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JXB Advance Access published online on May 21, 2009
Journal of Experimental Botany, doi:10.1093/jxb/erp161
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© 2009 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

Subcellular localization and expression of multiple tomato {gamma}-aminobutyrate transaminases that utilize both pyruvate and glyoxylate

Shawn M. Clark1, Rosa Di Leo1, Owen R. Van Cauwenberghe1 *, 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

{gamma}-Aminobutyric acid transaminase (GABA-T) catalyses the breakdown of GABA to succinic semialdehyde. In this report, three GABA-T isoforms were identified in the tomato (Solanum lycopersicum L.) plant. The deduced amino acid sequences of the three isoforms are highly similar over most of their coding regions with the exception of their N-terminal regions. Transient expression of the individual full-length GABA-T isoforms fused to the green fluorescent protein in tobacco suspension-cultured cells revealed their distinct subcellular localizations to the mitochondrion, plastid or cytosol, and that the specific targeting of the mitochondrion- and plastid-localized isoforms is mediated by their predicted N-terminal presequences. Removal of the N-terminal targeting presequences from the mitochondrion and plastid GABA-T isoforms yielded good recovery of the soluble recombinant proteins in Escherichia coli when they were co-expressed with the GroES/EL molecular chaperone complex. Activity assays indicated that all three recombinant isoforms possess both pyruvate- and glyoxylate-dependent GABA-T activities, although the mitochondrial enzyme has a specific activity that is significantly higher than that of its plastid and cytosolic counterparts. Finally, differential expression patterns of the three GABA-T isoforms in reproductive tissues, but not vegetative tissues, suggest unique roles for each enzyme in developmental processes. Overall, these findings, together with recent information about rice and pepper GABA-Ts, indicate that the subcellular distribution of GABA-T in the plant kingdom is highly variable.

Key words: {gamma}-Aminobutyrate, {gamma}-aminobutyrate transaminase, aminotransferase, gene expression, in silico analysis, recombinant expression, substrate specificity, subcellular localization, tomato, transient expression

* Present address: Lilly Analytical Research Laboratory, Eli Lilly Canada Inc., 3650 Danforth Avenue, Toronto, ON M1N 2E8, Canada.

Received 17 March 2009; Revised 24 April 2009 Accepted 27 April 2009


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