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Occurrence of Two Acetoacetyl-Coenzyme A Thiolases with Distinct Expression Patterns and Subcellular Localization in Tobacco

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Isoprenoid Synthesis in Plants and Microorganisms

Abstract

Acetoacetyl-coenzyme A thiolase (AACT, EC 2.3.1.9) catalyzes the condensation of two acetyl-CoA units into acetoacetyl-CoA. By screening a tobacco (Nicotiana tabacum L.) cDNA library using a radish probe, we isolated two genes encoding an AACT. The deduced protein sequences, referred to as NtAACT1 and NtAACT2, were found to be 77% identical and to share high homologies with other AACTs. Here, we report that both NtAACTs exhibited distinct expression patterns in planta and that the enzymes localize to separate cell compartments, suggesting different metabolic functions. At its C-terminus, NtAACT1 bears an SSL motif corresponding to a peroxisomal targeting signal of type 1. We present evidence that NtAACT1 is readily imported into glyoxysomes by demonstrating in vivo colocalization of the chimeric proteins, GFP-NtAACT1 and RFP-SKL, in transiently transformed tobacco BY-2 cells. The importance of the SSL motif was stressed by the absence of glyoxysomal delivery of the fusion protein lacking the tripeptide. In contrast, NtAACT2 is clearly a soluble enzyme, with no obvious targeting signal to a defined cell membrane compartment. Its participation in isoprenoid biosynthesis via the mevalonate pathway was attested by potato virus X-induced gene silencing experiments in Nicotiana benthamiana plants. Silencing of endogenous NbAACT2 was shown indeed to trigger a 50% reduction in the levels of sterol biosynthetic precursors. A spatiotemporal study of expression patterns of both genes in planta indicated that NtAACT2 was expressed throughout the plant, but more effectively in young tissues. In contrast, NtAACT1 displayed a significant expression only in senescent leaves and early stages of germination and appears to be functional in the glyoxysomal/peroxisomal β-oxidation of fatty acids, catalyzing the last step.

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Acknowledgments

We thank Prof. P. Benveniste for providing us the N. tabacum cDNA library prepared with mRNAs isolated from 8-week-old SH6 tobacco calli. We are grateful to Dr. M. Rodríguez-Concepción (University of Barcelona, Spain) and Dr. A. Hemmerlin (IBMP, Strasbourg) for making available the pGFP-MRC and pRFP vectors, respectively.

We also thank Valérie Cognat (IBMP, Strasbourg) for her help in phylogenetic analyses.

The Confocal Microscopy Platform was cofinanced by the Centre National de la Recherche Scientifique, the Université de Strasbourg, the Région Alsace, the Association pour la Recherche sur le Cancer (ARC), and the Ligue Nationale contre le Cancer.

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  1. Actelion Pharmaceuticals Ltd, Allschwil, Switzerland

    Laurent Wentzinger

  2. Department of Genetic Engineering, Deinove Company, Paris, France

    Esther Gerber

  3. Institut de Biologie Moléculaire des Plantes (CNRS UPR 2357), Département “Réseaux métaboliques”, Université de Strasbourg, 28 Rue Goethe, Strasbourg, F-67083, France

    Thomas J. Bach & Marie-Andrée Hartmann

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  1. Laurent Wentzinger
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  2. Esther Gerber
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  3. Thomas J. Bach
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  4. Marie-Andrée Hartmann
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Correspondence to Thomas J. Bach .

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  1. Institut de Biologie Moléculaire, Département Réseaux, Université de Strasbourg, 28 rue Goethe, Strasbourg, 67083, France

    Thomas J. Bach

  2. , Institut Le Bel, Université Louis Pasteur, 4 rue Blaise Pascal, Strasbourg CX, 67070, France

    Michel Rohmer

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Wentzinger, L., Gerber, E., Bach, T.J., Hartmann, MA. (2012). Occurrence of Two Acetoacetyl-Coenzyme A Thiolases with Distinct Expression Patterns and Subcellular Localization in Tobacco. In: Bach, T., Rohmer, M. (eds) Isoprenoid Synthesis in Plants and Microorganisms. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-4063-5_24

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