HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 4, 2780-2787, January 28, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/4/2780    most recent M407291200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Turner, J. E. Articles by Hooks, M. A. Search for Related Content PubMed PubMed Citation Articles by Turner, J. E. Articles by Hooks, M. A. Social Bookmarking                      What's this?

Characterization of Arabidopsis Fluoroacetate-resistant Mutants Reveals the Principal Mechanism of Acetate Activation for Entry into the Glyoxylate Cycle^*

James E. Turner, Karen Greville, Elaine C. Murphy, and Mark A. Hooks

From the School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, Wales, United Kingdom

The toxic acetate analogue monofluoroacetic acid was employed to isolate Arabidopsis tDNA-tagged plants deficient in their ability to utilize or sense acetate. Several tDNA-tagged lines were isolated, including two that were determined to be allelic to an EMS-mutagenized line denoted acn1 for ac non-utilizing. Following conventions, the tDNA-tagged mutants were designated acn1-2 and acn1-3. Both mutants displayed identical behavior to acn1-1 on a variety of fluorinated and nonfluorinated organic acids, indicating that resistance was specific to fluoroacetate. Thermal asymmetric interlaced PCR identified the sites of tDNA insertion in both mutants to be within different exons in a gene, which encoded a protein containing an AMP-binding motif. Reverse transcription-PCR confirmed that the gene was not expressed in the mutants, and quantitative reverse transcription-PCR showed that the gene is expressed in imbibed seeds and increases in amount during establishment. The wild type AMP-binding protein cDNA was cloned and expressed in Escherichia coli, and the expressed protein was purified by nickel chelate chromatography. The enzyme was identified as an acyl-CoA synthetase that was more active with acetate than butyrate and was not active with fatty acids longer than C-4. The enzyme was localized to peroxisomes by enzymatic analysis of organellar fractions isolated by sucrose density gradient centrifugation. Labeling studies with [¹⁴C]acetate showed that acn1 seedlings, like those of the isocitrate lyase mutant icl-1 (isocitrate lyase), are compromised in carbohydrate synthesis, indicating that this enzyme is responsible for activating exogenous acetate to the coenzyme A form for entry into the glyoxylate cycle.

Received for publication, June 30, 2004 , and in revised form, November 5, 2004.

^* This work was supported by British Biotechnology and Biological Sciences Research Council Grant 5/P14659 and postgraduate student funding from the University of Wales Bangor. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, Wales, UK. Tel.: 44-1248-382297; Fax: 44-1248-370731; E-mail: m.a.hooks{at}bangor.ac.uk.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. Lin and D. J. Oliver The Role of Acetyl-Coenzyme A Synthetase in Arabidopsis Plant Physiology, August 1, 2008; 147(4): 1822 - 1829. [Abstract] [Full Text] [PDF]

				S. Reumann, L. Babujee, C. Ma, S. Wienkoop, T. Siemsen, G. E. Antonicelli, N. Rasche, F. Luder, W. Weckwerth, and O. Jahn Proteome Analysis of Arabidopsis Leaf Peroxisomes Reveals Novel Targeting Peptides, Metabolic Pathways, and Defense Mechanisms PLANT CELL, October 1, 2007; 19(10): 3170 - 3193. [Abstract] [Full Text] [PDF]