Journal Article

Transgenic Tobacco (<i>Nicotiana tabacum</i> L.) Plants with Increased Expression Levels of Mitochondrial NADP<sup>+</sup>-dependent Isocitrate Dehydrogenase: Evidence Implicating this Enzyme in the Redox Activation of the Alternative Oxidase

Gordon R. Gray, Alicia R. Villarimo, Carmen L. Whitehead and Lee McIntosh

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 45, issue 10, pages 1413-1425
Published in print October 2004 | ISSN: 0032-0781
Published online October 2004 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pch162
Transgenic Tobacco (Nicotiana tabacum L.) Plants with Increased Expression Levels of Mitochondrial NADP+-dependent Isocitrate Dehydrogenase: Evidence Implicating this Enzyme in the Redox Activation of the Alternative Oxidase

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Many metabolic reactions are coupled to NADPH in the mitochondrial matrix, including those involved in thiol group reduction. One enzyme linked to such processes is mitochondrial NADP+-dependent isocitrate dehydrogenase (mtICDH; EC 1.1.1.42), although the precise role of this enzyme is not yet known. Previous work has implicated mtICDH as part of a biochemical mechanism to reductively activate the alternative oxidase (AOX). We have partially purified mtICDH from tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) cell suspension cultures and localized this to a 46-kDa protein on SDS–PAGE, which was verified by peptide sequencing. In the inflorescence of the aroid Sauromatum guttatum Schott (voodoo lily), mtICDH appears to be developmentally regulated, presenting maximal specific activity during the thermogenic period of anthesis when the capacity for AOX respiration is also at its peak. Transgenic tobacco plants were generated that overexpress mtICDH and lines were obtained that demonstrated up to a 7-fold increase in mtICDH activity. In isolated mitochondria, this resulted in a measurable increase in the reductive activation of AOX in comparison with wild type. When examined in planta in response to citrate feeding, a strong conversion of AOX from its oxidized to its reduced form was observed in the transgenic line. These data support the hypothesis that mtICDH may be a regulatory switch involved in tricarboxylic acid cycle flux and the reductive modulation of AOX.

Keywords: Keywords: Alternative oxidase — Isocitrate dehydrogenase — Mitochondria — Nicotiana tabacum L. — Sauromatum guttatum Schott — Tricarboxylic acid cycle.; Abbreviations: AA, antimycin A; AOX, alternative oxidase; CaMV, cauliflower mosaic virus; D-day, day of thermogenesis in the appendix region of the spadix; D–1, 1 d before D-day; D+2, 2 d after D-day; ICDH, isocitrate dehydrogenase; ICDH1, cytosolic NADP+-dependent ICDH; ICDH2, chloroplastic NADP+-dependent ICDH; NAD-IDH, mitochondrial NAD+-dependent ICDH; NAD-IDHa, catalytic subunit of NAD-IDH; MOPS, 3-(N-morpholino) propanesulfonic acid; mtICDH, mitochondrial NADP+-dependent ICDH; MS, Murashige–Skoog; SE, standard error of the mean; TBS, Tris-buffered saline; TBS-T, TBS containing polyoxyethylene-sorbitan monolaurate; TCA cycle, tricarboxylic acid cycle; UTR, untranslated region; WT, wild type.

Journal Article.  10272 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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