Biochemical characterization of a chloroplast localized fatty acid reductase from Arabidopsis thaliana Thuy T.P. Doan a, f, ⁎, Frédéric Domergue b , Ashley E. Fournier c , Sollapura J. Vishwanath c , Owen Rowland c , Patrick Moreau b , Craig C. Wood d , Anders S. Carlsson a , Mats Hamberg e , Per Hofvander a a Department of Plant Breeding and Biotechnology, Swedish University of Agricultural Sciences, P.O. box 101, 230 53 Alnarp, Sweden b Laboratoire de Biogenèse Membranaire, Université Victor Ségalen Bordeaux 2, CNRS, UMR5200, 146 rue Léo Saignat, Case 92, 33076 Bordeaux Cedex, France c Department of Biology and Institute of Biochemistry, Carleton University, Ottawa, Ontario, K1S 5B6, Canada d CSIRO Plant Industry, Canberra, ACT, Australia e Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden f Department of Biology, Nong Lam University, Ho Chi Minh City, Vietnam abstract article info Article history: Received 26 July 2011 Received in revised form 24 October 2011 Accepted 27 October 2011 Available online 30 November 2011 Keywords: Fatty alcohol Fatty aldehyde Fatty acyl-CoA reductase (FAR) Chloroplast Chloroplast transit peptide Primary long-chain fatty alcohols are present in a variety of phyla. In eukaryotes, the production of fatty al- cohols is catalyzed by fatty acyl-CoA reductase (FAR) enzymes that convert fatty acyl-CoAs or acyl-ACPs into fatty alcohols. Here, we report on the biochemical properties of a purified plant FAR, Arabidopsis FAR6 (AtFAR6). In vitro assays show that the enzyme preferentially uses 16 carbon acyl-chains as substrates and produces predominantly fatty alcohols. Free fatty acids and fatty aldehyde intermediates can be released from the enzyme, in particular with suboptimal chain lengths and concentrations of the substrates. Both acyl-CoA and acyl-ACP could serve as substrates. Transient expression experiments in Nicotiana tabacum showed that AtFAR6 is a chloroplast localized FAR. In addition, expression of full length AtFAR6 in Nicotiana benthamiana leaves resulted in the production of C16:0-alcohol within this organelle. Finally, a GUS reporter gene fusion with the AtFAR6 promoter showed that the AtFAR6 gene is expressed in various tissues of the plant with a distinct pattern compared to that of other Arabidopsis FARs, suggesting specialized functions in planta. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Primary fatty alcohols are found in plants, animals, and microbes, serving various unique functions. In plants, very long-chain fatty alcohols are, for example, components of plant cuticular waxes and suberin [1,2], which are hydrophobic barriers that protect plants from abiotic and biotic stresses [3,4]. In addition to being present in free form, fatty alcohols can also serve as the direct substrates for wax ester biosynthesis by esterification with a fatty acyl-CoA through the action of a wax synthase [4-6]. The resulting wax esters are often components of cuticular waxes or in the case of jojoba (Simmondsia chinensis), the primary storage compound of seeds [2,3,7]. Alcohol-forming fatty acyl-CoA reductases (FARs) catalyze the for- mation of fatty alcohols from fatty acyl-CoAs by two consecutive reac- tions. The fatty acyl-CoA is first reduced to a fatty aldehyde, which is then reduced into a fatty alcohol [8,9]. It has been found in plants that the reduction of fatty acyl-CoA into fatty alcohol is carried out by a NADPH-dependent FAR enzyme and it is thought to occur without release of the aldehyde intermediate [8,9]. The chain lengths and dis- tributions of fatty alcohols in plants are believed to be controlled by FAR substrate specificities and by their gene expression patterns [1,2,9-11]. However, since in vitro biochemical data of the properties of FAR are lacking, it is not known the relative importance of enzyme specificity per se in relation to the pool of acyl substrates available to the enzyme for determination of the fatty alcohol composition pro- duced in a given cell type. While alcohol-forming FARs from plants are usually around 490–500 amino acids in length [2], AtFAR6 is a 548 amino acid protein, containing a 71 amino acid N-terminal extension that is predicted to contain a chloroplast targeting sequence [12]. In a previ- ous study, expression of AtFAR6 in E. coli resulted in the production of alcohols with a substantial amount of C16:0-OH compared to C14:0- OH and C18:1-OH. However, even though the E. coli expression sys- tem is fast and efficient, this study revealed limitations such as that E. coli only contains endogenous fatty acyl chains up to 18 carbons in the length and that C18:1 is a positional isomer as compared to plants C18:1 (11c-C18:1 in bacteria vs. 9c-18:1 in plants) [13]. In ad- dition, the fatty acyl-ACP pool of E. coli is predominant over the fatty acyl-CoA pool [14,15]. Moreover, in an E. coli system, the results obtained from heterologous expression of FARs could be influenced Biochimica et Biophysica Acta 1821 (2012) 1244–1255 ⁎ Corresponding author at: P.O box 101, 230 53 Alnarp, Sweden. Tel.: + 46 40415559; fax: + 46 40415519. E-mail address: thuy.doan@slu.se (T.T.P. Doan). 1388-1981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2011.10.019 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip