Abstract
To assess the effectiveness of manipulating citrate metabolism with the aim of increasing citrate efflux from roots, we generated transgenic tobacco (Nicotiana tabacum L.) lines that either overexpressed mitochondrial citrate synthase (EC 4.1.3.7) activity or had reduced activity of cytosolic isocitrate dehydrogenase (EC 1.1.1.42). Despite increases in citrate synthase activities in transgenic lines of up to 5-fold, neither internal citrate concentrations nor citrate efflux were increased compared to controls suggesting that, in tobacco, citrate synthase activity does not directly determine citrate accumulation and efflux. Consistent with a lack of effect on citrate efflux, the increase in citrate synthase activity did not enhance the aluminium resistance of the transgenic lines. Preliminary data collected on two transgenic lines with cytosolic isocitrate dehydrogenase activities reduced to one-tenth and one third of the control for shoot and root tissues respectively, showed that while these changes in activities were associated with a 1.5-fold increase in internal citrate concentrations of both types of tissue, citrate efflux from roots was not increased. Further work is needed to establish whether the increase in internal citrate concentration is associated with enhanced aluminium resistance of these lines. We conclude that in tobacco internal citrate concentrations and citrate efflux are largely insensitive to large changes in either mitochondrial citrate synthase or cytosolic isocitrate dehydrogenase activities and suggest that other factors, such as transport out of the roots, control citrate efflux.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bradford M M 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
de la Fuente J M, Ramírez-Rodríguez V, Cabrera-Ponce J L and Herrera-Estrella L 1997 Aluminum tolerance in transgenic plants by alteration of citrate synthesis. Science 276, 1566–1568.
Delhaize E, Craig S, Beaton C D, Bennet R J, Jagadish V C and Randall P J 1993 Aluminum tolerance in wheat (Triticum aestivum L.) I. Uptake and distribution of aluminum in root apices. Plant Physiol. 103, 685–693.
Delhaize E, Hebb D M and Ryan P R 2000 Expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco is not associated with either enhanced citrate accumulation or efflux. Plant Physiol. 125, 2059–2067.
Fieuw S, Müller-Röber B, Gálvez S and Willmitzer L 1995 Cloning and expression analysis of the cytosolic NADP+ dependent isocitrate dehydrogenase from potato. Plant Physiol. 107, 905–913.
Gleave A P 1992 A versatile binary vector system with a T-DNA organizational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20, 1203–1207.
Hoffland E, Findenegg G R and Nelemans J A 1989 Solubilization of rock phosphate by rape. II. Local root exudation of organic acids as a response to P-starvation. Plant Soil 113, 161–165.
Horsch R B, Fry J E, Hoffman N L, Eichholtz D, Rogers S G and Fraley R T 1985 A simple and general method for transferring genes into plants. Science 227, 1229–1231.
Johnson J F, Allan D L and Vance C P 1994 Phosphorus stressinduced proteoid roots show altered metabolism in Lupinus albus. Plant Physiol. 104, 657–665.
Kamh M, Horst W J, Amer F, Mostafa H and Maier P 1999 Mobilization of soil and fertilizer phosphate by cover crops. Plant Soil 211, 19–27.
Keerthisinghe G, Hocking P J, Ryan P R and Delhaize E 1998 Effect of phosphorus supply on the formation and function of proteoid roots of white lupin (Lupinus albus L.). Plant Cell Environ. 21, 467–478.
Kihara T, Ohono T, Koyama H, Sawafuji T and Hara T 2002 Characterization of NADP-isocitrate dehydrogenase expression in a carrot mutant cell line with enhanced citrate excretion. Plant Soil (In This Issue).
Koyama H, Kawamura A, Kihara T, Hara T, Takita E and Shibata D 2000 Overexpression of mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphorus limited soil. Plant Cell Physiol. 41, 1030–1037.
Koyama H, Takita F, Kawamura A, Hara T and Shibata D 1999 Over expression of mitochondrial citrate synthase gene improves the growth of carrot cells in Al-phosphate medium. Plant Cell Physiol. 40, 482–488.
Kruse A, Fieuw S, Heineke D and Müller-Röber B 1998 Antisense inhibition of cytosolic NADP-dependent isocitrate dehydrogenase in transgenic potato plants. Planta 205, 82–91.
Lancien M, Gadal P and Hodges M 2000 Enzyme redundancy and the importance of 2-oxoglutarate in higher plant ammonium assimilation. Plant Physiol. 123, 817–824.
Landschütze V, Willmitzer L and Müller-Röber B 1995 Inhibition of flower formation by antisense repression of mitochondrial citrate synthase in transgenic potato plants leads to a specific disintegration of the ovary tissues of flowers. EMBO J. 14, 660–666.
Li X F, Ma J F and Matsumoto H 2000 Pattern of Al-induced secretion of organic acids differs between rye and wheat. Plant Physiol. 123, 1537–1543.
López-Bucio J, Martinez de la Vega O, Guevara-García A and Herrera-Estrella L 2000 Enhanced phosphorus uptake in transgenic tobacco plants that overproduce citrate. Nature Biotech. 18, 450–453.
Mugai E N, Agong S G and Matsumoto H 2000 Aluminium tolerance mechanisms in Phaseolus vulgaris L.: citrate sythase activity and TTC reduction are well correlated with citrate secretion. Soil Sci. Plant Nutr. 46, 939–950.
Murashige T and Skoog F 1962 A revised medium for a rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15, 473–497.
Neumann G, Massonneau A, Langlade N, Dinkelaker B, Hengeler C, Römheld V and Martinoia E 2000 Physiological aspects of cluster root function and development in phosphorus-deficient white lupin (Lupinus albus L.). Ann. Bot. 85, 909–919.
Neumann G, Massonneau A, Martinoia E and Römheld V 1999 Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin. Planta 208, 373–382.
Neumann G and Römheld V 1999 Root excretion of carboxylic acids and protons in phosphorus-deficient plants. Plant Soil 211, 121–130.
Rerie W G, Whitecross M and Higgins T J V 1991 Developmental and environmental regulation of pea legumin genes in transgenic tobacco. Mol. Gen. Genet. 225, 148–157.
Ruijter G, Panneman H, Xu D-B and Visser J 2000 Properties of Aspergillus niger citrate synthase and effects of citA overexpression on citric acid production. FEMS Lett. 184, 35–40.
Ryan P R, Delhaize E and Jones D L 2001 Function and mechanism of organic anion exudation from plant roots. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 527–560.
Sadka A, Dahan E, Or E, Roose M L, Marsh K B and Cohen L 2001 Comparative analysis of mitochondrial citrate synthase gene structure, transcript level and enzymatic activity in acidless and acid-containing Citrus varieties. Aust. J. Plant Physiol. 28, 383–390.
Sambrook J, Fritsch E F and Maniatis T 1989. Molecular Cloning: A Laboratory Manual, 2nd edn, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Srere PA 1967 Citrate synthase. Methods Enzymol. 13, 3–11.
Surin B, Boevink P, Keese P, Chu P, Larkin P, Llewellyn D, Kahn R, Ellacott G and Waterhouse P 1998.A suite of promoters and terminators for plant biotechnology. In Agricultural Biotechnology: Laboratory, Field and Market. Proceedings of the 4th Asia-Pacific Conference on Agricultural Biotechnology Ed. P J Larkin. pp. 121–122. Canberra UTC Publishing, Darwin, Australia.
Takita E, Koyama H and Hara T 1999 Organic acid metabolism in aluminum-phosphate utilizing cells of carrot (Daucus carota L.). Plant Cell Physiol. 40, 489–495.
Tesfaye M, Temple S J, Allan D L, Vance C P and Samac D A (2001) Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum. Plant Physiol. 127, 1836–1844.
Watt M and Evans J R 1999 Linking development and determinacy with organic acid efflux from proteoid roots of white lupin grown with low phosphorus and ambient or elevated atmospheric CO2 concentration. Plant Physiol. 120, 705–716.
Wesley S V, Helliwell C A, Smith N A, Wang M-B, Rouse D T, Liu Q, Gooding P S, Singh S P, Abbott D, Stoutjesdijk P A, Robinson S P, Gleave A P, Green A G and Waterhouse PM 2001 Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 27, 581–590.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Delhaize, E., Ryan, P.R., Hocking, P.J. et al. Effects of altered citrate synthase and isocitrate dehydrogenase expression on internal citrate concentrations and citrate efflux from tobacco (Nicotiana tabacum L.) roots. Plant and Soil 248, 137–144 (2003). https://doi.org/10.1023/A:1022352914101
Issue Date:
DOI: https://doi.org/10.1023/A:1022352914101