Received February 20, 2009
Accepted April 28, 2009
Functional Characterization of the Arabidopsis thaliana 
-Ketoacyl-CoA Reductase Candidates of the Fatty Acid Elongase
Frederic Beaudoin , Xianzhong Wu , Fengling Li , Richard P. Haslam , Jonathan E. Markham , Huanquan Zheng , Johnathan A. Napier , and Ljerka Kunst *
Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK; University of British Columbia, BC V6T 1Z4, Vancouver, Canada; Donald Danforth Plant Science Center, Saint Louis, Missouri, 63132, USA
* Corresponding author; email: kunst{at}interchange.ubc.ca.
In plants, very long chain fatty acids (VLCFAs; >18 carbon) are precursors of sphingolipids, triacylglycerols, cuticular waxes and suberin. VLCFA are synthesized by a multi-protein membrane-bound fatty acid elongation (FAE) system which catalyzes four successive enzymatic reactions: condensation, reduction, dehydration and a second reduction. A bioinformatics survey of the Arabidopsis genome has revealed two sequences homologous to YBR159w encoding a S. cerevisiae 
-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 RNAi 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.
