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Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana

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Abstract

Amidase 1 (AMI1) from Arabidopsis thaliana converts indole-3-acetamide (IAM), into indole-3-acetic acid (IAA). AMI1 is part of a small isogene family comprising seven members in A. thaliana encoding proteins which share a conserved glycine- and serine-rich amidase-signature. One member of this family has been characterized as an N-acylethanolamine-cleaving fatty acid amidohydrolase (FAAH) and two other members are part of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) or mitochondria (Tom complex) and presumably lack enzymatic activity. Among the hitherto characterized proteins of this family, AMI1 is the only member with indole-3-acetamide hydrolase activity, and IAM is the preferred substrate while N-acylethanolamines and oleamide are not hydrolyzed significantly, thus suggesting a role of AMI1 in auxin biosynthesis. Whereas the enzymatic function of AMI1 has been determined in vitro, the subcellular localization of the enzyme remained unclear. By using different GFP-fusion constructs and an A. thaliana transient expression system, we show a cytoplasmic localization of AMI1. In addition, RT-PCR and anti-amidase antisera were used to examine tissue specific expression of AMI1 at the transcriptional and translational level, respectively. AMI1-expression is strongest in places of highest IAA content in the plant. Thus, it is concluded that AMI1 may be involved in de novo IAA synthesis in A. thaliana.

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Abbreviations

DsRed:

Red fluorescent protein from Discosoma sp.

GFP:

Green fluorescent protein

IAA:

Indole-3-acetic acid

IAM:

Indole-3-acetamide

NAA:

1-Naphthaleneacetic acid

NAE:

N-acylethanolamine

NAM:

1-Naphthaleneacetamide

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Acknowledgements

We thank Dr. Rhidaya Shrestha and Dr. Kent D. Chapman (Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, Texas, USA) for providing the (His)6-tagged FAAH clone. Furthermore, the authors thank Dr. Andreas Wachter (Institute of Plant Sciences, Ruprecht-Karls-University, Heidelberg, Germany) for kindly providing the vectors pFF19-GFP and pFF19-RFP. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (Schwerpunktprogramm ‘Molecular Mechanisms of Phytohormone Action’, and subsequently Sonderforschungsbereich 480 ‘Molekulare Biologie komplexer Leistungen von botanischen Systemen’).

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  1. Department of Plant Physiology, Ruhr-University Bochum, Universitaetsstraße 150, ND 2/27, 44801, Bochum, Germany

    Stephan Pollmann, Daniel Neu, Thomas Lehmann & Elmar W. Weiler

  2. Institut für Anatomie, Universitätsklinikum Essen, Hufelandstraße 55, 45122, Essen, Germany

    Tina Schäfer

  3. The Australian National University, Research School of Biological Sciences (RSBS), Enviromental Biology Group, GPO Box 475, Canberra, ACT, 0200, Australia

    Oliver Berkowitz

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  1. Stephan Pollmann
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Correspondence to Stephan Pollmann.

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Pollmann, S., Neu, D., Lehmann, T. et al. Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana . Planta 224, 1241–1253 (2006). https://doi.org/10.1007/s00425-006-0304-2

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