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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Functional Characterization of the Arabidopsis β-Ketoacyl-Coenzyme A Reductase Candidates of the Fatty Acid Elongase^1,[W],[OA]

Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts AL5 2JQ, United Kingdom (F.B., R.P.H., J.A.N.); University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (X.W., F.L., H.Z., L.K.); and Donald Danforth Plant Science Center, St. Louis, Missouri 63132 (J.E.M.)

In plants, very-long-chain fatty acids (VLCFAs; >18 carbon) are precursors of sphingolipids, triacylglycerols, cuticular waxes, and suberin. VLCFAs are synthesized by a multiprotein membrane-bound fatty acid elongation system that catalyzes four successive enzymatic reactions: condensation, reduction, dehydration, and a second reduction. A bioinformatics survey of the Arabidopsis (Arabidopsis thaliana) genome has revealed two sequences homologous to YBR159w encoding a Saccharomyces cerevisiae β-ketoacyl reductase (KCR), which catalyzes the first reduction during VLCFA elongation. Expression analyses showed that both AtKCR1 and AtKCR2 genes were transcribed in siliques, flowers, inflorescence stems, leaves, as well as developing embryos, but only AtKCR1 transcript was detected in roots. Fluorescent protein-tagged AtKCR1 and AtKCR2 were localized to the endoplasmic reticulum, the site of fatty acid elongation. Complementation of the yeast ybr159 mutant demonstrated that the two KCR proteins are divergent and that only AtKCR1 can restore heterologous elongase activity similar to the native yeast KCR gene. Analyses of insertional mutants in AtKCR1 and AtKCR2 revealed that loss of AtKCR1 function results in embryo lethality, which cannot be rescued by AtKCR2 expression using the AtKCR1 promoter. In contrast, a disruption of the AtKCR2 gene had no obvious phenotypic effect. Taken together, these results indicate that only AtKCR1 is a functional KCR isoform involved in microsomal fatty acid elongation. To investigate the roles of AtKCR1 in postembryonic development, transgenic lines expressing RNA interference and overexpression constructs targeted against AtKCR1 were generated. Morphological and biochemical characterization of these lines confirmed that suppressed KCR activity results in a reduction of cuticular wax load and affects VLCFA composition of sphingolipids, seed triacylglycerols, and root glycerolipids, demonstrating in planta that KCR is involved in elongation reactions supplying VLCFA for all these diverse classes of lipids.

² These authors contributed equally to the article.

³ Present address: Department of Biology, McGill University, Montreal, Quebec, Canada H3A 1B1.

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) are: Johnathan A. Napier (johnathan.napier{at}bbsrc.ac.uk) and Ljerka Kunst (kunst{at}interchange.ubc.ca).

^[W] The online version of this article contains Web-only data.

^[OA] Open access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.137497

^* Corresponding author; e-mail kunst{at}interchange.ubc.ca.

Received February 20, 2009; accepted April 28, 2009; published May 13, 2009.