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JXB Advance Access originally published online on May 20, 2008
Journal of Experimental Botany 2008 59(9):2545-2554; doi:10.1093/jxb/ern123
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Identification and characterization of a plastid-localized Arabidopsis glyoxylate reductase isoform: comparison with a cytosolic isoform and implications for cellular redox homeostasis and aldehyde detoxification

Jeffrey P. Simpson1 *, Rosa Di Leo1 *, Preetinder K. Dhanoa2, Wendy L. Allan1, Amina Makhmoudova1, Shawn M. Clark1, Gordon J. Hoover1, Robert T. Mullen2 and Barry J. Shelp1,{dagger}

1Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada N1G 2W1
2Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1

{dagger} To whom correspondence should be addressed. E-mail: bshelp{at}uoguelph.ca

Enzymes that reduce the aldehyde chemical grouping (i.e. H-C=O) to its corresponding alcohol could be crucial in maintaining plant health. Recently, recombinant expression of a cytosolic enzyme from Arabidopsis thaliana (L.) Heynh (designated as glyoxylate reductase 1 or AtGR1) revealed that it effectively catalyses the in vitro reduction of both glyoxylate and succinic semialdehyde (SSA). In this paper, web-based bioinformatics tools revealed a second putative GR cDNA (GenBank Accession No. AAP42747 [GenBank] ; designated herein as AtGR2) that is 57% identical on an amino acid basis to GR1. Sequence encoding a putative targeting signal (N-terminal 43 amino acids) was deleted from the full-length GR2 cDNA and the resulting truncated gene was co-expressed with the molecular chaperones GroES/EL in Escherichia coli, enabling production and purification of soluble recombinant protein. Kinetic analysis revealed that recombinant GR2 catalysed the conversion of glyoxylate to glycolate (Km glyoxylate=34 µM), and SSA to {gamma}-hydroxybutyrate (Km SSA=8.96 mM) via an essentially irreversible, NADPH-based mechanism. GR2 had a 350-fold higher preference for glyoxylate than SSA, based on the performance constants (kcat/Km). Fluorescence microscopic analysis of tobacco (Nicotiana tabacum L.) suspension cells transiently transformed with GR1 linked to the green fluorescent protein (GFP) revealed that GR1 was localized to the cytosol, whereas GR2-GFP was localized to plastids via targeting information contained within its N-terminal 45 amino acids. The identification and characterization of distinct plastidial and cytosolic glyoxylate reductase isoforms is discussed with respect to aldehyde detoxification and the plant stress response.

Key words: Aldehyde detoxification, cytosol, glyoxylate reductase, plastid, recombinant expression, redox homeostasis, subcellular localization, succinic semialdehyde reductase, transient expression

* These authors contributed equally to the work.

Received 9 November 2007; Revised 29 March 2008 Accepted 1 April 2008


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