cucumber cotyledons.

The feeding behaviour of Schistocerca gregaria, the desert locust, on two starch mutants of Arabidopsis thaliana

Summary.Schistocerca gregaria, the desert locust, has been shown to regulate its dietary intake with respect to specific macronutrients in synthetic foods. This study examined the nutrients in the leaves of two starch mutants of Arabidopsis thaliana, and then compared the feeding behaviour of locusts on the two starch mutants. The high-starch mutant had c. 25 times more starch than the no-starch mutant. Newly molted 5th stadium locusts were preconditioned for 3 days on one of the mutants, and then observed for 90 min while exposed to the same or the alternative mutant. Locusts pretreated with the no-starch mutant fed longer during the first meal on high-starch mutants, spent more time feeding, and had the smaller latency to begin a meal when compared to the locusts pretreated on the high-starch mutant. The results of the study are interpreted in light of an integrative model of nutrient balancing.

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