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Regulation of carbohydrate metabolism
Carbohydrates are the major respiratory substrate in most plants, providing both the energy and metabolic precursors for growth. We are interested in understanding how the pathways of carbohydrate synthesis and degradation are regulated to meet the requirements of the plant in response to changing environmental and developmental demands. Our work focuses on integration of plastidic and cytosolic metabolism in both photosynthetic and non-photosynthetic tissues.
Our previous studies have established that fructose 2,6-bisphosphate (Fru-2,6-P2), a potent inhibitor of cytosolic fructose 1,6-bisphophatase, is important in regulating two aspects of photosynthesis; one is coordination of the rates of product formation and CO2 fixation, the other is the partitioning of photoassimilate between sucrose (formed in the cytosol) and starch (synthesised in the chloroplast). Transgenic tobacco plants expressing modified copies of a mammalian gene encoding the bifunctional enzyme responsible for Fru-2,6-P2 metabolism, and thus possessing altered steady-state levels of this metabolite, have allowed direct quantification of the contribution of Fru-2,6-P2 to the control of these processes. We are currently examining further aspects of metabolic integration between chloroplasts and cytosol by exploiting an Arabidopsis mutant that fails to accumulate the triose-phosphate translocator of the inner chloroplast envelope. Our latest results indicate that perturbing the pathways of carbon metabolism influences photosynthetic acclimation (the ability to respond to altered light intensity) through a novel mechanism. We are extending these studies through the use of recently identified mutations in the Arabidopsis gene for Fru-2,6-P2 synthesis and degradation.
In heterotrophic cells, the oxidative pentose phosphate pathway (oxPPP) is the principal source of NADPH for biosynthetic reductions and, using a novel technique based on metabolism of [1-14C] gluconate, we have demonstrated that this pathway is also the major source of reductant needed to protect against oxidative stress. In studies of maize mutants possessing null-activity alleles of each of the genes encoding the cytosolic isoforms of 6-phosphogluconate dehydrogenase, we have shown that the cytosolic and plastidic oxPPP cooperate in the provision of cellular reductant. Currently, we are developing techniques based on metabolic network analysis to discriminate between fluxes through pathways of carbohydrate oxidation that are duplicated in the cytosol and plastids. This work, performed in collaboration with Prof R.G. Ratcliffe, involves determining the specific abundance of 13C in individual carbon atoms of a range of metabolic end-products by NMR spectroscopy after feeding with 13C-glucose. Application of this technique to transgenic cell lines possessing altered levels of Fru-2,6-P2 has established that this metabolite regulates interconversion of hexose-phosphates and triose-phosphates in the cytosol through modulation of pyrophosphate:fructose 6-phosphate phosphotransferase activity. Furthermore, using this approach, we have discovered that plastidic oxPPP is the dominant route of carbohydrate oxidation in the root tips of maize seedlings. Techniques to resolve subcellular fluxes in vivo, which we are continuing to develop, will be used to establish the metabolic significance of the unique subcellular organisation of the pathways of carbohydrate oxidation in plants.
A.J. McCormick and N.J. Kruger (2015) Lack of fructose 2,6-bisphosphate compromises photosynthesis and growth in Arabidopsis in fluctuating environments. Plant Journal (in press).
N.J. Kruger and R.G. Ratcliffe (2015) Fluxes through plant metabolic networks: measurements, predictions, insights and challenges. Biochemical Journal 456, 27-38.
K.J. Gupta, K.H. Hebelstrup, N.J. Kruger and R.G. Ratcliffe (2014) Nitric oxide is required for homeostasis of oxygen and reactive oxygen species in barley roots under aerobic conditions. Molecular Plant 7, 747-750.
杨亦扬，马立锋，黎星辉，R.G.Ratcliffe，N.J. Kruger, 阮建云 (2013) 氮素水平对茶树新梢叶片代谢谱及其昼夜变化的影响. 茶叶科学 33, 491-499.
Full Publication List (while at this department)
Kruger, N.J, Ratcliffe, R.G. (2015) Fluxes through plant metabolic networks: Measurements, predictions, insights and challenges Biochemical Journal. 465: pp 27-38.
McCormick, A.J, Kruger, N.J. (2015) Lack of fructose 2,6-bisphosphate compromises photosynthesis and growth in Arabidopsis in fluctuating environments Plant Journal. 81 (5): pp 670-683.
Kruger, N.J, Masakapalli, S.K, Ratcliffe, R.G. (2014) Optimization of steady-state 13C-labeling experiments for metabolic flux analysis Methods in Molecular Biology. Eds. Dieuaide-Noubhani M.Alonso A.P.. ISBN: 9781627036870. pp 53-72.
Masakapalli, S.K, Bryant, F.M, Kruger, N.J, Ratcliffe, R.G. (2014) The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a flexible balance between the cytosolic and plastidic contributions to carbohydrate oxidation in response to phosphate limitation Plant Journal. 78 (6): pp 964-977.
Masakapalli, S.K, Kruger, N.J, Ratcliffe, R.G. (2013) The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a complex response to changes in nitrogen supply Plant Journal..
Morgan, M.J, Osorio, S, Gehl, B, Baxter, C.J, Kruger, N.J, George, Ratcliffe R, Fernie, A.R, Sweetlove, L.J. (2013) Metabolic engineering of tomato fruit organic acid content guided by biochemical analysis of an introgression line Plant Physiology. 161 (1): pp 397-407.
Kruger, N.J, Ratcliffe, R.G. (2012) Pathways and fluxes: Exploring the plant metabolic network Journal of Experimental Botany. 63 (6): pp 2243-2246.
Kruger, N.J, Masakapalli, S.K, Ratcliffe, R.G. (2012) Strategies for investigating the plant metabolic network with steady-state metabolic flux analysis: Lessons from an Arabidopsis cell culture and other systems Journal of Experimental Botany. 63 (6): pp 2309-2323.
Smith, A.M, Kruger, N.J, Lunn, J.E. (2012) Source of sugar nucleotides for starch and cellulose synthesis Proceedings of the National Academy of Sciences of the United States of America. 109 (14):.
Beste, D.J.V, Bonde, B, Hawkins, N, Ward, J.L, Beale, M.H, Noack, S, Noh, K, Kruger, N.J, Ratcliffe, R.G, McFadden, J. (2011) 13c metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation PLoS Pathogens. 7 (7):.
Howard, T.P, Fryer, M.J, Singh, P, Metodiev, M, Lytovchenko, A, Obata, T, Fernie, A.R, Kruger, N.J, Quick, W.P, Lloyd, J.C, Raines, C.A. (2011) Antisense suppression of the small chloroplast protein cp12 in tobacco alters carbon partitioning and severely restricts growth Plant Physiology. 157 (2): pp 620-631.
Andriotis, V.M.E, Kruger, N.J, Pike, M.J, Smith, A.M. (2010) Plastidial glycolysis in developing Arabidopsis embryos New Phytologist. 185 (3): pp 649-662.
Masakapalli, S.K, le, Lay P, Huddleston, J.E, Pollock, N.L, Kruger, N.J, Ratcliffe, R George. (2010) Subcellular flux analysis of central metabolism in a heterotrophic Arabidopsis cell suspension using steady-state stable isotope labeling Plant Physiology. 152 (2): pp 602-619.
Couldwell, D.L, Dunford, R, Kruger, N.J, Lloyd, D.C, Ratcliffe, R.G, Smith, A.M.O. (2009) Response of cytoplasmic pH to anoxia in plant tissues with altered activities of fermentation enzymes: Application of methyl phosphonate as an NMR pH probe Annals of Botany. 103 (2): pp 249-258.
Kruger, N.J, Ratcliffe, R.G. (2009) Insights into plant metabolic networks from steady-state metabolic flux analysis Biochimie. 91 (6): pp 697-702.
Troncoso-Ponce, M.A, Kruger, N.J, Ratcliffe, R.G, Garces, R, Martinez-Force, E. (2009) Characterization of glycolytic initial metabolites and enzyme activities in developing sunflower (Helianthus annuus L.) seeds. Phytochemistry. 70 (9): pp 1117-1122.
Harrison, P.W, Kruger, N.J. (2008) Validation of the design of feeding experiments involving [14C]substrates used to monitor metabolic flux in higher plants Phytochemistry..
Kruger, N.J, Troncoso-Ponce, M.A, Ratcliffe, R.G. (2008) 1H NMR metabolite fingerprinting and metabolomic analysis of perchloric acid extracts from plant tissues Nature Protocols. 3 (6): pp 1001-1012.
Kruger, N.J., Ratcliffe, R.G. (2008) Metabolic organization in plants: a challenge for the metabolic engineer. Advances in Plant Biochemistry and Molecular Biology : Bioengineering and Molecular Biology of Plant Pathways. 1: pp 1-27.
Williams, T.C.R, Miguet, L, Masakapalli, S.K, Kruger, N.J, Sweetlove, L.J, Ratcliffe, R.G. (2008) Metabolic network fluxes in heterotrophic arabidopsis cells: Stability of the flux distribution under different oxygenation conditions Plant Physiology. 148 (2): pp 704-718.
Bieniawska, Z, Paul, Barratt D.H, Garlick, A.P, Thole, V, Kruger, N.J, Martin, C, Zrenner, R, Smith, A.M. (2007) Analysis of the sucrose synthase gene family in Arabidopsis Plant Journal. 49 (5): pp 810-828.
Kruger, N.J, Ratcliffe, R.G. (2007) Dynamic metabolic networks: Going with the flow Phytochemistry. 68: pp 2136-2138.
Kruger, N.J, Huddleston, J.E, Le, Lay P, Brown, N.D, Ratcliffe, R.G. (2007) Network flux analysis: Impact of 13C-substrates on metabolism in Arabidopsis thaliana cell suspension cultures Phytochemistry. 68: pp 2176-2188.
Kruger, N.J, Le, Lay P, Ratcliffe, R.G. (2007) Vacuolar compartmentation complicates the steady-state analysis of glucose metabolism and forces reappraisal of sucrose cycling in plants Phytochemistry. 68: pp 2189-2196.
Malone, J.G, Mittova, V, Ratcliffe, R.G, Kruger, N.J. (2006) The response of carbohydrate metabolism in potato tubers to low temperature Handbook of Environmental Chemistry, Volume 5: Water Pollution. 47 (9): pp 1309-1322.
Krishnan, P, Kruger, N.J, Ratcliffe, R.G. (2005) Metabolite fingerprinting and profiling in plants using NMR Journal of Experimental Botany. 56 (410): pp 255-265.
Walters, R.G, Ibrahim, D.G, Horton, P, Kruger, N.J. (2004) A mutant of arabidopsis lacking the triose-phosphate/phosphate translocator reveals metabolic regulation of starch breakdown in the light Plant Physiology. 135 (2): pp 891-906
Kruger, N.J, Ratcliffe, R.G, Roscher, A. (2003) Quantitative approaches for analysing fluxes through plant metabolic networks using NMR and stable isotope labelling Phytochemistry Reviews. 2: pp 17-30
Kruger, N.J, Von, Schaewen A. (2003) The oxidative pentose phosphate pathway: Structure and organisation Current Opinion in Plant Biology. 6 (3): pp 236-246.
Fernie, A.R, Roscher, A, Ratcliffe, R.G, Kruger, N.J. (2002) Activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase by fructose 2,6-bisphosphate stimulates conversion of hexose phosphates to triose phosphates but does not influence accumulation of carbohydrates in phosphate-deficient tobacco cells Physiologia Plantarum. 114 (2): pp 172-181.
Garlick, A.P, Moore, C, Kruger, N.J. (2002) Monitoring flux through the oxidative pentose phosphate pathway using [1-14C]gluconate Planta. 216 (2): pp 265-272.
Markham, J.E, Kruger, N.J. (2002) Kinetic properties of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves European Journal of Biochemistry. 269 (4): pp 1267-1277.
Fernie, A.R, Roscher, A, Ratcliffe, R.G, Kruger, N.J. (2001) Fructose 2,6-bisphosphate activates pyrophosphate: Fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling heterotrophic cells Planta. 212 (2): pp 250-263.
Sweetlove, L.J, Kruger, N.J, Hill, S.A. (2001) Starch synthesis in transgenic potato tubers with increased 3-phosphoglyceric acid content as a consequence of increased 6-phosphofructokinase activity Planta. 213 (3): pp 478-482.
Theodorou, M.E, Kruger, N.J. (2001) Physiological relevance of fructose 2,6-bisphosphate in the regulation of spinach leaf pyrophosphate:fructose 6-phosphate 1-phosphotransferase Planta. 213 (1): pp 147-157.
Roscher, A, Kruger, N.J, Ratcliffe, R.G. (2000) Strategies for metabolic flux analysis in plants using isotope labelling Journal of Biotechnology. 77 (1): pp 81-102.
Scott, P, Lange, A.J, Kruger, N.J. (2000) Photosynthetic carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing decreased amounts of fructose 2,6-bisphosphate Planta. 211 (6): pp 864-873
Sweetlove, L.J, Dunford, R, Ratcliffe, R.G, Kruger, N.J. (2000) Lactate metabolism in potato tubers deficient in lactate dehydrogenase activity Plant, Cell and Environment. 23 (8): pp 873-881.
Kruger, NJ, Hill, SA, Ratcliffe, RG. (1999) Regulation of Primary Metabolic Pathways in Plants Proceedings of the Phytochemical Society of Europe (volume 42). Kluwer Academic Publishers, Dordrecht, The Netherlands. ISBN: 0-7923-5494-X.
Runquist, M, Kruger, N.J. (1999) Control of gluconeogenesis by isocitrate lyase in endosperm of germinating castor bean seedlings Plant Journal. 19 (4): pp 423-431.
Averill, R.H, Bailey-Serres, J, Kruger, N.J. (1998) Co-operation between cytosolic and plastidic oxidative pentose phosphate pathways revealed by 6-phosphogluconate dehydrogenase-deficient genotypes of maize Plant Journal. 14 (4): pp 449-457.
Kruger, N.J. (1995) Errors and artifacts in coupled spectrophotometric assays on enzyme activity Phytochemistry. 38 (5): pp 1065-1071.
Scott, P, Kruger, N.J. (1995) Influence of elevated fructose-2,6-bisphosphate levels on starch mobilization in transgenic tobacco leaves in the dark Plant Physiology. 108 (4): pp 1569-1577
Scott, P, Lange, A.J, Pilkis, S.J, Kruger, N.J. (1995) Carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing elevated fructose 2,6-bisphosphate levels Plant Journal. 7 (3): pp 461-469
Kruger, N.J. (1994) Detection of polypeptides on immunoblots using secondary antibodies or protein A. Methods in molecular biology (Clifton, N.J.). 32: pp 215-226
Kruger, N.J. (1994) The Bradford method for protein quantitation. Methods in molecular biology (Clifton, N.J.). 32: pp 9-15
Kruger, N.J, Scott, P. (1994) Manipulation of fructose-2,6-bisphosphate levels in transgenic plants Biochemical Society Transactions. 22 (4): pp 904-909
Scott, P, Kruger, N.J. (1994) Fructose 2,6-bisphosphate levels in mature leaves of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) Planta. 193 (1): pp 16-20
Thomas, S, Kruger, N.J. (1994) Source of apparent ADP-dependent phosphofructokinase activity in plant extracts Plant Science. 95 (2): pp 133-139.
Montavon, P, Kruger, N.J. (1993) Essential arginyl residue at the active site of pyrophosphate:fructose 6-phosphate 1-phosphotransferase from potato (Solanum tuberosum) tuber Plant Physiology. 101 (3): pp 765-771
Castrillo, M, Kruger, N.J, Whatley, F.R. (1992) Sucrose metabolism in mango fruit during ripening Plant Science. 84 (1): pp 45-51
Montavon, P, Kruger, N.J. (1992) Substrate specificity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase from potato tuber Plant Physiology. 99 (4): pp 1487-1492
Rowntree, E.G, Kruger, N.J. (1992) Inhibition of pyrophosphate:Fructose-6-phosphate 1-phosphotransferase by imidodiphosphate Plant Science. 86 (2): pp 183-189