Biochimica et Biophysica Acta 1821 (2012) 1244–1255

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Biochimica et Biophysica Acta

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Biochemical characterization of a chloroplast localized fatty acid reductase fromArabidopsis 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, Swedenb Laboratoire de Biogenèse Membranaire, Université Victor Ségalen Bordeaux 2, CNRS, UMR5200, 146 rue Léo Saignat, Case 92, 33076 Bordeaux Cedex, Francec Department of Biology and Institute of Biochemistry, Carleton University, Ottawa, Ontario, K1S 5B6, Canadad CSIRO Plant Industry, Canberra, ACT, Australiae Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Swedenf Department of Biology, Nong Lam University, Ho Chi Minh City, Vietnam

⁎ Corresponding author at: P.O box 101, 230 53 Alnarpfax: +46 40415519.

E-mail address: thuy.doan@slu.se (T.T.P. Doan).

1388-1981/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.bbalip.2011.10.019

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 July 2011Received in revised form 24 October 2011Accepted 27 October 2011Available online 30 November 2011

Keywords:Fatty alcoholFatty aldehydeFatty acyl-CoA reductase (FAR)ChloroplastChloroplast 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 intofatty 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 andproduces predominantly fatty alcohols. Free fatty acids and fatty aldehyde intermediates can be releasedfrom the enzyme, in particular with suboptimal chain lengths and concentrations of the substrates. Bothacyl-CoA and acyl-ACP could serve as substrates. Transient expression experiments in Nicotiana tabacumshowed that AtFAR6 is a chloroplast localized FAR. In addition, expression of full length AtFAR6 in Nicotianabenthamiana leaves resulted in the production of C16:0-alcohol within this organelle. Finally, a GUS reportergene fusion with the AtFAR6 promoter showed that the AtFAR6 gene is expressed in various tissues of theplant with a distinct pattern compared to that of other Arabidopsis FARs, suggesting specialized functionsin 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 fattyalcohols are, for example, components of plant cuticular waxes andsuberin [1,2], which are hydrophobic barriers that protect plantsfrom abiotic and biotic stresses [3,4]. In addition to being present infree form, fatty alcohols can also serve as the direct substrates forwax ester biosynthesis by esterification with a fatty acyl-CoA throughthe action of a wax synthase [4-6]. The resulting wax esters are oftencomponents of cuticular waxes or in the case of jojoba (Simmondsiachinensis), 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 isthen reduced into a fatty alcohol [8,9]. It has been found in plants thatthe reduction of fatty acyl-CoA into fatty alcohol is carried out by aNADPH-dependent FAR enzyme and it is thought to occur without

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release of the aldehyde intermediate [8,9]. The chain lengths and dis-tributions of fatty alcohols in plants are believed to be controlled byFAR substrate specificities and by their gene expression patterns[1,2,9-11]. However, since in vitro biochemical data of the propertiesof FAR are lacking, it is not known the relative importance of enzymespecificity per se in relation to the pool of acyl substrates available tothe enzyme for determination of the fatty alcohol composition pro-duced in a given cell type.

While alcohol-forming FARs from plants are usually around490–500 amino acids in length [2], AtFAR6 is a 548 amino acidprotein, containing a 71 amino acid N-terminal extension that ispredicted to contain a chloroplast targeting sequence [12]. In a previ-ous study, expression of AtFAR6 in E. coli resulted in the production ofalcohols 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 thatE. coli only contains endogenous fatty acyl chains up to 18 carbonsin the length and that C18:1 is a positional isomer as compared toplants 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 fattyacyl-CoA pool [14,15]. Moreover, in an E. coli system, the resultsobtained from heterologous expression of FARs could be influenced

1245T.T.P. Doan et al. / Biochimica et Biophysica Acta 1821 (2012) 1244–1255

by the presence of endogenous fatty aldehyde reductase activitiesthat reduce fatty aldehyde into fatty alcohol [16,17]. As a conse-quence, the fatty alcohol profiles of genes expressed in E. coli mightdeviate from the substrate specificity of the FAR enzymes in planta[1,2,12]. Other studies have also revealed discrepancies between thesubstrate specificities of FARs expressed in E. coli utilizing endoge-nous acyl-CoA pools and the apparent specificities of FARs expressedin planta (e.g. in seeds) [10,11].

In the present work, we show by heterologous expression in yeastthat the truncated version of AtFAR6, which lacks the N-terminal ex-tension, produced C16:0 primary fatty alcohol much more efficientlythan the full length protein. We therefore expressed this version ofAtFAR6 in E. coli, purified the recombinant protein to near homogene-ity and characterized its enzyme activity and substrate specificityusing in vitro assays. Our results show that, in addition to the produc-tion of fatty alcohols, the enzyme can release both free fatty acids andfatty aldehydes. By transient expression in Nicotiana sp. leaves, weshow that AtFAR6 is chloroplast localized and that it generates mainlyC16:0 primary alcohol, which remains within this organelle. Finally,analysis of transgenic plants containing an AtFAR6 promoter-GUSfusion indicated that the gene is expressed in various tissues of theplant, but in a distinct pattern compared to the other AtFAR genes,thus implying that AtFAR6 has unique roles in planta.

2. Materials and methods

2.1. Chemicals

All chemicals, solvents, and lipid standards/references were re-agent grade and, if not otherwise stated, purchased from Merck,Darmstadt, Germany; Sigma, St Louis, MO, USA; or Larodan Fine Che-micals, Malmö, Sweden. All restriction endonucleases were fromFermentas International Inc. Burlington, Ontario, Canada. [14C]-fattyacyl-CoAs were synthesized from their mixed anhydrides accordingto the method of Sanchez et al. [18]. C18:0-aldehyde was kindly pro-vided by Prof. Reinhard Jetter (University of British Columbia, Van-couver, Canada). 1-Tetradecanol was prepared by refluxing methyltetradecanoate (100 mg) for 2 h with LiALH4 (200 mg) in 30 mL ofdiethyl ether followed by isolation by silica gel chromatography.The mass spectrum recorded on the trimethylsilyl (Me3Si) derivativeshowed a prominent ion at m/z 271 (M+ - CH3) and weaker ions atm/z 286 (M+), 255, 185 and 103 (Me3SiO+=CH2). Tetradecanalwas prepared by stirring 1-tetradecanol (25 mg) for 1 h at 23 °Cwith Dess-Martin periodinane (40 mg) in 3 mL of dry methylenechloride. Hexane was added and the solution washed four timeswith water. Pure aldehyde was obtained following Silica gel chroma-tography. The mass spectrum showed a weak ion at m/z 212 (M+)and more prominent ions at m/z 194, 168, 152 and 96. The benzylox-ime (BO) derivative, which on GC-MS appeared as a doublet due tosyn/anti isomerism of the oxime function, showed mass spectralions at m/z 317 (M+), 300, 149, 110 and 91 ([CH2=C6H5]+).

2.2. Expression of AtFAR6 and mAtFAR6 in yeast

The DNA sequences encoding full length AtFAR6 and truncatedAtFAR6 (mAtFAR6), which lacks the predicted sequence for thechloroplast transit peptide, were amplified by PCR from pET15b har-boring AtFAR6 [12] using forward primers FAR6GWYf and mFAR6G-WYf for AtFAR6 and mAtFAR6, respectively, along with reverseprimer mFAR6GWYr for both amplicons (see Supplemental Table S1for primer sequences). The resulting fragments were cloned intopDONR221 entry vector (Invitrogen) via BP reactions and thensubcloned into pYES-DEST52 destination vector (Invitrogen) via LRreactions according to manufacturer's procedure. pYES-DEST52harbouring AtFAR6 and mAtFAR6 were transformed into Saccharomy-ces cerevisiae W303-1A strain and selected on synthetic drop out

medium containing 2% (v/v) glucose, 0.17% (w/v) yeast nitrogenbase, 0.5% ammonium sulphate and 0.07% (w/v) drop out supplementlacking uracil. The induction of protein expression was carried out asdescribed by Rowland et al. [2]. In this method, a single colony waspicked and streaked on selective media agar plates containing 2%(v/v) galactose. After 6 days of induction at 30 °C, yeast cells werescraped from the plates. The cells were freeze dried and dry weightsmeasured. Fatty acid methyl esters (FAMEs) were prepared by incu-bating the dried yeast cells with 1 ml of methanolic-H2SO4 2% (v/v)for 90 min at 90 °C. Lipids were then extracted by the addition of2 ml of hexane and 3 ml of water. The extracted lipids were recov-ered, dried under nitrogen gas and separated on TLC Silica 60 plates(Merck) with hexane/diethyl ether/acid acetic (55:45:0.5, v/v/v) asthe developing solvent. Lipids were visualized by spraying the platesevenly with water. The spots on the TLC plate corresponding to fattyalcohol were located based on the migration of heptadecanol (C17:0-OH) standard. These spots were subsequently scraped from the plateand eluted from the gel matrix with methanol/chloroform (2:1, v/v)by the method of Bligh and Dyer [19]. Alcohols were dried undernitrogen gas at room temperature and dissolved in 50 μl of hexanefor GC analysis.

2.3. GC analysis

GC analysis was performed using a Shimadzu gas chromatographequipped with a flame ionization detector and a capillary column(WCOT fused silica 50 m×0.32 mm, CP-wax 58 (FFAP)-CB). Heliumwas used as carrier gas at a column flow rate of 7.7 ml/min. Six micro-liters of the hexane sample was injected into the column. Theinjection and detector temperatures were 240 °C and 250 °C, respec-tively. Initial temperature was set at 100 °C for 5 min, then the tem-perature was raised at a rate of 15 °C/min up to 240 °C, and thenheld at 240 °C for 10 min. The identification of fatty alcohols was per-formed by comparing the retention times with authentic standards.Alcohol quantifications were performed by the internal standardmethod using heptadecanol (50 nmol), which was added prior tolipid extraction.

2.4. GC-MS analysis

The products from in vitro assayswere derivatized using 4 volume of50 mMbenzylhydroxylamine hydrochloride in methanol and 1 volumeof 0.5 M sodium acetate in water. The vortexed mixture was kept atroom temperature overnight and then acidified to pH 3 and extractedwith diethyl ether. The ether phase was washed with water andevaporated to dryness. The residue was dissolved in a small volume ofmethanol and treated for about 30 sec with an excess of ethereal diazo-methane. The material obtained following evaporation was treatedwith amixture of trimethylchlorosilane/hexamethyldisilazane/pyridine(2:1:2 by vol.) at room temperature for 15 min. Excess reagentwas removed in a vacuum and the residue dissolved in hexane. Thismethod will convert fatty acids to methyl esters, alcohols to Me3Siderivatives, and aldehydes to BO derivatives, all suitable for analysisby GLC or GC-MS. GC-MS was carried out with a Hewlett-Packardmodel 5970B mass-selective detector connected to a Hewlett-Packardmodel 5890 gas chromatograph equipped with a capillary column of5% phenylmethylsiloxane (12 m, 0.33 μm film thickness). Helium wasused as the carrier gas, and the column temperature was raised at10 °C per min from 120 °C to 300 °C. The instrument was operatedeither in the full scan mode (m/z 50–600) or in the selected-ion moni-toring mode.

2.5. Purification of AtFAR6 and mAtFAR6 expressed in E. coli

The DNA sequences encoding full-length AtFAR6 and matureAtFAR6 (mAtFAR6), which lacks the predicted chloroplast transit

1246 T.T.P. Doan et al. / Biochimica et Biophysica Acta 1821 (2012) 1244–1255

peptide sequence, were amplified by PCR using pET15b harboringAtFAR6 [12] as template. Forward primer FAR6GWNf2 and reverseprimer FAR6GWNr were used to amplify the AtFAR6 coding regionand forward primer mFAR6GWNf2 and reverse primer FAR6GWNrwere used to amplify the mAtFAR6 coding region (primer sequencesare listed in Supplementary Table S1). The forward primers containedcodons for a TEV cleavage site at their 5’ ends. In a subsequentamplification, the second forward primer attB1TEVcontaining anattB1 site was used together with reverse primer FAR6GWNr. PurifiedPCR products were subjected to a BP-reaction for introduction intopDONR221 (Invitrogen) to create Entry vectors pEntry-FAR6 andpEntry-mFAR6. The modified coding regions were subsequentlymoved into pDESTHisMBP [20] via a LR-reaction according to themanufacturer's protocols. pDESTHisMBP harboring AtFAR6 or mAt-FAR6 (pHisMBP-AtFAR6 or pHisMBP-mAtFAR6) were transformedinto E. coli strain Rosetta (DE3) (Novagen) for heterologous proteinexpression.

A single colony was picked and used to inoculate 40 ml of Luria-Bertani broth (LB) media supplemented with 50 μg/ml carbenicillinand 34 μg/ml chloramphenicol. The cultures were grown overnightat 37 °C with 240 rpm agitation. Subsequently, the overnight cultureswere diluted in 800 ml of antibiotic-supplemented LB media to a finalOD600 of 0.1 and then incubated at 37 °C, 240 rpm agitation until theOD600 reached 0.5 to 0.8. The cultures were transferred to room tem-perature for 30 min followed by the addition of isopropyl−1-thio-D-galactopyranoside (IPTG) to a final concentration of 0.5 mM to inducegene expression. Bacteria were grown for an additional 4 to 6 hours atroom temperature and 240 rpm agitation before being harvested bycentrifugation at 5000 g and 4 °C for 20 min. The cell pellets werethen flash frozen in liquid nitrogen and stored at −80 °C for subse-quent protein purification.

For protein purification, the bacterial pellets were resuspended in36 ml of 100 mM ice cold phosphate buffer, pH 7.0 containing 40 mMimidazole, 10% (v/v) glycerol, 1x Complete Protease Inhibitor (RocheApplied Science), 5 mM MgCl2, 2 mM ATP, 150 mM KCl and 18 μlLysonase (Novagen). Prior to homogenization, 1 volume of 0.1 mmglass beads were added to the cell suspensions and incubated on icefor 30 min. Cells were homogenized using a FastPrep 24 homogenizer(MP Bio) at 4.0 m/s for 3 pulses of 30 seconds with 2 min rest be-tween pulses. The cell lysates were clarified by centrifugation at3000 g and 4 °C for 5 min. The supernatants were further centrifugedat 10,000 g and 4 °C for 20 min. Collected supernatants were loadedonto a 1 ml HisTrap HP column (GE Healthcare) at a flow rate of1 ml/min using BioLogic LP liquid chromatography system (Bio-Rad). The loaded column was then washed with 15 ml of bindingbuffer (100 mM Phosphate buffer, pH 7.0 containing 40 mM imidazoland 10% (v/v) glycerol) at a flow rate of 1 ml/min. Subsequently, a lin-ear gradient of 40 mM to 500 mM imidazole in 20 ml of 100 mMphosphate buffer, pH 7.0 containing 10% glycerol was applied at aflow rate of 1 ml/min to elute His-tagged proteins from the column.Eluted proteins were aliquoted, flash frozen in liquid nitrogen andstored at −80 °C for further analysis and enzyme assays. Proteinconcentration was determined using a BCA protein assay (ThermoScientific) according to the manufacturer's recommendations. Corre-sponding fractions from E.coli extracts harboring empty plasmidpET15b were used as controls in enzyme assays.

2.6. In vitro enzyme assays

The activity of enzyme was determined by measuring the forma-tion of [14C]-fatty aldehyde and [14C]-fatty alcohol from its corre-sponding [14C]-fatty acyl-CoA or -ACP. To verify the identities of theobserved fatty aldehydes, fatty acids and alcohols on thin layer chro-matography plate (TLC), the products were derivatized as describedabove for GC-MS analysis in comparison to authentic compounds.To avoid substrate depletion, the assay conditions were adjusted

with regard to incubation time and enzyme concentration, such thatless than 50% of substrate was utilized in each assay. Unless otherwisespecified, the assays were conducted in 50 μl of 100 mM phosphatebuffer, pH 7.0 containing 10 mM NADPH, 50 μM [14C] fatty acyl-CoA,3 mg/ml BSA and 0.3 to 0.6 μg of protein. After incubation for30 min at 30 °C, the reactions were stopped by adding 125 μl of chlo-roform and the total lipids were extracted by the method of Bligh andDyer [19]. Extracted lipids were subsequently separated on TLC Silica60 plates (Merck) with hexane/diethyl ether/ acid acetic (55:45:0.5,v/v/v) as the developing solvent. [14C] on the TLC plates was mea-sured for 15 hours using electronic autoradiography (Instant Imager,Canberra Packard) with 3250 dpm of [14C]-C18:1-OH used as thestandard control for the quantification.

2.7. Transient expression of AtFAR6 and mAtFAR6 in Nicotianabenthamiana leaves

Nicotiana benthamiana plants were grown at 26 °C±0.5, 60% rela-tive humidity with 12 h light/12 h dark photoperiod (320 μmol/m2/s).The 4 to 6 week old plants were used for infiltration.

For binary vector constructions,AtFAR6 andmAtFAR6were amplifiedby PCR using reverse primer FAR6_Bn7_R containing a recognitionsite for XbaI, and forward primers FAR6_Bn7_F and mFAR6_Bn7_F forAtFAR6 and mAtFAR6, respectively, containing a recognition site forXhoI (Supplementary Table S1). The amplified fragments were digestedwith XhoI and XbaI, and then inserted between the corresponding sitesof vector pART7 [21]. The plasmids harboring AtFAR6 and mAtFAR6,pART7-AtFAR6 and pART7-mAtFAR6, were subsequently digestedwith NotI. The resulting expression cassettes, which contain AtFAR6 ormAtFAR6 downstream of the cauliflower mosaic 35S promoter, werethen gel purified and ligated into the NotI site of the expression vectorpART27 [21]. The binary vector harboring either AtFAR6 or mAtFAR6,pART27-AtFAR6 or pART27-mAtFAR6, as well as the P19 and GFP con-structs [22] were introduced into Agrobacterium tumefaciens strainGV3101::pMP90 [23] by electro-transformation.

The A. tumefaciens cells were grown overnight at 28 °C, 240 rpmagitation in 5 ml LB medium supplemented with 50 μg/ml rifampicin,50 μg/ml gentamycin and 50 μg/ml spectinomycin. For the culture ofbacteria harbouring P19, 50 μg/ml of kanamycin was used instead ofspectinomycin. When the OD reached 0.5 to 1, acetosyringone wasadded to a final concentration of 100 μM to induce the vir operon ofA. tumefaciens. Bacteria were grown for an additional 3 hours andthen harvested by centrifugation at 3000 g for 5 min at room temper-ature. The harvested cells were subsequently resuspended in 1 ml ofinfiltration medium containing 5 mM MgCl2, 5 mM MES, pH 5.7 and100 μM freshly added acetosyringone. Prior to infiltration, bacteriaharbouring either AtFAR6 or mAtFAR6 were mixed with bacteria har-bouring P19 and GFP constructs (also in the infiltration medium),such that the final OD of each bacteria was 0.2. Bacteria only harbour-ing P19 and GFP constructs were used as the control. Leaves wereinfiltrated from the underside using a 1 ml syringe without a needle.After 4 to 5 days in a growth chamber, the infiltrated leaves were ex-amined for GFP expression. The leaf areas with GFP fluorescencewere excised and fresh weight measured. The harvested leaveswere homogenized in 3.75 ml methanol/chloroform (2:1 v/v), fol-lowed by lipid extraction using the method of Bligh and Dyer [19].The extracted lipids corresponding to 100 mg of fresh weight leaf tis-sue was loaded and separated on TLC Silica 60 plates (Merck) withhexane/diethyl ether/ acid acetic (85:15:1, v/v/v) as the developingsolvent. The alcohol spots were located and then scraped for GC andGC-MS analysis as previously described. For the separation of wax es-ters, the TLC plates was developed in hexane/diethyl ether/acetic acid(95:5:1, v/v/v) following by elution of the wax ester area, trans-methylation and GC analysis as described in the analysis of fattyalcohols produced in yeast.

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The leaves of N. benthamiana after 3 days of infiltration were usedfor chloroplast isolation as described by Morré et al. [24].

2.8. Subcellular localization

AtFAR6 and mAtFAR6 were amplified from Arabidopsis cDNA byPCR using reverse primer AtFAR6r and forward primers AtFAR6fand mAtFAR6f for AtFAR6 and mAtFAR6, respectively, while the N-terminal extension corresponding to the 71 first amino acids was am-plified, using primers AtFAR6f and AtFAR6r2 listed in SupplementalTable S1. The corresponding PCR fragments were cloned intopDONR™207 ENTRY vector by GATEWAY® recombination cloningtechnology using the attB x attP (BP) recombination sites. Fragmentswere then transferred into the pK7YWG2 DESTINATION vector [25]by LR cloning, resulting in a 3’-transcriptional fusion between thecDNA and the eYFP gene. Constructs were transferred into the Agro-bacterium tumefaciens GV3101 and used for transient expression inNicotiana tabacum leaves according to Charte et al. [26].

2.9. FAR6 promoter:GUS fusion and GUS histochemical assay

A 2187 bp fragment of AtFAR6 upstream sequence was amplifiedusing BAC T8M16 as template and primers FAR6_Prom_forward andFAR6_Prom_reverse (Supplemental Table S1). The forward and re-verse primers contained SalI and BamHI recognition sites, respective-ly. The amplified products were digested with SalI and BamHI andcloned between the corresponding sites of pBI101 (Clontech) fusingthe first seven codons from the AtFAR6 coding region in frame withthe coding region of the β-glucuronidase (GUS) gene. The constructwas introduced into wild-type Col-0 plants by Agrobacterium-mediated transformation using the floral dip method [27].

Various plant tissues of third generation lineswere analyzed for GUSactivity at different stages of the plant's life cycle. Plant tissues were col-lected in 24-well culture plates, submerged in cold heptane, incubatedon ice for 5 minutes, and then allowed to air dry for 5 minutes. The tis-sues were rinsed with staining buffer (50 mM NaPO4 pH 7.0, 0.5 mMK4Fe(CN)6, 0.5 mMK3Fe(CN)6, and 0.1% TritonX-100) and then coveredin staining buffer containing 1.12 mM 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-Glucuronide). The samples were then incubated forvarious times at 37 °C: root tissues were stained for 1–2 hours, stem tis-sues were stained for 14–17 hours, and other aerial tissues (e.g. flower)were stained for 7–12 hours.Whole tissue samples were examined andphotographed using a Zeiss Discovery V20 stereomicroscope equippedwith an Axiocam MRC camera. Sectioning of stem tissue embedded inparaffin was done as described in Domergue et al. [1]. For sectioningof anthers, GUS-stained floral tissues were incubated in 25 mM Sodiumphosphate (pH6.8) containing gluteraldehyde (2%) at 4 °C overnight.Following fixation, samples were rinsed for 2 min in 50% ethanol anddehydrated in an ethanol series of 50%, 70%, 95%, and two changes of100% ethanol for 30 min each. Dehydrated samples were then infiltrat-edwith 100% LRWhite resin (ElectronMicroscopy Sciences) on a rockerwith three changes of 100% resin, each for an hour. The resin-infiltratedtissues were then mixed with an appropriate quantity of accelerator(supplied with the resin). The mixture was immediately poured intoa capsule and allowed to polymerize at room temperature. Ten-micrometer-thick sections were cut on a microtome and imaged usinga compound microscope (Zeiss Axio Imager M2).

3. Results

3.1. Fatty alcohol production by AtFAR6 in yeast

AtFAR6 is a 548 amino acid protein containing a 71 amino acid N-terminal extension in contrast to most other alcohol-forming FARsfrom plants, which are typically around 490–500 amino acids in

length [2]. Among AtFAR homologues, AtFAR6 had highest aminoacid sequence similarity to AtFAR2/MS2, a FAR enzyme responsiblefor production of C16:0 alcohol in chloroplast [2,28,29]. Similar toAtFAR2/MS2, AtFAR6 is predicted to be chloroplast localized by sub-cellular localization programs, albeit with low reliability scores [12].ChloroP identifies a sequence corresponding to the 47 N-terminalamino acids of the translated AtFAR6 cDNA sequence as a putativechloroplast transit peptide (ctp) [30]. To investigate the activity ofAtFAR6 in yeast, the coding regions of a full length version (AtFAR6)and a truncated version (mAtFAR6), which lacks the region corre-sponding to the predicted 47 amino acid chloroplast transit peptidesequence (Fig. 1A), were each subcloned into the pYES-DEST52 plas-mid downstream of the GAL1 promoter and then transformed intoS. cerevisiae W303-1A strain. After induction with galactose, yeastcells expressing full-length AtFAR6 produced C16:0 primary fattyalcohol, albeit at very low levels, which were not observed in the con-trol yeast harboring empty pYES-DEST52 plasmid. In contrast, cellsexpressing mAtFAR6 accumulated much higher levels of C16:0 aswell as C18:0 fatty alcohols. For both mAtFAR6 and AtFAR6 expres-sions, the C16:0-OH produced was much more than the C18:0-OHproduced, 15.4 and 3.9 times, respectively (Fig. 1B). In addition,expression in yeast of a version of AtFAR6 lacking the entire N-terminal extension relative to most FARs (i.e. the first 71 aminoacids) and mAtFAR6 both resulted in fatty alcohol production(Supplemental Fig. S1).

3.2. Activity and substrate specificity of purified FAR enzyme in vitro

We then expressed both AtFAR6 and mAtFAR6 in E. coli in order toperform in vitro assays with the purified proteins. To improve theirsolubility, both enzymes were fused at the C-terminus to maltosebinding protein (MBP), which had been shown to have a positive ef-fect on protein solubility [31]. Enzymes were then purified using Ni2+

resin from E. coli extracts and fractions showed a strong band on SDSgel electrophoresis (Fig. 2A). Removal of the maltose binding part se-verely decreased the stability of the enzyme, making it not possible todo accurate enzyme assays with this protein (data not shown). Itshould be pointed out that the specificities obtained from in vitro as-says were consistent with the in vivo specificities of the enzyme with-out the maltose binding part in yeast and N. benthamiana (datapresented below). Therefore, it is not likely that the maltose bindingpart of the protein affected the specificities of the enzyme.

In vitro assays in the presence of [14C]-16:0-CoA resulted in theproduction in C16:0-OH with both proteins AtFAR6 and mAtFAR6.Nevertheless, since similar to the yeast expression results, the activityof AtFAR6 was significantly lower than that of mAtFAR6 (Fig. 2B), wedecided to use only mAtFAR6 for further in vitro characterization. Itwas found that under in vitro condition besides fatty alcohol pro-duced as an end product (Fig. 2 C and supplemental Fig. S2 A, E, F),mAtFAR6 also released fatty aldehyde (Fig. 2C and supplemental Fig.S2 A-D) and free fatty acids (Fig. 2C and supplemental Fig. S2 A).Moreover, we observed that mAtFAR6 could utilize C18:0- aldehydefor producing fatty alcohol (Supplemental Fig. S3). Purified mAtFAR6utilized NADPH as a cofactor and no significant activity was observedwhen NADH was used as cofactor (Fig. 2D). The purified enzyme dis-played similar activity under a wide range of pH (4 to 10) and the ac-tivity was linear with time for up to 30 min of incubation at 30 °C(data not shown). Due to the presence of some extra protein bandsin the purified fraction, the same protein purification procedure wasapplied to an extract of E. coli harboring empty pET15b plasmid,similar eluted protein fractions were collected and used for in vitroassay as negative control. No metabolism products (i.e. fatty alde-hyde, fatty alcohol, fatty acid) were detected with this negativecontrol reaction upon addition of acyl-CoA (data not shown).

The substrate specificity of the enzyme was determined by usingsaturated [14C]-fatty acyl-CoA substrates with carbon chain lengths

AtFAR6

Putative transit peptide H2N COOH

mAtFAR6 H2N COOH

47 amino acids 501 amino acidsH2N COOH

RRVQTS C

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Fig. 1. Heterologous expression of AtFAR6 in yeast. A. Structures of the proteins AtFAR6 (full length), mAtFAR6 (mature, without transit peptide) and the putative chloroplast transitpeptide predicted by ChloroP program. B. Fatty alcohols produced in yeast expressing AtFAR6 or mAtFAR6. Error bars indicate 95% confidence limits.

AldehydeAlcohol

Fatty acid

D

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kDa200

150120

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60-70

50

4030

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Fig. 2. Activity of purified mAtFAR6 and AtFAR6 in vitro. A. SDS-PAGE of purified MBP-mAtFAR6. L1: Protein ladder, L2: purified fraction containing MBP-mAtFAR6 B. Activity ofmAtFAR6 and AtFAR6 in vitro. Assays were conducted in 100 mM potassium phosphate buffer (pH 7.0), 50 μM [14C] 16:0–CoA, 10 mM NADPH and 3 mg/ml BSA at 30 °C. Errorbars indicate 95% confidence limits. C. Electronic radiochromatogram of TLC showing the alcohol, aldehyde and free fatty acid produced by mAtFAR6. Assays were conducted in100 mM potassium phosphate buffer (pH 7.0), 50 μM [14C] 16:0–CoA, 10 mM NADPH and 3 mg/ml BSA at 30 °C. L1: [14C] 18:1-OH standard, L2: assay without adding enzyme,L3: assay using 50 μg/ml mAtFAR6 D. Effects of cofactors on activity of mAtFAR6. Assays were conducted in 100 mM potassium phosphate buffer (pH 7.0), using 1.25 μM [14C]16:0–CoA as substrate in the presence of either 10 mM NADPH or NADH. Assays were incubated at 30 °C. Error bars indicate 95% confidence limits.

1248 T.T.P. Doan et al. / Biochimica et Biophysica Acta 1821 (2012) 1244–1255

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Fig. 3. Substrate specificity of purified mAtFAR6. Assays were conducted in 100 mM po-tassium phosphate buffer (pH 7.0), 50 μM [14C] acyl–CoA, 10 mM NADPH and 3 mg/mlBSA at 30 °C. Error bars indicate 95% confidence limits.

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Fig. 5. Influence of [14C] 16:0–CoA on the activity of purified mAtFAR6 in vitro. Assayswere conducted in 100 mM potassium phosphate buffer (pH 7.0), 10 mMNADPHwith-out the addition of BSA. Error bars indicate 95% confidence limits.

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ranging from C14 to C20. Among the tested acyl-CoAs, the utilizationof [14C]-16:0-CoA yielded the greatest reduction to its correspondingfatty alcohol and aldehyde (Fig. 3). When [14C]-16:0-CoA or [14C]-18:0-CoA were used as substrates, fatty alcohols were the principalproduct and with aldehydes representing about half of the fatty alco-hols produced and free fatty acids being only minor products (Fig. 3).In contrast, the reduction of [14C]-20:0-CoA generated about equalproportions of fatty alcohols and fatty aldehydes, while that of[14C]-14:0-CoA produced equal proportions of fatty aldehydes andfree fatty acids and very low amount of fatty alcohol (Fig. 3). It shouldbe noted that the total reduction of C14:0-CoA was only approximate-ly 5% of the formation of alcohols from 16:0-CoA (Fig. 3). The highestratio of reductase activity (production of fatty alcohol and aldehyde)to thioesterase activity (production of free fatty acid) was observedwhen using C16:0-CoA as the substrate (Fig. 3). No productswere detected when using C18:1-CoA, 2-methyl- 16:0-CoA, 2-methyl_18:0-CoA or C16:0 free fatty acid as substrates (data notshown).

Because AtFAR6 was found to be a plastid localized protein inthe transient expressions system (see section below), the kinetics ofenzyme activity with C16:0-ACP and C16:0-CoA substrates weredetermined with non purified enzyme in E. coli extracts. The resultshowed that the maximum specific activity of enzyme towardC16:0-ACP was reached at a Km of 1.48±0.60 μM as compared toKm of 3.36±0.67 μM when using C16:0-CoA as substrate (Fig. 4Aand B). The results were later compared with purified enzyme forthe acyl-CoA substrate, and gave approximately the same Km values(2.58±0.87 μM) as that for the non-purified enzyme but with an

[14C] C16:0-CoA (µM) [14C] C16:0-

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Vmax = 293.0 ± 34.1Km = 1.48 ± 0.60 µM

A BNon purified protein Non purif

Fig. 4. Kinetics of AtFAR6, based on total reduction products of enzyme (i.e. fatty alcohol andpotassium phosphate buffer (pH 7.0), 10 mM NADPH at 30 °C. Data was analyzed using Grappurified enzyme with [14C] 16:0–CoA. B. Michaelis-Menten plot for non purified enzyme wi

expected higher Vmax value (Fig. 4C). Due to limitation of [14C]acyl-ACP substrates available, it was not possible to repeat the kinet-ics with acyl-ACP using the purified enzyme but the results fromincubations of the purified enzyme with two different acyl-ACP con-centrations were consistent with the results obtained with the non-purified enzyme (data not shown).

The critical micelle concentration of 16:0-CoA range from 7 μM-250 μM depending on pH, mixtures of salts, buffer and other compo-nents [32]. We observed that when no BSA was added to the assays,the reduction activity of AtFAR6 was increased up to 5 μM C16:0-CoA but at higher concentration the activity of enzyme was decreased(Fig. 5) and with no activity at all observed at 20 μM C16:0-CoA(Fig. 6B). Therefore we hypothesized that the micelle of acyl-CoAswas inhibitory to the enzyme. In order to further investigate this,we carried out enzyme assays using C14:0-CoA, C16:0-CoA andC18:0-CoA at concentration of 7.5 times above the Km for C16:0-CoA and in the presence of BSA at a concentration that ranged from0 to 2 mg/ml for C14:0-CoA and C18:0-CoA; for C16:0-CoA substrate,0 to 9 mg/ml of BSA was used. No reduction was seen in the assaywithout adding BSA with any of the substrates, indicating that sub-strates were present in micelles that inhibited enzyme activity. Con-version to both alcohols and aldehydes were observed when BSAwas present in the assays (Fig. 6A-C). When C14:0-CoA was used,fatty aldehyde and fatty acids were almost exclusively produced inthe presence of BSA and the fatty alcohol/fatty aldehyde ratio wasonly slightly changed with the increasing of BSA concentration(Fig. 6A). At the C16:0-CoA substrate concentration tested, the great-est alcohol production was reached at a BSA concentration of 1 to2 mg/ml (Fig. 6B) and the specific activity was about the same asfound at optimal 16:0-CoA concentrations (5 μM) without addition

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Cied protein Purified mFAR6

aldehyde). Assays were conducted using different substrate concentrations in 100 mMhpadPrism. Error bars indicate 95% confidence limits. A. Michaelis-Menten plot for nonth [14C] 16:0–ACP. C. Michaelis-Menten plot for purified mAtFAR6 with [14C] 16:0–CoA.

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Fig. 6. Influence of BSA on the activity of purified mAtFAR6 in vitro. Assays were con-ducted in 100 mM potassium phosphate buffer (pH 7.0), 10 mM NADPH and 20 μM[14C] acyl–CoA at 30 °C for 30 min. Error bars indicate 95% confidence limits. A. Assaysusing [14C] 14:0–CoA as the substrate. B. Assays using [14C] 16:0–CoA as the substrate.C. Assays using [14C] 18:0–CoA as the substrate.

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of BSA (Fig. 5). The same trend was found when C18:0-CoA was usedas the substrate, although the greatest alcohol and aldehyde produc-tion was in this case reached at a BSA concentration of 0.5 mg/ml to1 mg/ml (Fig. 6C). It was also observed that for the preferred sub-strate (C16:0-CoA), the ratio of alcohol to aldehyde increased withfurther increasing BSA concentration but overall production of alco-hols decreased (Fig. 6B).

3.3. Subcellular localization of AtFAR6

In order to gain insights into the subcellular localization of AtFAR6,we performed transient expression experiments in Nicotiana tabacumleaves using constructs carrying various parts of AtFAR6 fused to the

30µm 25µm

FAR6wFAR6-YFPA B

Fig. 7. Subcellular localization of full-length AtFAR6 and truncated variants. Yellow fluoresamino acids (B), and the N-terminal 71 amino acids of AtFAR6 (C), were transiently expremicroscopy.

coding region of yellow fluorescent protein (YFP). When the full-length AtFAR6 sequence was fused at the N-terminus of YFP, fluores-cence appeared in a distinct punctuate pattern that is typical of plas-tidic localization (Fig. 7A). In contrast, when a sequence lacking thefirst 71 amino acids of the AtFAR6 protein was used, the YFP fluores-cence was more diffuse and resembled that of cytosolic proteins(Fig. 7B). To confirm that the N-terminal sequence of AtFAR6 couldtarget a protein to the chloroplast, we then expressed a fusion ofthe first 71 amino acids of the AtFAR6 protein to YFP. Expression ofthis fusion protein resulted in a similar punctate pattern observedwith the full-length AtFAR6 sequence (Fig. 7C). Plastidial localisationwas confirmed by co-expression studies using the N-terminal portionof the N. tabacum monogalactosyltransferase fused to green fluores-cent protein (GFP) (Supplemental Fig. S4).

3.4. Transient expression in Nicotiana benthamiana leaves

The transient expression of genes in Nicotiana benthamiana leavesis a valuable tool for functional studies of genes in planta. Recently, thetransient expression of genes involved in lipid synthesis has been suc-cessfully implemented [22,33]. In the present study, we expressedeither AtFAR6 or mAtFAR6 together with the P19 and GFP genes. P19is a viral protein that acts as an inhibitor of silencing in order to pro-long the expression of transgenes in the host cells [22,33,34]. GFPwas used as an expression marker to monitor the infiltration andexpressed leaf areas used for further analysis. Analysis of lipids fromleaves 4 to 5 day post infiltration showed that in contrast to the insig-nificant alcohol production detected with infiltrations involving nega-tive control and mAtFAR6 (Fig. 8A and B), a substantial amount ofprimary alcohols were produced in leaves infiltrated with AtFAR6(Fig. 8C). For AtFAR6-infiltrated leaves, the primary alcohols constitut-ed between 1.5 and 3% of total acyl groups, while the control leavesand the leaves transiently expressing mAtFAR6 contained only 0.05%and 0.11% primary alcohols out of total acyl groups, respectively(data not shown). Among the primary alcohols in leaves expressingAtFAR6, C16:0-OH and C18:0-OH were the predominant produced al-cohols, which were 74% and 16% of total primary alcohol production,respectively (Fig. 8C). We also found that expression of AtFAR6 in-duced the production of the wax ester containing C16:0-OH whichaccounted for 20% of C16:0 alcohol produced. This was not observedin control infiltrated tissue or from leaves expressing mAtFAR6 (datanot shown). The alcohol analysis of intact chloroplasts isolated fromN. benthamiana leaves showed that, contrary to a trace amount of alco-hols found in the chloroplasts frommAtFAR6 or control leaves (Fig. 9Aand B), a significant accumulation of C16:0-OH and C18:0-OH wasdetected in the chloroplasts of leaves infiltrated with AtFAR6 (Fig. 9C).

3.5. Domains of AtFAR6 gene expression

Previous work using quantitative RT-PCR indicated that AtFAR6 isexpressed in stems [1] and DNA microarrays indicated enrichment

30µm

/oNterm-YFP NtermFAR6-YFPC

cent protein (YFP) fusions of full-length AtFAR (A), AtFAR6 lacking the N-terminal 71ssed in Nicotiana tabacum leaves and fluorescence imaged by laser scanning confocal

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Fig. 8. GC analysis of fatty alcohol produced in Nicotiana benthamiana leaves expressing mAtFAR6 and AtFAR6. C17:0-OH (3 nmol) was added as internal standard. The identifiedalcohol peaks were confirmed by GC-MS. A. Control: leaves expressed GFP and P19. B. mAtFAR6: leaves expressed mAtFAR6, GFP and P19. C. AtFAR6: leaves expressed AtFAR6,GFP and P19.

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in the epidermis [35]. We investigated the gene expression pattern ofAtFAR6 using transgenic Arabidopsis plants harboring a translationalfusion of the predicted AtFAR6 promoter (2187 bp upstream of thestart codon) fused to the GUS reporter gene (Fig. 10). Histochemicalstaining for GUS activity was performed on twelve T1 transformants,and then repeated in several T2 or T3 transgenic lines that showedrepresentative expression patterns. The AtFAR6 promoter drove re-porter gene expression in various tissues throughout the plant, in-cluding the stem epidermal layer and the underlying few cell layers,but not in the inner cortex or vascular bundles (Fig. 10A-C). Inflowers, the AtFAR6 promoter was active in the epidermis, endotheci-um and tapetum of anthers, but not in the microspores (Fig. 10D-E).The AtFAR6 promoter also drove GUS expression in the replum andreceptacle of siliques (Fig. 10F). In roots, GUS activity was observedin emerging root primordia and expression remained in the root capthroughout lateral and primary root development (Fig. 10G-I).

4. Discussion

4.1. Activity of AtFAR6

It has been found that plant FARs catalyze the conversion of fattyacyl-CoA into primary alcohol via a presumed fatty aldehyde interme-diate [1,2,7,9]. However, the aldehyde intermediate was not detectedin enzyme assays and was proposed not to be released by the FAR [9].In contrast to this, in vitro assays using purified AtFAR6 enzymeshowed that in addition to alcohols produced as the final product,fatty aldehydes were also released as well as free fatty acids. Theratio of produced alcohol, aldehyde and fatty acids depended stronglyon the carbon chain length and the saturation status of the substrateas well as on the assay conditions (e.g. available substrate concentra-tion). BSA was utilized to reduce the inhibitory effect caused by mi-celle formation of fatty acyl-CoAs [9]. The stimulatory effect of BSA

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Fig. 9. GC analysis of fatty alcohol produced in chloroplasts of Nicotiana benthamiana leaves expressing mAtFAR6 and AtFAR6. C17:0-OH (3 nmol) was added as internal standard.A. Control: chloroplasts of leaves expressed GFP and P19. B. mAtFAR6: chloroplasts of leaves expressed mAtFAR6, GFP and P19. C. AtFAR6: chloroplasts of leaves expressed AtFAR6,GFP and P19.

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on enzyme activity was also reported by Pollard et al. [36]. In our ex-periments, the reduction activity of AtFAR6 was stimulated in thepresence of BSA up to an optimal concentration, after which it wasdecreased. The result indicates that AtFAR6 utilize free monomericform of acyl-CoA and is inhibited by micelles of acyl-CoAs, which isremoved by the binding of the substrate to BSA, but it cannot usethe BSA bound substrates. When using C14:0-CoA as a substrate,only fatty aldehydes and free fatty acids were produced and thiswas independent of substrate/BSA ratios. This would suggest thatC14:0-CoA is accepted by the enzyme as a substrate but a substantialportion of the acyl groups are not bound to the protein after cleavagefrom CoA and thus released as free fatty acids. The proportion of the14:0 acyl chains that is bound to the enzyme only undergo the first

reduction step, with release of the fatty aldehyde. Therefore, our re-sults illustrate that besides being an alcohol-producing enzyme,AtFAR6 acts both as a thioesterase and an aldehyde-producingenzyme, at least in vitro. Under in vitro condition, whether AtFAR6acts as primarily an aldehyde-forming FAR or an alcohol-formingFAR is determined by the acyl chain length of available substrates aswell as the substrate concentration.

4.2. Substrate specificity of AtFAR6

Several studies on plant FARs have revealed that the fatty alcoholpatterns produced by in vivo expression depend highly on the relativeamounts of substrates present in the expression system. This applies

Fig. 10. Gene expression pattern of AtFAR6. GUS expression pattern under the control of the 2187 bp upstream region, relative to the start codon, of AtFAR6. A representative trans-genic line is shown, depicting staining in stem (A-C), anther (D-E), silique receptacle (F), and root (G-I).

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to the spatial availability of substrate as well as the available panel ofsubstrates. As an example, expression of jojoba FAR in E. coli resultedin the accumulation of C16:0-OH and C18:1-OH while mostly C22:1-OH besides minor amounts of C20:1 and C24:1-OH were producewhen the jojoba FAR was expressed in seeds of high erucic acidrape (HEAR) Brassica napus [10]. Consistent with the seed expressionsystem, the naturally occurring wax esters of jojoba seeds consist ofC20 to C24 monounsaturated fatty alcohols [37]. Discrepancies insubstrate specificities among different host expression systems werealso found in the case of a FAR from wheat [11], as well as CER4/AtFAR3 and AtFAR1 from Arabidopsis [1,2,12]. This illustrates the ca-pacity of the studied FARs to utilize a wide range of substrates. Incontrast to these examples, AtFAR6 consistently showed specificitytoward only C16:0 and C18:0 substrates under in vitro conditionsand in all tested expression systems (E. coli, yeast, N. benthamianaleaves). Therefore, it is highly likely that AtFAR6 generates C16:0-OH and potentially C18:0-OH in Arabidopsis.

We detected a small amount of wax ester comprised of C16:0-OHwhen AtFAR6 was transiently expressed in N. benthamiana leaves,which suggests a possibility that at least a portion of the fatty alcoholproduced by AtFAR6 is utilized for wax ester biosynthesis in Arabi-dopsis. It has been found that the seed coat- associated suberin ofBrassica napus contains C14, C15 and C16 primary alcohols [38]whereas in Arabidopsis seed coat as well as root, suberin consists pri-marily alcohols C18, C20, C22 chain length primary alcohols [1]. In cu-ticular waxes of Arabidopsis, the carbon chain lengths of primaryalcohols are 24 to 30 carbons in length [2,39]. Accordingly, AtFAR6,which has preference for C16:0 length substrates, is unlikely to be

involved in the production of primary alcohols for suberin or cuticularwaxes in Arabidopsis. Also, the gene expression pattern deducedfrom promoter:GUS transgenic lines is inconsistent with the typicalexpression patterns of cuticle or suberin-associated genes. In addi-tion, analyses of suberin and cuticular waxes of Arabidopsis lineswith T-DNA insertions in AtFAR6 did not show any difference infatty alcohols and/or wax ester levels (data not shown).

4.3. Subcellular localization of AtFAR6

AtFAR6 was determined via a computer assisted prediction pro-gram to contain a chloroplast transit peptide sequence [12]. In plantcells, de novo lipid synthesis takes place in the chloroplast or othertypes of plastids and results in the production of saturated fattyacyl-ACPs with 16 to 18 carbons. These fatty acyl-ACPs are then desa-turated (in case of C18:0-ACP) and/or acylated to glycerolipid in theplastids or hydrolyzed from ACP by acyl-ACP thioesterases, exportedto the cytosol and activated to fatty acyl-CoAs for further utilizationin the cytosol [40]. Using in vitro assays, we could show that mAtFAR6has the capacity for utilizing acyl-ACP as substrates and the affinityfor this substrate was somewhat higher than for acyl-CoA. These find-ings are consistent with previous experiments showing that AtFAR6was the most efficient enzyme in producing alcohols in E. coli,where acyl-ACP substrates are more likely to be available [12,15].The similar Km values for acyl-CoA and acyl-ACP by FAR6 is insharp contrast to the report of Km values for another chloroplastlocalized FAR (DPW) producing C16:0 alcohol [29]. The Km forC16:0-ACP (3.6 μM) with DPW is in the same range as we found for

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FAR6 whereas the Km for C16:0-CoA with DPW is reported to be 280times higher (1010 μM). This indicates that either DPW uses micellebound acyl-CoAs or that the detergent CHAPS used in these assayseffectively reduces the amount of monomeric acyl-CoA available forthe enzyme by forming mixed micelles with the substrate. Whenusing yeast as a heterologous system, only the expression of mAtFAR6yielded high levels of fatty alcohols suggesting that the presenceof the N-terminal extension is deleterious for AtFAR6 activity inyeast. Conversely, in N. benthamiana, the expression of AtFAR6, butnot that of mAtFAR6, showed significant accumulation of C16:0-OHand C18:0-OH. In contrast to the yeast system, the full lengthAtFAR6 protein is most probably correctly targeted and importedinto the chloroplast in the leaves, where its targeting signal is cleaved.The presence of significant amounts of alcohols within isolated chlo-roplasts of N. benthamiana leaves expressing AtFAR6 strongly sug-gested that AtFAR6 is a chloroplast localized enzyme and is muchmore active in this compartment than in the cytosol and that atleast a large proportion of the produced alcohols are contained inthis organelle. Finally, the chloroplast localization of AtFAR6 is clearlysupported by the experiments conducted using AtFAR6-YFP fusionproteins. These experiments also showed that the N-terminal 71amino acids are necessary and sufficient for importation into theplastids.

It had previously been shown in DNA microarray and quantitativeRT-PCR analysis that AtFAR6 is expressed at relatively high levels instem epidermis of Arabidopsis [1,35]. A significant role for AtFAR6 incuticle synthesis is unlikely, however, as discussed above. C16:0fatty alcohols have not yet been detected, either in free or combinedform, in cuticle extracts or in any other Arabidopsis tissues analyzedthus far. AtFAR6 gene expression in anthers overlaps with AtFAR2/MS2, a gene with relatively high amino acid sequence similarity toAtFAR6. In anthers AtFAR2/MS2 is responsible for producing C16:0alcohol [2,28], and therefore together these FARs may be producingfatty alcohols associated with pollen exine. A plastid-localized FARfrom rice called DEFECTIVE POLLENWALL (DPW) that has activity to-wards C16:0 acyl-ACP and acyl-CoA has recently been reported [29].AtFAR6, along with FAR2/MS2, may fulfill analogous roles of DPWduring anther and microspore development in Arabidopsis. Sincepromoter-GUS fusion expression was detected in other tissues ofthe plant, such as the root tip, it is probable that C16:0-OH producedby AtFAR6 also has unique, yet to be determined, roles in planta.

Supplementary materials related to this article can be found on-line at doi:10.1016/j.bbalip.2011.10.019.

Acknowledgements

The Swedish International Development Cooperation Agency(SIDA/SAREC) and the National Science and Engineering ResearchCouncil of Canada (NSERC) are gratefully acknowledged for financialsupport. This work is part of ICON, a European Commission sponsoredFP7 project. F.D. thanks S. Laloi for participating in this work,Bordeaux Imaging Center of its support, and G. Holzl (University ofBonn) for providing the N. tabacum monogalactosyltransferase. Prof.Sten Stymne is greatly acknowledged for discussion and advice inpreparing the manuscript.

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