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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

NAD-Dependent Isocitrate Dehydrogenase Mutants of Arabidopsis Suggest the Enzyme Is Not Limiting for Nitrogen Assimilation^1,[C]

Institute de Biotechnologie des Plantes, Unité Mixte de Recherche, Centre National de la Recherche Scientifique 8618, Université de Paris Sud-XI, 91405 Orsay cedex, France (T.L., V.F., E.B., M.H.); and Abteilung Willmitzer, Max-Planck-Institut für Molekulare Pflanzenphysiologie, 14476 Postdam-Golm, Germany (E.U.-W., A.R.F.)

NAD-dependent isocitrate dehydrogenase (IDH) is a tricarboxylic acid cycle enzyme that produces 2-oxoglutarate, an organic acid required by the glutamine synthetase/glutamate synthase cycle to assimilate ammonium. Three Arabidopsis (Arabidopsis thaliana) IDH mutants have been characterized, corresponding to an insertion into a different IDH gene (At5g03290, idhv; At4g35260, idhi; At2g17130, idhii). Analysis of IDH mRNA and protein show that each mutant lacks the corresponding gene products. Leaf IDH activity is reduced by 92%, 60%, and 43% for idhv, idhi, and idhii, respectively. These mutants do not have any developmental or growth phenotype and the reduction of IDH activity does not impact on NADP-dependent isocitrate dehydrogenase activity. Soil-grown mutants do not exhibit any alterations in daytime sucrose, glucose, fructose, citrate, ammonium, and total soluble amino acid levels. However, gas chromatography-mass spectrometry metabolic profiling analyses indicate that certain free amino acids are reduced in comparison to the wild type. These data suggest that IDH activity is not limiting for tricarboxylic acid cycle functioning and nitrogen assimilation. On the other hand, liquid culture-grown mutants give a reduced growth phenotype, a large increase in organic acid (citrate is increased 35-fold), hexose-phosphate, and sugar content, whereas ammonium and free amino acids are moderately increased with respect to wild-type cultures. However, no significant changes in 2-oxoglutarate levels were observed. Under these nonphysiological growth conditions, pyridine nucleotide levels remained relatively constant between the wild-type and the idhv line, although some small, but significant, alterations were measured in idhii (lower NADH and higher NADPH levels). On the other hand, soil-grown idhv plants exhibited a reduction in NAD and NADPH content.

² Present address: Unité de Nutrition Azotée des Plantes, Institut National de la Recherche Agronomique, Route de St. Cyr, 78026 Versailles, France.

³ Present address: Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Michael Hodges (michael.hodges{at}u-psud.fr).

^[C] Some figures in this article are displayed in color online but in black and white in print.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.100677

^* Corresponding author; e-mail michael.hodges{at}u-psud.fr; fax 33–1–69153424.

Received April 6, 2007; accepted April 25, 2007; published April 27, 2007.

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