Planta (2007) 226:381–394 DOI 10.1007/s00425-007-0489-z
O RI G I NAL ART I C LE
Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes wax-esters Andrew King · Jeong-Won Nam · Jixiang Han · Josh Hilliard · Jan G. Jaworski
Received: 18 November 2006 / Accepted: 29 January 2007 / Published online: 24 February 2007 © Springer-Verlag 2007
Abstract The surface of plants is covered by cuticular wax, which contains a mixture of very long-chain fatty acid (VLCFA) derivatives. This wax surface provides a hydrophobic barrier which reduces non-stomatal water loss. One component of the cuticular wax is the alkyl esters, which typically contain a VLCFA esteriWed to an alcohol of a similar length. As part of an EST project, we recently identiWed an acyltransferase with 19% sequence identity (amino acid) to a bacterial ‘bifunctional’ wax-ester synthase/diacylglycerol acyltransferase (WS/DGAT). Northern analysis revealed that this petunia homologue was expressed predominantly within the petals. The cDNA encoding the WS/DGAT homologue was introduced into a yeast strain deWcient in triacylglycerol biosynthesis. The expressed protein failed to restore triacylglycerol biosynthesis, indicating that it lacked DGAT activity. However, isoamyl esters of fatty acids were detected, which suggested that the petunia cDNA encoded a wax-synthase. Waxes were extracted from petunia petals and leaves. The petal wax extract was rich in
Electronic supplementary material The online version of this article (doi:10.1007/s00425-007-0489-z) contains supplementary material, which is available to authorized users. A. King · J.-W. Nam · J. Han · J. Hilliard · J. G. Jaworski Donald Danforth Plant Science Center, 975 N Warson Road, St Louis, MO 63132, USA A. King (&) CNAP, Department of Biology, University of York, Heslington, York, YO10 5DD, UK e-mail:
[email protected]
VLCFA esters of methyl, isoamyl, and short-tomedium straight chain alcohols (C4–C12). These low molecular weight wax-esters were not present in leaf wax. In-vitro enzymes assays were performed using the heterologously expressed protein and 14C-labelled substrates. The expressed protein was membrane bound, and displayed a preference for medium chain alcohols and saturated very long-chain acyl-CoAs. In fact, the activity would be suYcient to produce most of the low molecular wax-esters present in petals, with methyl-esters being the exception. This work is the Wrst characterization of a eukaryotic protein from the WS/DGAT family. Keywords Acyltransferase · Cuticle · Petal · Petunia · Wax · Wax synthase Abbreviations BHT Butlylated hydroxytoluene BSA Bovine serum albumin BCA Bicinchonic acid BSTFA N,O-bis-(trimethylsilyl)-triXuoroacetamide CoA Coenzyme A DAG Diacylglycerol DGAT Diacylglycerol acyltransferase EST Expressed sequence tag FAMES Fatty-acid methyl esters MGAT Monoacylglycerol acyltransferase TAG Triacylglycerides TMSC Trimethylchlorosilane TMS Trimethylsilyl VCLFA Very long chain fatty acid VLC Very long chain WS Wax synthase
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Introduction The surface of plants, referred to as the cuticle, can be sub-divided into two components: cutin and cuticular waxes. Cutin is a polymer of interesteriWed hydroxylated C16 and C18 fatty acid derivatives, which overlays the epidermal cell wall (Kolattukudy 1980; Nawrath 2002). This cutin layer is covered by and embedded with the cuticular waxes (Kunst and Samuels 2003), an organic solvent extractable layer of lipids derived primarily from VLCFAs (C20–C32). This cuticular wax layer provides a coating to the plant which restricts water loss (Riederer and Schreiber 2001), reXects UV radiation (Holmes and Keiller 2002), regulates the transport of solutes (Baur et al. 1999), and can also play a role in speciWc plant-insect interactions (Markstädter et al. 2000). The composition of cuticular waxes varies greatly between species and between diVerent surfaces of the same plant. Typical components are alkanes, free fatty acids, alcohols, aldehydes, ketones, alkyl-esters, diterpenoids and triterpenoids. The alkylesters (wax-esters) have been found in most cuticular waxes analysed, and range from being a minor component to the major constituent of the wax. The wax from leaves of A. thaliana, for example, contains only 0.1–0.2% esters (Jenks et al. 1995), whereas the wax from fronds of Copernicia cerifera contain up to 85% esters (Kolattukudy 1976). In addition to plant cuticular lipids, wax-esters are found widely in nature. They are present on the cuticle of insects, as storage lipids in jojoba seeds, in beeswax, in sperm-whale oil and as lipid inclusions in some n-alkane degrading bacteria (Kolattukudy 1976; Ishige et al. 2003). Three unrelated families of wax (ester) synthases (WS) have been identiWed so far, all of which are acylCoA dependent transferases. The Wrst identiWed WS was from jojoba (Lardizabal et al. 2000). This desert shrub is unusual in that the main storage lipid that accumulates within its seeds is wax-esters (>98%) rather than triacylglycerol (Ohlrogge et al. 1978). Arabidopsis thaliana contains twelve homologues of the jojoba wax synthase, though none have yet been characterized. The second WS characterized was that from the bacterium Acinetobacter calcoaceticus. This enzyme was in fact bifunctional in vivo, acting as a diacylglycerol acyltransferase (DGAT) and a WS (Kalscheuer and Steinbüchel 2003). In-vitro, this enzyme is capable of using a wide range of substrates as acyl acceptors including alcohols of many chain lengths, 1,2 and 1,3-diacylglycerols, monoacylglycerol, ,-diols and even thiols (Kalscheuer and Steinbüchel 2003; Kalscheuer et al. 2003, 2004; Stöveken et al. 2005; UthoV et al. 2005). This acyltransferase family is not widely
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distributed amongst the prokaryotes. It is almost exclusively restricted to the Actinomycetes, and particularly expanded within the Mycobacterium genus. M. tuberculosis for example has 15 homologues of the Acinetobacter WS/DGAT, and recent studies suggest that the mycobacterial genes may be involved in diverse functions. The activity of DGAT has recently been demonstrated for a number of these homologues, and it has been proposed that these enzymes may play an important role in the accumulation of triacylglycerol within the bacterium under hypoxic conditions (Daniel et al. 2004). Two homologues are located on an operon containing genes involved in cell-wall biosynthesis, suggesting that they could be involved in esteriWcations necessary for the production of the mycobacterial cellwall (Singh et al. 2003). The sequenced genomes of A. thaliana and Oryza sativa contain 11 and 3 homologues of the Acinetobacter WS/DGAT, respectively. Additionally, a recent tBlastn search we conducted of plant EST databases indicated that this gene family is present in many angiosperm and gymnosperm plant species. In mammals, a third WS was recently identiWed which is involved in the synthesis of lipids present in sebum and meibum (Cheng and Russell 2004). This WS is a member of the acyltransferase family, which includes mammalian monoacylglycerol acyltransferases (MGAT) and DGATs (Cheng and Russell 2004), fungal DGATs, and DGAT2 from A. thaliana (Lardizabal et al. 2001; Oelkers et al. 2002). There are no further homologues of this protein in A. thaliana. Cuticular wax biosynthesis has been the focus of much study, particularly in A. thaliana, maize and barley. A number of genes involved in cuticular wax synthesis have been identiWed, usually via screens for mutants which display a visibly altered plant surface, such as glossy rather than glacous surface (Jenks et al. 1995; Post-Beittenmiller 1996). These mutants are typically referred to as glossy (gl) in maize and eceriferum (cer) in Arabidopsis and barley. The precise function for many of the genes identiWed by this approach is still unknown, but none encode proteins with homology to any of the known wax-ester synthases. Furthermore, the chemical analysis that has been performed on these mutants does not suggest that any of the genes identiWed by this approach encode wax-ester synthases. In summary, no plant cuticular wax-ester synthase has yet been identiWed, though based on the number of homologues present from both the jojoba WS and Acinetobacter WS/DGAT families in A. thaliana, one or more of these would seem likely candidates. During a recent EST project within our laboratory, we were attempting to identify acyltransferases from petunia Xower which may be involved in the biosynthesis of lipids in the
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stigma exudates (Matsuzaki et al. 1986; Koiwai and Matsuzaki 1988). One of the acyltransferases we identiWed had signiWcant homology to the bifunctional Acinetobacter WS/DGAT. Expression analysis of this gene revealed that is was highly expressed within the petals. We present evidence that this acyltransferase is involved in the synthesis of a range of wax-esters found in the petunia petal, which are comprised of a VLCFA esteriWed to a short-to-medium chain alcohol (·12 carbons).
Materials and methods Materials Behenoyl-CoA was obtained from Avanti Polar Lipids (Alabaster, AL, USA). 1-14C palmitic acid (1,961 kBq mol¡1) and 1-14C-n-octanol (141 kBq mol¡1) were from American Radiolabelled Chemicals (St Louis, MO, USA). All other chemicals, including acylCoAs and alcohol substrates were obtained from Sigma unless indicated otherwise. EST database construction and analysis One milligram of total RNA was extracted from the stigma of petunia Xowers at the 1–2 cm unopened bud developmental stage. A custom cDNA library which contained 9.0 £ 107 primary plaques was constructed from this RNA by Stratagene (La Jolla, CA, USA) in the Uni-Zap vector. Plasmid DNA (pBlueScript SK-) was produced via in vivo mass excision according to Stratagene’s protocols. The resulting plasmid DNA was used for the production of an EST library at Michigan State University. The EST library was produced from a total of 8,737 high-quality 5⬘-sequencing runs which yielded 1,111 multi-sequence clusters and 4,578 singletons. Sequences were annotated using results from a tBlastn search of GenBank. RNA extraction and blotting Total RNA was extracted from plant tissues using the Qiagen (Valencia, CA, USA) Plant RNA Miniprep kit or alternatively, for large-scale preparations, the trizol method (Chomczymski and Saachi 1987). DIG labelled cDNA probes were made via PCR from a plasmid cDNA library template using primers 5⬘-TTCG GCAGGAATTGAAGATT-3⬘ and 5⬘-ATTAAC TTAAATGCAATTTTGTAA-3⬘. Total RNA (2 g) was separated on a 1.2% agarose gel (containing 2% formaldehyde) and then blotted by upward capillary
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transfer onto Hybond N+ (Amersham, Piscataway, NJ, USA). Transfer of RNA was veriWed by staining blots with 0.02% methylene blue. RNA blots were then developed following the manufacturer’s hybridization and detection protocols (Roche Applied Science, Indianapolis, IN, USA). Yeast strains and growth conditions Saccharomyces cerevisiae JCY500 (MAT trp1-1 ade21 ura3-1 his3-11,15 leu2-3,112 are1::HIS3 are2:: LEU2 dga1::TRP1 lro1::Kanr) was the kind gift of Dennis Voelker (Quittnat et al. 2004). Cells were grown on SD medium (complete or without uracil) containing 2% glucose (maintenance on medium solidiWed with 2% agar), raYnose (precultures) or 2% galactose (induced cultures) as the sole carbon source. The nitrogen source was either 0.5% (NH4)2SO4 or leucine as indicated. The yeast cultures were grown at 30°C in an orbital shaker at 250 r.p.m. Heterologous expression of the wax synthase homologue The coding region of the petunia wax synthase cDNA was ampliWed from a library clone containing the fulllength cDNA via PCR using primers 5⬘-CACCATGA AGTCACTTGCCACA-3⬘ and 5⬘-TGATCATTTCA GATTCTT-3⬘ and Pfu polymerase (Stratagene). The PCR product was gel puriWed and transferred into pTOPO/SD using Invitrogen’s Topo cloning kit to create a Gateway entry vector. This entry vector was used to create a galactose inducible yeast expression vector in pYES-DEST52. This expression vector was transformed into S. cerevisiae JCY500 via the lithium acetate method (Geitz and Woods 2002). For induction of protein expression, overnight starter cultures grown in raYnose medium were used to inoculate galactose medium at an OD600 of 0.1. These cultures were harvested during mid-exponential phase (OD600 of 1-2) and the cells pelleted at 3,000 g for 5 min. All subsequent steps were performed 4°C, using ice-cold buVers. Cells were washed once in sterile water, and then resuspended in extraction buVer (100 mM HEPESNaOH pH 7.1, 1 mM EDTA, 1 mM DTT, 1.2 M sorbitol, 1 mM PMSF, 1 g ml¡1 leupeptin, 1 g ml¡1 pepstatin A and 1.2 M sorbitol). Cells were then broken in 2 ml screw-cap tubes with 0.5 mm glass beads using four 30 s cycles in the Biospec Minibeadbeater (Biospec Products Inc., OK, USA), with 1 min cooling in ice-water between cycles. The caps of the 2 ml tubes were then pierced with a 25-gauge needle and then placed inverted in 15 ml Falcon tubes. Cell lysates were
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recovered by centrifugation at 4,000g for 1 min. The supernatant was then centrifuged for a further 5 min at 2,000g to removed any intact cells and cell ghosts. The lysate was then centrifuged at 400,000g for 15 min in an ultracentrifuge to obtain a soluble (supernatant) and microsomal (pellet) fraction. The microsomal fraction was washed once and then resuspended in extraction buVer by gentle agitation with a glass rod. Aliquots of the microsomal and soluble fractions were Xash frozen in liquid nitrogen and then stored at ¡86°C. Protein concentrations were determined using the BCA assay (Pierce, Rockford, IL, USA) with BSA as a standard. Radiolabelled acyl-coenzyme A synthesis [1-14C]-palmitoyl-CoA was synthesized enzymatically (Taylor et al. 1990) using acyl-coenzyme A synthetase from Pseudomonas putida obtained from MP Biomedicals (Irvine, CA, USA). A two-step puriWcation process was used to remove unreacted free fatty acids and coenzyme A. The reaction (Wnal volume of 800 l) was acidiWed with 10 l of HCl and washed three times with 1.6 ml of diethyl ether. 1.6 ml of H2O and 0.6 ml of 4 M NaCl was then added, and the acyl-CoA extracted twice with 1.5 ml of H2O saturated n-butanol. The combined butanol extracts were then extracted twice with 3 ml of H2O to remove salt. The butanol was then evaporated under a stream of nitrogen and the acylCoA dissolved in 1 ml of 80 mM NaHCO3. The acylCoA was then loaded onto a Sep-Pak C18 cartridge and eluted with 3 ml of 0.4 M NH4OH in 80% methanol and neutralized immediately with 150 l of glacial acetic acid. The acyl-CoA was then dried under N2 and dissolved in 1 ml 80 mM NaHCO3. Enzyme assays Enzyme assays were conducted at 32°C in a volume of 25 l and contained Wnal concentrations of 100 mM HEPES-NaOH, pH 7.1, 1 mM DTT, 1 mM EDTA, 0.02% (by vol) Triton X-100 and 1 mg ml¡1 lipid-free BSA. For comparison of reactions rates with diVerent alcohols, the palmitoyl-CoA concentration was kept at 50 M. Alcohols were prepared as double concentration stock solutions in 0.04% (v/v) Triton X-100 and 2 mg ml¡1 BSA by soniWcation. Reactions were preincubated at 32°C for 5 min and initiated via the addition of enzyme (5 l of 5 g l¡1 microsomal protein). Reactions were terminated after 20 min by the addition of 500 l of chloroform/methanol (2/1, v/v). The reaction was then partitioned with 200 l of 1 M NaCl and the chloroform layer recovered and evaporated under nitrogen. The lipids were then dissolved in a
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minimal volume of chloroform and separated on a 250 M thick silica gel K60 TLC plate (EMD Biosciences, Gibbstown, NJ, USA) developed with hexane/diethyl ether/formic acid (70/30/1, by vol). The wax-ester products were then quantiWed using a Packard Instant Imager (Meriden, CT, USA). For comparison of reaction rates with diVerent acyl-CoAs, 250 M [1-14C]-n-octanol and 50 M acyl-CoAs were used. Extraction of yeast lipids Totals lipids were extracted from 50 ml stationary phase cultures of yeast which were resuspended in water to a Wnal volume of 1 ml and then broken with glass beads as described above. The cell lysate (including debris) was then extracted with 5 ml of chloroform/ methanol (2/1, v/v). The extract was then partitioned by the addition of 2 ml of 1 M NaCl and the chloroform layer dried over anhydrous sodium sulphate. The chloroform was then evaporated under a stream of nitrogen and the lipids resuspened in a 100 l of chloroform. A 20 l aliquot of the lipids were then applied to a 250 M thick silica Gel K60 TLC plate which was developed with hexane/diethyl ether/formic acid (80/20/1, by vol). Lipids were located by UV exposure after spraying with 0.02% primulene in 80% acetone (w/v/v). Plant maintenance and extraction and analysis of cuticular waxes Petunia hydrida W115 (cv ‘Mitchell’, Ball Seed Co, West Chicago, IL, USA) was grown in the greenhouse with day cycles of 14 h at 23–24°C followed by 10 h night cycles at 22–23°C. Cuticular waxes were extracted from 8–12 g of petal or leaf tissue by washing once with 100 ml and once with 50 ml of hexane for 30 s. A third hexane wash did not recover a signiWcant amount of wax (supplementary Fig. 1). The hexane was then removed from the combined extracts by rotary evaporation at 40°C under reduced pressure. The extract was then placed in a vacuum desiccator overnight to remove any low molecular weight volatile components. The wax fraction (<30 mg) was then dissolved in hexane and applied to a 1 g silica gel (Iatrobeads, Mitsubishi Raguka Iatron Inc., Tokyo, Japan) column which had been equilibriated with hexane. Lipids were then eluted with 10 ml of (A) hexane, (B) pentene stabilized chloroform, (C) acetone and (D) methanol. Under these conditions which are routinely used in our laboratory, wax-esters are eluted in the CHCl3. The mass of each fraction were determined by performing triplicate measurements on a Mettler Toledo XS105 balance, recording each mass to within
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the tenth of a milligram range. Fractions were then analysed by GC-MS with or without the derivitization treatments described below.
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nate esters straight chain alcohols (C4–C8), isoalcohols (C4–C6) and anteisoamyl alcohol were also produced via this method to serve as references for GC-MS analysis (retention times and mass spectra).
Production of FAMES and TMS derivatives GC-MS analysis One hundred micrograms of the wax-ester containing CHCl3 fraction obtained from silica gel chromatography was dissolved in 1.3 ml of transmethylation reagent (19/6/1, by vol, methanol/toluene/conc. H2SO4 + 50 mg l¡1 BHT) and reXuxed for 2 h at 80 C. After cooling, 5 ml of 1 M NaCl was added and the FAMES and fatty alcohols extracted into 2 £ 2 ml of hexane. The combined hexane layers were evaporated under N2 and dissolved in 0.5 ml of CHCl3. The CHCl3 was then applied to a 1 g silica gel column and the FAMEs eluted with 10 ml CHCl3 and the fatty alcohols with 10 ml of acetone. The FAMEs fraction was concentrated by evaporation before GC-MS analysis. The acetone fraction containing the fatty alcohols was evaporated under N2 and dissolved in 100 l of a 1/1 (by vol) mix of hexane and 99/1 BSTFA/TMCS and heated to 70 C for 1 h. The reagent was then evaporated under N2 before dissolving the reaction products in hexane for GC-MS analysis.
All analyses were performed using a ThermoWnnigan GCQ coupled to a Polaris MS and AS2000 autosamper. Sample injections volumes were 1 l. Plant waxes, FAMES and TMS derivatives were analysed using a Rtx-5MS column (30 m, 0.25 mm ID, 0.25 m Wlm; Restek, Bellefonte, PA, USA) using a temperature program of 100°C for 1 min, to 300°C at 20°C min¡1, hold at 300°C for 10 min, to 350°C at 20°C min¡1, hold at 350°C for 10 min. Yeast lipids and nicotinate esters were analysed on a DB23 column (20 m, 0.2 mm ID, 0.2 m Wlm; J&W ScientiWc, Folsom, CA, USA), using a temperature program of 100°C for 3 min, to 250°C at 20°C min¡1, hold at 250°C for 10 min.
Results A putative WS/DGAT is expressed predominantly within the petals
Production of nicotinates An aliquot of 0.5–1 mg of the wax-ester containing fraction obtained from the column chromatography was dissolved in 500 l of transmethylation reagent. The wax-ester fractions were then reXuxed at 80 C for 2 h to produce FAMES and free alcohols. The reaction was then cooled on ice and 16 ml of 1 M NaCl added. The FAMES and free alcohols were extracted into 4 ml of chloroform, which was then dried over anhydrous sodium sulphate. A 1 ml aliquot of the chloroform layer was transferred to a vial for GC-MS analysis. The remaining 3 ml of chloroform was cooled on ice and converted to nicotinates (Christie 2003). The reaction was then evaporated under a stream of nitrogen and dissolved in 2 ml hexane. Insoluble material was removed by Wltration through a glass-wool plugged Pasteur pipette, and then the Wltrate was added to a 1 g silica gel column that had been prepared in hexane. The column was washed with the 10 ml of chloroform, and the nicotinate esters of the alcohols eluted in 10 ml of acetone. The acetone fraction was the evaporated under nitrogen and dissolved in 1 ml of chloroform prior to GC-MS analysis. This clean-up procedure was suYcient to remove the bulk of the unreacted N,N-dicyclohexylcarbodiimide which otherwise had a retention time which masked peaks of nicotinate esters. Nicoti-
We have identiWed a cDNA, designated PhWS1, which encoded a putative WS/DGAT. This cDNA was one of a number of acyltransferase gene candidates from a petunia stigma EST project. The aim of this stigma EST project was to identify genes which may be involved in the synthesis of estolides in the stigma exudates in Solanaceae (Matsuzaki et al. 1983, 1986; Koiwai and Matsuzaki 1988). The cDNA encoded a 522 amino acid protein with a sequence that was 19% identical to an A. calcoaceticus WS/DGAT. The protein had a predicted molecular weight of 59.3 kDa and a pI of 6.52. An alignment of protein encoded by PhWS1, the A. calcoaceticus WS/DGAT, three putative DGATs from M. tuberculosis (Daniel et al. 2004), and three A. thaliana homologues of unknown function is shown in Fig. 1, with the putative catalytic HHXXXDG motif highlighted. Interestingly, the phylogenic analysis of PhWS1 and the A. thaliana WS/ DGAT homologues reveals that the petunia protein does not cluster with any of the Arabidopsis proteins (supplementary Fig. 2). To determine the spatial expression pattern of the petunia WS/DGAT gene we probed a blot that contained RNA from petunia stem, leaves, and a variety of Xoral tissues (Fig. 2). The mRNA encoding the putative WS/DGAT was detected only within Xoral tissues, and in particular,
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Fig. 1 Sequence alignment of protein encoded by PhWS1 with A. calcoaceticus WS/ DGAT (AcWS/DGAT), three homologues from M. tuberculosis with reported DGAT activity (MtTGS1, MtTGS2 and MtTGS4), and three homologues from A. thaliana of unknown function. The putative catalytic HHXXXDG motif is highlighted by the black bar
Fig. 2 Northern analysis of expression of PhWS1 in various plant tissues. Blot contains 2 g of total RNA probed with a DIG-labelled cDNA probe (WS1). Ribosomal RNA bands stained with 0.02% (w/v) methylene blue are also shown. Tissue samples are as follows: lane a stem, b leaves, c unopened Xowers 1–2 cm less stigma, d unopened 5–6 cm Xowers less stigma, e stigma from 1– 2 cm Xowers, f stigma from 5 to 6 cm Xowers, g style, h anther and Wlament, i ovaries, j petal and k sepals. Tissues in lanes g–k were also from 5–6 cm Xowers
within the petals and, to a much lesser level, within the stigma. Expression of PhWS1 directs isoamyl wax-ester biosynthesis in yeast Because the putative WS/DGAT had signiWcant homology to a family of proteins which includes bacterial DGATs, we examined whether the protein had any
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DGAT activity. Therefore we introduced this cDNA into a S. cerevisiae strain which is deWcient in TAG (and steryl ester) biosynthesis (Oelkers et al. 2002; Sandager et al. 2002; Quittnat et al. 2004). S. cerevisiae is generally a good model system for analysing plant DGAT activity because it produces diacylglycerides containing long chain saturated and monounsaturated fatty acids like those found in plant oils. TLC analysis of the lipids extracted from S. cerevisiae JCY500 revealed that the expression of the petunia cDNA did not restore TAG biosynthesis in this yeast strain. However, a novel lipid product, with equivalent polarity to steryl and wax esters, was detected (Fig. 3a). GC-MS analysis revealed that these lipids were isoamyl esters of palmitic (C16:0), stearic (C18:0), arachidic (C20:0), behenic (C22:0) and lignoceric acid (C24:0). Isoamyl arachidate was the most abundant of these lipids (Fig. 3b). S. cerevisiae produces isoamyl alcohol via catabolism of leucine (Dickinson et al. 1998). To elevate the intracellular isoamyl alcohol concentration in the yeast, we replaced the 0.5% ammonium sulphate in the SD growth medium with 0.5% leucine. As a result, the level of isoamyl esters produced increased, and the wax-ester proWle
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Fig. 3 Wax ester production by S. cerevisiae JCY500 containing either pYES-DEST52 (null plasmid, ¡) or pYESPhWS1 (+). a TLC analysis of total lipids produced by yeasts grown in SC medium in which either (NH4)2SO4 or leucine are provided as a nitrogen source at a concentration of 0.5% w/v. Lipids were located by exposure to UV after spraying with 0.05% primulene. The spots located at the top of the plates for the yeast cells expressing PhWS1 correspond to isoamyl esters. b, c GC chromatographs showing isoamyl esters produced by S. cerevisiae cells grown in (NH4)2SO4 or leucine nitrogen source SC medium (b, c, respectively). The numbers above the peaks indicate the number of carbons present in the saturated fatty acid of the isoamyl esters. d Comparison of the mass spectra produced from an authentic isoamyl ester standard, and the ester produced by S. cerevisiae expressing the petunia wax synthase cDNA
now included monoenoic fatty acids, although these were less abundant than their saturated counterparts (Fig. 3c). Yeast grown on leucine as a sole nitrogen source produced isoamyl stearate as the most abundant wax-ester. The structures of each of the wax-esters was conWrmed by comparing the GC retention times and mass spectra with those of synthetic standards (Fig. 3d). PhWS1 encodes a membrane bound enzyme alcohol-acyltransferase The production of the isoamyl esters of fatty acids in yeasts suggested that PhWS1 encodes an alcohol-acyl-
transferase. The A. calcoaceticus DGAT/WS has been shown to be localized mainly to the plasma membrane and the surface of the lipid bodies which accumulate in this bacterium (Stöveken et al. 2005). A Kyte-Doolittle plot of protein corresponding to PhWS1 revealed that it contained two domains with a hydrophobicity score above 1.5 (Kyte and Doolittle 1982). These regions were predicted to be transmembrane with TMAP software (Persson and Argos 1996) using a multiple alignment of petunia protein and the eleven A. thaliana homologues as input data (Fig. 4a). The Nterminal of the protein, which includes the putative catalytic HHXXXDG motif, is predicted to be cytosolic.
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Petunia petals produce signiWcant amounts of mediumchain alcohol wax-esters in their cuticular wax
Fig. 4 a Kyte–Doolittle plot of the protein encoded by PhWS1 obtained using a window size of 19 (Kyte and Doolittle 1982). The two bars shown indicate transmembrane domains predicted by TMAP (Persson and Argos 1996) from a multiple sequence alignment of the petunia WS and the eleven A. thaliana homologues. b Wax-ester synthase assay conducted with soluble (Sol) and membrane (Mbe) protein fractions of S. cerevisiae JCY500 carrying plasmid pYES-DEST52 (¡ve) or pYES-PhWS1 (+WS). Assays were conducted with 20 g microsomal protein, 250 M isoamyl alcohol, 50 M 14C palmitoyl-CoA in 25 l reactions at 32°C for 20 min. Reaction products were extracted into chloroform and applied to a TLC plate which was developed with hexane/diethyl ether/formic acid (70/30/1, by vol). The image was obtained by autoradiometry. Only the region of the TLC plate corresponding to wax-esters is shown
To determine whether petunia protein was membrane localized, acyltransferase assays were conducted on soluble and membrane protein extracts from S. cerevisiae JCY500 cells expressing PhWS1. Isoamyl alcohol and 14C-palmitoyl-CoA were used as substrates (Fig. 4b). Activity was found only in the membrane fraction of yeast cells expressing PhWS1. Using isoamyl alcohol and 14C-palmitoyl-CoA as substrates, the assay was also optimised with respect to pH. The assay was conducted between pH 6.8 and 8.0, with the highest activity being obtained at pH 7.1 (data not shown). However, it should be noted that diversion of the 14Cpalmitoyl-CoA into phospholipids and free fatty acids were competing reactions in this crude microsomal assay, and the higher activity at pH 7.1 was partly due to the lower activity of these competing reactions at this pH.
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Acyl-esters of alcohols containing long-chain fatty acids are not volatile. Therefore, the preliminary analysis of the PhWS1 suggested that it may encode a protein responsible for the synthesis of cuticular waxesters rather than the volatile esters responsible for petal aroma. However, the cuticular wax-esters of plants typically contain VLC fatty alcohols from the acyl-reduction pathway, though wax-esters containing smaller alcohols (including aromatic) have been reported in a number of species (Gulz 1993; Sümmchen et al. 1995; Goodwin et al. 2003). In an example relevant to this study, Goodwin et al. (2003) reported the presence of fatty-acid esters of an alcohol which they tentatively identiWed as 3-hydroxy-heptan-2-ol in wax from the petals of snapdragon. To determine whether the alcohol-acyltransferase activity that was observed for the enzyme encoded by PhWS1 had any biological relevance to the plant from which it was obtained, we analysed the cuticular waxes of petunia petals and leaves. To partially purify the wax-esters, we performed silica gel column chromatography. Under the conditions used, wax-esters were eluted in the chloroform fraction, as conWrmed by GC-MS. Fractionation of a wax-extract from petunia petals via silica gel chromatography yielded a chloroform fraction accounting for 39.0% of the recovered material (Table 1). The total recovered material was typically >95% (data not shown). The chloroform fraction from the leaf waxes only accounted for 6.1% of the waxes. GC-MS analysis of the chloroform fraction from petunia petals produced a chromatogram in which 83.9 § 0.6% of the peak area corresponded to lowmolecular weight esters (C21–C36) consisting of saturated VLCFA and short chain alcohols (Fig. 5a). Production of FAMES conWrmed that these were saturated VLCFA, with the order of predominance being C24 > C22 > C26 > C20 > C28 (Fig. 5b). Analysis of the mass-spectral data indicated that the alcohol components of the wax-esters included methanol, and amyl, hexyl and octyl alcohols. To determine the structures of these 5–8 carbon alcohols present within the wax-ester, the alcohols were liberated from the fatty acids by transmethylation and converted to nicotinate esters. By comparing the retention times and mass spectra of the petal-wax derived nicotinates with those produced from alcohol standards, we were able to identify these alcohols as isoamyl, n-hexyl and n-octyl alcohols (Fig. 5c). Additionally, minor amounts of nbutyl, n-decyl and n-dodoceyl nicotinates were also observed. Esters of these minor alcohols were not
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Table 1 Fractionation of petunia cuticular wax extracts by silica gel chromatography. Lipids (<30 mg) were fractionated on a 1 g silica gel column (Iatrobeads) by elution with 10 ml portions of solvents of increasing polarity. Amounts indicated are percentages of the total wax recovered (§SD, n = 3), which was typically >95% of the loaded material Eluant
Hexane Chloroform Acetone Methanol
% of total wax Leaf wax
Petal wax
14.1 § 5.3 6.1 § 1.8 72.1 § 4.7 7.8 § 1.0
6.6 § 2.1 39.0 § 3.8 50.6 § 3.6 3.8 § 0.8
observed in the GC traces of the underivitized waxesters due to peak overlaps. Detailed analysis of the mass spectra of the wax-esters shown in Fig. 5b indicated that methyl, isoamyl and n-hexyl esters were more abundant than the n-octyl esters. GC-MS analysis of the leaf waxes revealed that they did not contain any low-molecular weight wax-esters (data not shown). To determine whether any high molecular weight wax esters (i.e., those containing VLC alcohols) were present in leaf and petal waxes, the chloroform fractions obtained by silica gel chromatography were transmethylated in the presence of a heptadecanoyl heptadecanoate standard, and then separated by a further round of silica gel chromatography into FAME and free alcohol fractions. The alcohols were then converted into TMS derivatives. GC-MS analysis revealed that 13.4 § 2.2 mol% of the petal wax-esters contained
VLC alcohols (C22–C28). Leaf waxes also contained C20–C28 fatty acids esteriWed to C28–C32 alcohols (data not shown). The use of hexane is well-established for the removal of surfaces waxes from leaves. Petals, however, are much thinner than leaves. To determine whether this extraction process removed any intracellular lipids from petals, we analysed silica gel chromatography fraction by TLC for the presence of phospholipids (supplementary Fig. 1). Phospholipids, which would have been eluted from the silica gel with methanol, were not visible on TLC plates sprayed with primulene. They were, however, visible in extracts of the hexane washed petals ground in chloroform/methanol (2/1, v/v). Additionally, we were able to recovered additional wax-esters from the hexane-washed petals, indicating that wax-esters were not completely extractable by hexane. This may represent cuticular waxes that have not yet been exported from the cells. The enzyme encoded by PhWS1 displays a preference for medium-chain alcohols Preliminary analysis of the reaction with n-octanol as a substrate at concentrations from 100–1,000 M revealed that the reaction was still linear at 20 min. However, the reaction appeared to be somewhat inhibited at higher concentrations of octanol, perhaps due to its limited solubility (Fig. 6). All subsequent reactions were therefore carried out using 250 M of alcohol as
Fig. 5 a Gas chromatograph (GC) of the chloroform fraction of petunia petals wax-extract. Peaks corresponding to the low molecular weight wax-esters are indication by Cx, where x refers to the total number of carbons present in the saturated wax-ester species. b GC of FAMES produced via the transmethylation of the waxester fraction displayed in (a). Numbers above the peaks indicate the number of carbons present in the saturated fatty acid of the methyl ester. c GC of nicotinate esters produced from the free alcohols liberated from the transmethylated petunia petal wax-esters
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Planta (2007) 226:381–394 Table 2 Comparison of wax-synthase activity in the presence of diVerent acyl acceptors with 14C-palmitoyl-CoA as acyl donor. Microsomal wax synthase assays performed with 50 M of 14Cpalmitoyl-CoA and 250 M of an alcohol acceptor as indicated. Values are the mean of at least three independent assays §SD
Fig. 6 a Time course of wax-ester synthesis using 50 M palmitoyl-CoA and varying concentrations of n-octanol. Assays contained 25 g of microsomal protein. The R2 values of each of the slopes was >0.99, indicating that the reactions were still linear after 20 min. b (inset) Reaction velocity plotted against substrate concentration for assays shown in (a)
an acyl acceptor. The proWle of the waxes obtained from the petunia petals suggested that the wax-synthase encoded by PhWS1 may display a preference for medium chain alcohols. In-vitro assays were conducted using 14C-palmitoyl-CoA and a range of straight-chain, branched, aromatic and terpenoids alcohols (Table 2). The highest activity was detected with n-dodecanol, which is only a minor component of the petal waxes. Acyltransferase activity with n-octanol and n-decanol was also relatively high. Activity with isoamyl and n-hexyl alcohols was about eight-fold lower than observed with n-octanol, and a further substantial reduction in activity when was observed when n-butanol was the acyl acceptor. No activity was detected with methanol. There was a very substantial drop in activity (26-fold) observed when the alcohol chainlength was increased from 12 to 14 carbons. This decrease was not due to the relative insolubility of n-tetradecanol compared to n-dodecanol. The assay performed at a 0.2% (v/v) Triton X-100 concentration in which the n-tetradecanol was clearly solublized resulted in a similar activity (data not shown). The acylglycerides 1-monoolein, 2-monoolein and 1,2 diolein were not used as substrates, which is consistent with the observation that expression of PhWS1 did not restore TAG synthesis in the yeast quadruple knock-out strain. Glycerol also could not be used as a substrate. The terpenoid alcohols geraniol and E,Efarnesol were used as substrates. Activity with geraniol was about Wvefold lower than n-decanol, and the activity with farnesol equivalent to that of n-tetradecanol.
123
Acceptor
SpeciWc activity (pmol min¡1 mg¡1)
No alcohol Methanol n-butanol n-amyl alcohol Isoamyl alcohol Anteiso-amyl alcohol n-hexanol Isohexanol n-octanol 2-octanol n-decanol n-dodecanol n-tetradecanol n-hexadecanol Geraniol E,E-farnesol 1,2-diolein 1-monoolein 2-monoolein Glycerol Benzyl alcohol 2-phenylethanol
not detected not detected 6.0 § 1.4 34.8 § 6.4 48.2 § 12 10.7 § 1.4 45.1 § 9.2 56.2 § 6.7 405 § 95 not detected 597 § 128 919 § 118 35.1 § 9.1 not detected 129 § 26 33 § 11 not detected not detected not detected not detected 26.7 § 3.7 not detected
Benzyl alcohol could also act as an acyl-acceptor, but not 2-phenylethanol. The enzyme encoded by PhWS1 prefers saturated long and very long chain fatty acyl-CoA esters To conWrm that the reactions were dependent on acylCoAs rather than free fatty acids, we performed the reaction using 14C-octan-1-ol as an acyl acceptor, and C16:0, C18:0 and C18:1 free fatty acids and acyl-CoA esters as substrates. For each fatty acid, the activity observed with the CoA-esters ranged from 300–600 pmol min¡1 mg¡1 whereas the activity for of the free fatty acids was barely above the background activity (supplementary Table 1). This ‘background’ activity may occur due presence of trace amounts of acyl-CoAs present in the microsomal protein preparations. The proWle of wax-esters produced by the S. cerevisiae strain expressing PhWS1 was somewhat enriched in saturated fatty acids. When the yeast was grown on ammonium as a nitrogen source, the isoamyl esters were also enriched in VLCFAs. These results suggested that that petunia wax-synthase may prefer saturated VLC acyl-CoAs as substrates. We conWrmed this in vitro by performing assays with various acyl-CoAs as shown in Table 3. Activity was threefold and 1.7fold less for C16 and C18 monoenoic acyl-CoA
Planta (2007) 226:381–394
391
Table 3 Comparison of wax-synthase activity in the presence of diVerent acyl-CoA donors with 14C-n-octanol as acyl acceptor. Microsomal wax synthase assays performed with 250 M of n-octanol and 50 M of an acyl-CoA as indicated. Values are the mean of three independent assays §SD Acceptor
No acyl-CoA Myristoyl-CoA Palmitoyl-CoA Palmitoleoyl-CoA Stearoyl-CoA Oleoyl-CoA Linoleoyl-CoA Arachidoyl-CoA Behenoyl-CoA
SpeciWc activity (pmol min¡1 mg¡1)
(14:0) (16:0) (16:1) (18:0) (18:1) (18:2) (20:0) (22:0)
46.3 § 4.8 238 § 17 348 § 26 118 § 13 751 § 38 450 § 52 61.0 § 9.9 1,942 § 335 1,035 § 206
compared to saturated C16 and C18 acyl-CoAs, respectively. Activity with linoleoyl-CoA was only slightly higher than observed with no exogenous acylCoA addition. This background activity is possibly due to the presence of trace amounts of acyl-CoAs in the microsomal protein preparation. Arachidoyl and behenoyl-CoAs gave the greatest amount of activity. The preference for these longer-chain saturated acyl-CoAs is consistent with the observation that the petunia petal wax-esters contain saturated VLCFA.
Discussion We have demonstrated that a petunia homologue of the A. calcoaceticus WS/DGAT is capable of synthesizing a range of low molecular weight wax esters. This is the Wrst characterization of a member of this gene family from plants, and the Wrst identiWcation of a cuticular wax-ester synthase. The preferred in vitro substrates for the petunia enzyme appear to be saturated VLC acyl-CoAs (C20 and C22), and medium chain alcohols (C8–C12). The in vitro preference for the saturated VLC acyl-CoAs is supported by the observation that the wax-esters obtained from the petunia petal contained saturated VLCFA. Additionally, when expressed in S. cerevisiae, the predominant wax-ester species produced were isoamyl esters of saturated fatty acids. When yeast was grown on ammonium sulphate as a nitrogen source, the levels of the VLCFA (C20– C24) in the wax-esters were particularly enriched relative to the typical internal acyl-CoA pools. When intracellular acyl-CoA proWles of yeast grown in SD galactose medium have been analysed, the only acyl-CoA esters that have been detected are 14:0, 16:0, 16:1, 18:0, 18:1 and 22:1 (Aharoni et al. 2000; Gaigg et al. 2001; Tonon
et al. 2005). Therefore, saturated VLC acyl-CoAs such as 20:0, 22:0 and 24:0 probably make up less than one percent of the available acyl-CoAs within the yeast cells used in this study, which were grown under similar conditions. The proWle of isoamyl esters produced when the yeast was grown on leucine was diVerent to that of the yeast grown on ammonium sulphate. There was still a strong bias towards the saturated acyl species, but stearic acid was the most predominant fatty acid. This was probably due to a depletion of the VLC acyl-CoAs under conditions of enhanced wax-ester synthesis at the higher internal isoamyl alcohol concentrations. The preference of the PhWS1 encoded acyltransferase for short-chain alcohols also support the conclusion that it is involved in petal wax-esters biosynthesis. However, the petunia protein preferred n-decanol and n-dodecanol rather than shorter chain (C4–C6) alcohols. n-Decanol and n-dodecanol were only minor components of the wax esters compared to n-hexanol and isoamyl alcohol. The petal waxes contained methyl-esters. The wax-synthase encoded by PhWS1 was not able to produce methyl esters in vitro. This suggests that these methyl-esters are likely to be the product of another acyltransferase. High molecular weight wax-esters were also present within the petunia petal (i.e., those containing VLC alcohols). Synthesis of these esters would also require an additional WS activity, as the wax synthase encoded by PhWS1 could not acylate VLC alcohols. The observation that petunia wax-synthase prefers C10–C12 alcohols which are only minor components of the petal wax-esters highlights the role of substrate availability in determining the in vivo functionality of the acyltransferase. The structurally unrelated soluble alcohol acyltransferases (AAT) involved in the synthesis of volatile esters of fruits have also been shown to have a wider range of in vitro capabilities in respect of the alcohol substrates they will use (Aharoni et al. 2000; Yahyaoui et al. 2002; Beekwilder et al. 2004). In fact, the in vitro substrate preferences of the soluble AATs does not always directly correlate with the in vivo functionality in that higher speciWc activities are often reported for non-native substrates compared to those that would be encountered in planta (Beekwilder et al. 2004). As the petunia wax-synthase appears to have somewhat promiscuous activities towards primary alcohols, it would suggest that in-vitro analysis of other members of the WS/DGAT family alone would be of limited value in determining their function. In analogy with the soluble AATs, consideration must be given to the biochemistry of the tissues in which the acyltransferases are expressed.
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Although PhWS1 belongs to an acyltransferase family which includes DGATs, the acyltransferase encoded by this cDNA did not have the capacity to restore TAG biosynthesis in a neutral-lipid deWcient yeast strain. Additionally, no acyltransferase activities were observed with either monoolein or diolein as substrates in in vitro assays. Therefore, it would appear that PhWS1, unlike it’s counterpart in A. calcoaceticus, is not involved in glycerolipid biosynthesis. The lack of DGAT activity in petunia wax synthase is not without precedence in the WS/DGAT family of proteins. All 15 homologues from M. tuberculosis H37Rv have been analysed for both DGAT and WS activity (Daniel et al. 2004). Only four of the proteins displayed signiWcant levels of DGAT activity, and none appeared to be wax-ester synthases. Furthermore, the three other protein families containing enzymes that synthesize TAG also contain members which acylate substrates other than DAG. The DGAT1 family contains sterol acyltransferases with no detectable DGAT activity (Cases et al. 1998). The DGAT2 family includes the mammalian wax-synthases, which also lack DGAT activity (Cheng and Russell 2004). The phospholipid:diacylglycerol acyltransferase from S. cerevisiae belongs to a protein family which includes the phospholipid:sterol acyltransferases and phospholipases (Dahlqvist et al. 2000; Thomaeus et al. 2001; Noiriel et al. 2004; Ståhl et al. 2004; Banas et al. 2005). Based on searches of the public EST databases, it appears that the WS/DGAT gene family is broadly extended in terrestrial plants. WS/DGAT homologues have been found in many other plant species, including monocots, (Triticum aestivum, GenBank accession CK163450) and gymnosperms (Pinus taeda, GenBank accession CV032126). Arabidopsis thaliana has eleven members of the WS/DGAT family. To date, none of the A. thaliana members of this gene family have been characterized. Some of these may be wax-ester synthases, but this species is not known to produce signiWcant amounts of these esters in its cuticular wax. Therefore, it is unlikely that these are all proteins involved in the synthesis of wax-esters. Expression data for Wve of the A. thaliana WS/DGAT genes is already available in the Arabidopsis Lipid Gene Database (Beisson et al. 2003). This includes highly detailed microarray data obtained as part of the AtGenExpress project (Schmid et al. 2005). These data reveal that many of the A. thaliana WS/DGAT genes are highly upregulated in speciWc plant tissues. Of the Wve sequences for which microarray expression data is available, only one of these (At3g49210) appears to be upregulated within the petal. The gene appears to be upregulated within petals of stage 15 petals (2 days
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after Xower opening) but not within stage 12 petals (just prior to Xower opening) (Smyth et al. 1990). It should be noted that this gene is also upregulated within a number of other tissues, including the sepals of stage 15 Xowers, senescing leaves, roots and stamens. At3g49210 displays 45% sequence identity to PhWS1, which is not signiWcantly highly than the sequence identity observed between PhWS1 and the other A. thaliana WS/DGAT homologues (supplementary Fig. 2). Therefore, despite being expressed in the petals, there is little to suggest that At3g49210 is functionally equivalent to PhWS1. The availability of the A. thaliana microarray data should serve as a useful starting point in determining the functions of other members of this gene family. Other possible functions for the A. thaliana proteins include triacylglycerol, cutin or suberin biosynthesis. The types of wax-ester observed in the petunia petals were unusual in that they contain low molecular weight alcohols in contrast to the VLC fatty alcohols that are typically observed in cuticular wax. These shorter-chain wax-esters were absent from the leaf wax. It is quite possible that these unusual esters may be present within the petal wax to confer some property speciWc to the role of the petal. The petal waxes would need to be permeable to volatile semiochemicals such as methyl benzoate (Underwood et al. 2005). Petal waxes may also need to be more UV-transparent than leaf waxes, as petals often have demarcations within the UV spectrum visible to pollinators (Gronquist et al. 2001). As there is wide variation in wax composition however, it is diYcult to draw conclusions how these unusual waxes relate to petal function. The VLC fatty alcohols usually found in cuticular wax (either as free alcohols, or esteriWed to fatty acids) are produced by the reduction of VLC acyl-CoA species produced by an ER-localized elongation system (Kunst and Samuels 2003). Stearoyl-CoA is typically the primer for the production of these VLC acyl-CoAs. Therefore, the small-to-medium chain alcohol species present in the petunia petal waxes are clearly produced via a pathway distinct from that involved in VLC acylCoA biosynthesis. The straight-chained alcohols present in the petal wax-esters were all even-numbered in chain length. This two-carbon elongation series strongly suggests that they are produced via the reduction of acyl-intermediates from a FAS-type fatty acid biosynthesis reaction. Indeed, pathways for the production of medium chain fatty acids have already been shown to operate in petunia and other solanaceous plants for the production of acylated sugars in trichome exudates (van der Hoeven and SteVens 2000; Kroumova
Planta (2007) 226:381–394
and Wagner 2003). The only branched-chain alcohol found in the wax-esters was isoamyl alcohol. There are a number of possible routes for the biosynthesis of this alcohol, including the transamination and decarboxylation of leucine, as observed in S. cerevisiae (Dickinson et al. 1998). The enzymes assays conducted with the heterologously expressed protein indicated that the petunia wax-synthase is a membrane-bound protein. This was in agreement with prediction obtained using TMAP software (Persson and Argos 1996). As the ER membranes are thought to be the site of cuticular wax biosynthesis in plants (Kunst and Samuels 2003), this would seem a likely location for the wax synthase. None of the known C-terminal ER retrieval signals were found within wax-synthase peptide sequence; i.e., the H/KDEL, dilysine motifs (Pagny et al. 1999), or the more cryptic aromatic amino-acid rich domains recently characterized in plants (McCartney et al. 2004). However, the lack of these known ER retention sequences does not rule out the possibility of the petunia wax-synthase protein being ER-bound. In summary we report here the Wrst characterization of a member of the WS/DGAT family in plants. The protein encoded by PhWS1 lacks DGAT activity, and functions in the Xower petals to produce low-molecular weight cuticular wax-esters. Acknowledgments This work was funded by Dow Chemical Company (Midland, MI, USA) and Dow Agrosciences LLC (Indiapolis, IN, USA). J. Hilliard was sponsored by a National Science Foundation summer internship program. The nucleotide sequence corresponding to the PhWS1 cDNA has been deposited in GenBank (DQ093641).
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