R. G. Ratcliffe

Glycolytic Enzymes Associate Dynamically with Mitochondria in Response to Respiratory Demand and Support Substrate Channeling

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using 13C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from 13C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells

Abstract. The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P2) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P2 were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P2 had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-13C]glucose, in the lines possessing the highest amounts of Fru-2,6-P2, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of 14C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P2 can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates.

Revealing metabolic phenotypes in plants: inputs from NMR analysis.

Assessing the performance of the plant metabolic network, with its varied biosynthetic capacity and its characteristic subcellular compartmentation, remains a considerable challenge. The complexity of the network is such that it is not yet possible to build large‐scale predictive models of the fluxes it supports, whether on the basis of genomic and gene expression analysis or on the basis of more traditional measurements of metabolites and their interconversions. This limits the agronomic and biotechnological exploitation of plant metabolism, and it undermines the important objective of establishing a rational metabolic engineering strategy. Metabolic analysis is central to removing this obstacle and currently there is particular interest in harnessing high‐throughput and/or large‐scale analyses to the task of defining metabolic phenotypes. Nuclear magnetic resonance (NMR) spectroscopy contributes to this objective by providing a versatile suite of analytical techniques for the detection of metabolites and the fluxes between them.

Strategies for metabolic flux analysis in plants using isotope labelling.

Flux measurements through metabolic pathways generate insights into the integration of metabolism, and there is increasing interest in using such measurements to quantify the metabolic effects of mutation and genetic manipulation. Isotope labelling provides a powerful approach for measuring metabolic fluxes, and it gives rise to several distinct methods based on either dynamic or steady-state experiments. We discuss the application of these methods to photosynthetic and non-photosynthetic plant tissues, and we illustrate the different approaches with an analysis of the pathways interconverting hexose phosphates and triose phosphates. The complicating effects of the pentose phosphate pathway and the problems arising from the extensive compartmentation of plant cell metabolism are considered. The non-trivial nature of the analysis is emphasised by reference to invalid deductions in earlier work. It is concluded that steady-state isotopic labelling experiments can provide important information on the fluxes through primary metabolism in plants, and that the combination of stable isotope labelling with detection by nuclear magnetic resonance is particularly informative.

In vivo NMR studies of higher plants and algae

Publisher Summary This chapter discusses in vivo nuclear magnetic resonance (NMR) studies of higher plants and algae. As the opportunity arose for biologists to apply the emerging techniques of NMR spectroscopy to systems of biological interest, it was perhaps inevitable that they would first use NMR to study the properties of water in cells and tissues. The advantages of studying the water non-invasively, in an unperturbed system, were apparently only partly offset by the problems of interpretation that arose from the heterogeneity of living systems and a considerable literature developed in this field. High-resolution multinuclear NMR spectroscopy permits the detection of certain ions and metabolites in vivo, as well as the tissue water, and thus increases the potential enormously for tackling biochemical and physiological problems non-invasively; while NMR imaging, although still relying on the detection of the water signal, provides a method for mapping the spatial distribution of the water in the sample. The potential importance of these techniques to biologists and physiologists meant that their interests and requirements began to be reflected in the design of NMR equipment and this accelerated the application of the new techniques to physiological problems. In vivo NMR studies have always emphasized the non-invasive character of the investigation and the chapter illustrates how this important property is exploited in studies of higher plants and algae.

Ammonium Assimilation and the Role of [gamma]-Aminobutyric Acid in pH Homeostasis in Carrot Cell Suspensions

In vivo 15N NMR spectroscopy was used to monitor the assimilation of ammonium by cell-suspension cultures of carrot (Daucus carota L. cv Chantenay). The cell suspensions were supplied with oxygen in the form of either pure oxygen (“oxygenated cells”) or air (“aerated cells”). In contrast to oxygenated cells, in which ammonium assimilation had no effect on cytoplasmic pH, ammonium assimilation by aerated cells caused a decrease in cytoplasmic pH of almost 0.2 pH unit. This led to a change in nitrogen metabolism resulting in the accumulation of [gamma]-aminobutyric acid. The metabolic effect of the reduced oxygen supply under aerated conditions could be mimicked by artificially decreasing the cytoplasmic pH of oxygenated cells and was abolished by increasing the cytoplasmic pH of aerated cells. The activity of glutamate decarboxylase increased as the cytoplasmic pH declined and decreased as the pH recovered. These findings are consistent with a role for the decarboxylation of glutamate, a proton-consuming reaction, in the short-term regulation of cytoplasmic pH, and they demonstrate that cytoplasmic pH influences the pathways of intermediary nitrogen metabolism.

Manipulating cytoplasmic pH under anoxia: A critical test of the role of pH in the switch from aerobic to anaerobic metabolism

Ethanol production by maize (Zea mays L.) root tips, measured by an enzymic assay of the suspending medium, was correlated with changes in the cytoplasmic pH, determined by in-vivo 31P nuclear magnetic resonance (NMR) spectroscopy, following the onset of anoxia. Strong evidence for the role of the cytoplasmic pH in triggering the switch to ethanol production under anoxia was obtained by: (i) varying the pH of the suspending medium between pH 4 and pH 10; and (ii) using the permeant weak base methylamine to combat the acidification of the cytoplasm induced by the anoxic conditions. Experimentally, it proved to be much easier to manipulate the cytoplasmic pH under anoxia after the pH had stabilised, rather than during the initial rapid acidification that occurred following the onset of anoxia, and in the presence of methylamine, it was possible to impose a normal aerobic cytoplasmic pH value on tissue that was metabolising anaerobically. By this means it was possible to demonstrate the reversibility of the pH effect on ethanol production under anoxia and thus to provide good evidence in support of the biochemical pH-stat model of the anoxic response. The NMR measurement of the cytoplasmic pH in the presence of methylamine was achieved by using a manganese pretreatment technique to eliminate interference between the cytoplasmic and vacuolar Pi signals, and it seems likely that the experimental approach used here will have further applications in studies of the metabolic response to anoxia.

OBSERVATIONS ON THE SUBCELLULAR DISTRIBUTION OF THE AMMONIUM ION IN MAIZE ROOT TISSUE USING IN-VIVO 14N-NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

We show that the pH dependence of the base-catalysed exchange rate of the ammonium ion provides a basis for discriminating between the cytoplasmic and vacuolar pools of ammonium in plant tissues. In vivo, 14N-nuclear magnetic resonance spectra were recorded with and without 1H-decoupling and information on the subcellular distribution of NH4+was obtained from a lineshape analysis of the 1H-coupled spectrum. We applied this method to maize (Zea mays L.) root tissues and found that: (i), the cytoplasmic ammonium concentration was low, which was in accord with the large activity of glutamine synthetase present in the roots; and (ii), inhibition of glutamine synthetase with methionine sulphoximine increased the cytoplasmic ammonium concentration, and led to the appearance of ammonium in the xylem sap.

Interpretation of the pH dependence of protein adsorption on clay mineral surfaces and its relevance to the understanding of extracellular enzyme activity in soil

Abstract The pH dependence of the adsorption of bovine serum albumin (BSA) and Aspergillus niger β- d -glucosidase on montmorillonite and of its structural consequences was studied by several methods in order to understand the mechanisms of interaction between extracellular enzymes and clay minerals in soils. The relative influence of electrostatic and hydrophobic interactions on adsorption was deduced from experiments involving the coadsorption of BSA and methylated BSA. Also, the surface coverage of the clay by the BSA was studied by following the release of a paramagnetic charge-compensating cation on adsorption of the protein. This method is based on the line broadening effect of the released cation on the 31P NMR signal from phosphate in the solution, and the specific interfacial area of the BSA was deduced from the ratio of the surface covered by the quantity of protein adsorbed. Information on the secondary and overall structure of adsorbed BSA was obtained by Fourier transform IR spectroscopy from the frequency range and line width of the amide I/I′ signal and from the intensity of the COOH band. Finally, the catalytic activity of the β- d -glucosidase adsorbed on the clay was compared with its activity in solution, and the pH dependence of the adsorption was measured. The following general conclusions could be drawn from these experiments. (i) Below the isoelectric point (IEP), proteins unfold on the clay surfaces in response to electrostatic attractive interactions, a phenomenon which inhibits enzyme activity. (ii) Near the IEP proteins are adsorbed with little modification of conformation and thus enzymes preserve their catalytic activities. (iii) Above the IEP the proportion of adsorbed proteins decreases due to electrostatic repulsive interactions, permitting the diffusion of enzymes in the liquid-filled pore network of the soil.

A 31P NMR study of the adsorption of bovine serum albumin on montmorillonite using phosphate and the paramagnetic cation Mn2+: modification of conformation with pH

Abstract The specific interfacial area of bovine serum albumin (BSA) adsorbed on montmorillonite was deduced from the ratio between the quantity of cations exchanged on adsorption of BSA and the quantity of protein bound. A paramagnetic cation, Mn2+, was used, and its release from the clay surface was followed by measuring the line broadening effect of displaced Mn2+ on the 31P NMR signal from orthophosphate in the solution. From experiments conducted at different protein/clay ratios and different pH, it was deduced: (i) that no more than one monolayer was adsorbed; (ii) that the specific interfacial area was the same at low and at high surface coverage; (iii) that below the isoelectric point (i.e.p.) the specific interfacial area of BSA increased with decreasing pH, with a constant surface coverage of the clay surface; and (iv) that above the i.e.p. the surface coverage of the clay decreased with further increases in pH. It is assumed that electrostatic interactions between the protein and the clay surface play a major role in these phenomena. The correlation with the behavior of extracellular enzymes in soils is emphasized.