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First published online July 11, 2008; 10.1104/pp.108.123471 Plant Physiology 148:97-107 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Identification of the Wax Ester Synthase/Acyl-Coenzyme A:Diacylglycerol Acyltransferase WSD1 Required for Stem Wax Ester Biosynthesis in Arabidopsis1,2,[W],[OA]Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (F.L., X.W., P.L., D.B., H.Z., L.S., R.J., L.K.); and Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 (R.J.)
Wax esters are neutral lipids composed of aliphatic alcohols and acids, with both moieties usually long-chain (C16 and C18) or very-long-chain (C20 and longer) carbon structures. They have diverse biological functions in bacteria, insects, mammals, and terrestrial plants and are also important substrates for a variety of industrial applications. In plants, wax esters are mostly found in the cuticles coating the primary shoot surfaces, but they also accumulate to high concentrations in the seed oils of a few plant species, including jojoba (Simmondsia chinensis), a desert shrub that is the major commercial source of these compounds. Here, we report the identification and characterization of WSD1, a member of the bifunctional wax ester synthase/diacylglycerol acyltransferase gene family, which plays a key role in wax ester synthesis in Arabidopsis (Arabidopsis thaliana) stems, as first evidenced by severely reduced wax ester levels of in the stem wax of wsd1 mutants. In vitro assays using protein extracts from Escherichia coli expressing WSD1 showed that this enzyme has a high level of wax synthase activity and approximately 10-fold lower level of diacylglycerol acyltransferase activity. Expression of the WSD1 gene in Saccharomyces cerevisiae resulted in the accumulation of wax esters, but not triacylglycerol, indicating that WSD1 predominantly functions as a wax synthase. Analyses of WSD1 expression revealed that this gene is transcribed in flowers, top parts of stems, and leaves. Fully functional yellow fluorescent protein-tagged WSD1 protein was localized to the endoplasmic reticulum, demonstrating that biosynthesis of wax esters, the final products of the alcohol-forming pathway, occurs in this subcellular compartment.
The cuticle is a thin hydrophobic layer that covers the outermost surface of the primary aerial tissues of terrestrial plants. It controls nonstomatal water loss (Riederer and Schreiber, 2001  ), protects plants from UV radiation (Krauss et al. 1997; Solovchenko and Merzlyak, 2003; Pfündel et al., 2006), minimizes deposition of dust, pollen, and pathogen spores (Barthlott and Neinhuis, 1997), and mediates a variety of plant interactions with insects (Eigenbrode and Espelie, 1995; Müller, 2006), as well as bacterial and fungal pathogens (Carver and Gurr, 2006; Leveau, 2006). In addition, the cuticle prevents fusions between plant organs during development (Lolle et al., 1998; Sieber et al., 2000).
The cuticle framework is provided by cutin, an insoluble plant-specific polyester composed of C16 and C18 hydroxy and epoxy fatty acids and glycerol (Heredia, 2003
Cuticular wax biosynthesis involves elongation of saturated C16 and C18 fatty acyl-CoAs to VLCFA wax precursors between 24 and 34 carbons in length and their subsequent modification by either the alkane-forming (decarbonylation) or the alcohol-forming (acyl reduction) pathway. The alkane pathway results in the synthesis of aldehydes, alkanes, secondary alcohols, and ketones, whereas the alcohol pathway yields primary alcohols and wax esters (Kunst et al., 2006
The ester content in plant cuticular waxes varies greatly among species and, to some extent, even within species. For example, Arabidopsis leaf and stem cuticular waxes contain only 0.1% to 0.2% and 0.7% to 2.9% of wax esters, respectively (Jenks et al., 1995
Even though major steps in plant cuticular wax biosynthesis have been proposed, our knowledge of the enzymes involved and the type of biochemical reactions that they catalyze is incomplete. Forward genetic approaches using a collection of wax-deficient Arabidopsis eceriferum (cer) mutants and maize (Zea mays) glossy mutants resulted in the identification of several genes encoding wax biosynthetic enzymes, including CER6, CER10, GL8A, and GL8B (Millar et al., 1999
For the alcohol-forming pathway, only the first of the two steps, the conversion of VLCFAs to primary alcohols by the fatty acyl-CoA reductase CER4, has been investigated at the molecular level (Rowland et al., 2006
In an attempt to clone and characterize the gene/enzyme responsible for wax ester biosynthesis in Arabidopsis stem epidermis, we focused our attention on gene families exhibiting high sequence homology with the three types of WS enzymes known to catalyze wax ester formation in other organisms. The first type, the mammalian WS enzymes, have the highest activity with acyl-CoAs between C12 and C16 in length and efficiently use alcohols shorter than C20 (Cheng and Russell, 2004
To narrow down the number of candidate genes, we decided to concentrate on the jojoba-type WS and WS/DGAT genes up-regulated in the stem epidermis during active wax synthesis (Suh et al., 2005
Among the Arabidopsis genes annotated as WS/DGAT, WSD1 showed the highest expression level in the stem epidermis and a high expression ratio of epidermis to total stem in a microarray experiment (Suh et al., 2005 ). To determine whether this gene is involved in wax ester formation in Arabidopsis stems, we obtained mutant lines wsd1-1 (SALK_067714) and wsd1-2 (SALK_118165; Alonso et al., 2003) from the Arabidopsis Biological Resource Center (ABRC) and analyzed them for disruption of the WSD1 gene using PCR. The wsd1-1 allele had a T-DNA insertion in the second exon of the WSD1 gene, 643 bp downstream from the predicted translation start site, and wsd1-2 carried a T-DNA insertion in the 5'-untranslated region of the WSD1 gene, 180 bp upstream of the predicted translation start site (Fig. 1A
). Semiquantitative reverse transcription (RT)-PCR analyses conducted to assess steady-state levels of WSD1 gene transcript in mutant alleles revealed no detectable WSD1 transcript in wsd1-1 and reduced transcript accumulation in wsd1-2 (Fig. 1B).
Stem Cuticular Wax Phenotypes of wsd1 Mutants Insertions in the WSD1 gene did not result in a glossy stem phenotype or altered appearance and density of epicuticular wax crystals on the stem surface (scanning electron micrographs not shown), characteristic of a number of wax-deficient mutants studied to date. To further investigate the effect of WSD1 disruption on wax accumulation and specifically wax ester formation, we therefore extracted total stem waxes from the wsd1-1 and wsd1-2 mutants and wild-type plants and analyzed them by gas chromatography (GC). No differences in the absolute amounts and the relative proportions of most wax components, including aldehydes, alkanes, secondary alcohols, ketones, fatty acids, primary alcohols, phenyl-ethyl esters, and triterpenoids, were found in mutant lines in comparison to the wild type (Fig. 2A ). The only compound class affected by wsd1 mutations was the alkyl esters, which were severely reduced in both mutants (Fig. 2). The alkyl ester profile detected in the wild-type wax included chain lengths ranging from C38 to C48 (Fig. 2B). None of these wax esters was detectable in the total wax extracts from the mutant stems. Therefore, a more detailed analysis of wax esters from wild-type and mutant stem cuticular wax mixtures was carried out after separation of wax compound classes by thin-layer chromatography (TLC). In the wild-type wax, ester chain lengths varied between C38 and C54, with a predominance of even-numbered homologs and a maximum at C44 (Fig. 1; Table I ). Only trace amounts of even-numbered wax esters and no odd-numbered wax esters were detected in either wsd1-1 or wsd1-2 mutant. The chain length distribution of mutant esters was shifted relative to the wild type, resulting in a predominance of the C40 homolog.
In Vitro Characterization of WSD1 Using Heterologous Expression in Escherichia coli
Because WSD1 has significant sequence similarity to the WS/DGAT from A. calcoaceticus ADP1, the question arises as to whether the WSD1 protein has both WS and DGAT activity. To test this, we heterologously expressed the WSD1 cDNA in E. coli BL21 (DE3). WS assays were carried out with crude protein extract using radiolabeled [1-14C]palmitoyl-CoA as the acyl donor and 1-octadecanol (C18 alcohol) as acyl acceptor, whereas [1-14C]palmitoyl-CoA and 1,2-dipalmitoylglycerol were used as substrates in DGAT activity assays. The reaction products were detected by autoradiography following TLC separation and the specific activities of WSD1 were determined by scintillation counting. Our results showed that WSD1 is a bifunctional enzyme, which has high WS activity (84.4 ± 5.5 pmol/mg min) and substantially lower DGAT activity (7.7 ± 1.0 pmol/mg min; Fig. 3
). The ratio of 10.9 between WS and DGAT activity of WSD1 was similar to the ratio of these activities (11.1) determined for the A. calcoaceticus ADP1 bifunctional enzyme (Kalscheuer and Steinbüchel, 2003
In Vivo Characterization of WSD1 Using Heterologous Expression in Yeast
To verify that WSD1 is indeed a bifunctional enzyme with WS and DGAT activity, we introduced the WSD1 cDNA into the yeast (Saccharomyces cerevisiae) mutant H1246 deficient in storage lipid biosynthesis (Sandager et al., 2002
Organ- and Tissue-Specific Expression of WSD1 To determine the organ-specific expression of the WSD1 gene, quantitative PCR (qPCR) and RT-PCR were performed. In both experiments, the highest WSD1 mRNA accumulation was detected in flowers, followed by stem tops and leaves. Very low levels of transcript were found in roots and seeds only by qPCR (Fig. 6, A and B ). To confirm these results and more precisely define the tissue specificity of WSD1 expression, the 1,978-kb genomic fragment upstream of the WSD1 coding region was fused to the GUS reporter gene (PWSD1:GUS). Consistent with qPCR and RT-PCR results, histochemical analysis of GUS activity in transgenic plants harboring PWSD1:GUS showed a high level of expression in the top 3 cm of stems (Fig. 7A ). Cross sections indicated WSD1 expression in all cell types (Fig. 7B), and this result was verified by in situ hybridization (Fig. 7C; Supplemental Fig. S1). Furthermore, GUS activity was apparent in the hydathodes and veins of 20-d-old cauline leaves (Fig. 7D), filaments of stamens (Fig. 7E), and siliques (Fig. 7F), but not in seeds, roots, and young seedlings (data not shown).
To investigate the subcellular localization of the WSD1 enzyme in Arabidopsis, the WSD1 cDNA was fused with the yellow fluorescent protein (YFP) coding sequence at its 5' end and expressed in the wsd1-1 mutant under the control of the 35S promoter. Expression of the P35S:YFP:WSD1 construct complemented the wax ester deficiency of the wsd1-1 mutant (Fig. 8 ) by restoring stem wax ester levels to 30% to 50% of the wild type, thus confirming that the mutant phenotype was caused by the T-DNA insertion in the WSD1 gene. At the same time, the complementation of the wsd1 phenotype by the YFP-tagged WSD1 demonstrated that the addition of YFP did not interfere with WSD1 folding or function and allowed us to localize the functional protein within epidermal cells. Protein sequence analysis using the DAS transmembrane prediction server (http://www.sbc.su.se/  miklos/DAS) classified WSD1 as an integral membrane protein containing at least four transmembrane domains (Supplemental Fig. S2). This predicted membrane association was experimentally verified by examination of the P35S:YFP:WSD1 transgenic plants in which YFP fluorescence uniformly labeled a reticulate network typical of the endoplasmic reticulum (ER; Fig. 9A
). The YFP fluorescence signal colocalized with the signal from hexyl rhodamine B, a dye that labels the ER in plants (Boevink et al., 1996, Fig. 9, B–D).
Alkyl esters are the final products of the alcohol pathway of cuticular wax biosynthesis and account for up to 3% of total wax on Arabidopsis inflorescence stem. These wax esters consist mainly of C16 acyl and C26 to C30 alkyl moieties (Lai et al., 2007 ), but the identity of the WS that catalyzes their formation was previously unknown. Based on sequence similarities to enzymes that mediate wax ester biosynthesis in other organisms and high levels of expression at the time of maximal wax deposition (Suh et al., 2005), we predicted that WSD1, encoded by a WS/DGAT-type gene At5g37300, is the WS involved in stem wax ester production. Several lines of evidence discussed below demonstrate that this is indeed the case. Phenotypic analysis of two independent wsd1 mutants with T-DNA insertions in the WSD1 gene revealed deficiencies in the accumulation of wax esters in both lines. These compounds were not detectable in the total wax extracts from either the loss-of-function allele wsd1-1 or the partial-loss-of-function line wsd1-2 (Fig. 2). Cuticular wax ester deficiencies in wsd1 mutants were successfully complemented by the introduction of the wild-type WSD1 (Fig. 8), indicating that inactivation of this gene was responsible for the observed mutant phenotypes. Collectively, these results support the conclusion that the WSD1 enzyme is required for wax ester biosynthesis in Arabidopsis stem.
In contrast to the jojoba WS, which could not be functionally expressed in microorganisms (Lardizabal et al., 2000
WSD1 expression was detected in Arabidopsis shoots, most prominently in flowers, in the top 3 cm of inflorescence stems and in leaves, consistent with a role for this gene in cuticular wax production (Figs. 6 and 7). Surprisingly, WSD1 expression was not restricted to the epidermal cells like most other genes involved in wax deposition (Xia et al., 1997
The subcellular localization of the active YFP-WSD1 by confocal microscopy demonstrated that this enzyme resides in the ER membranes (Fig. 9). Because the biosynthesis of fatty acyl and alkyl substrates for wax ester production also takes place at this site (Lessire et al., 1985 Based on all the evidence discussed above, we conclude that WSD1 plays a major role in the formation of wax esters in the stem of Arabidopsis. This enzyme is involved in cuticle formation, and in this physiological function acts as WS by using primary alcohols as acyl acceptors. This raises the question of whether wax ester formation is the only function of WSD1 or whether the enzyme can also use diacylglycerols as acyl acceptors and thus function as a DGAT. Results from three of our experiments address this question.
First, unlike the ADP1 A. calcoaceticus enzyme, which restored TAG biosynthesis when expressed in the quadruple mutant of yeast H1246 deficient in storage lipid production (Kalscheuer et al., 2004
A large family of 11 sequences, including WSD1, has been tentatively annotated as WS/DGATs in the Arabidopsis genome (Fig. 10A
; Supplemental Fig. S4), so it is conceivable that at least some of these predicted WSD enzymes can work as DGATs. They have similar sequence (16.0%–20.5% amino acid sequence identity over their entire length) and size, ranging from 476 to 523 residues in length with short N-terminal or C-terminal extensions in a few cases. In addition, like the A. calcoaceticus WS/DGAT, the WSD1-WSD11 sequences contain a highly conserved condensing domain with a proposed active-site motif (146HHXXXDG152) in their N-terminal region (Fig. 10B; Supplemental Fig. S4) suggested to be essential for catalytic activity in the acyl-CoA acyltransferase reactions involved in wax ester and TAG formation (Kalscheuer and Steinbüchel, 2003
In summary, we have shown that WSD1, a member of the Arabidopsis WS/DGAT enzyme family, is the WS that catalyzes the biosynthesis of wax esters, dimeric cuticular wax components found on Arabidopsis shoots. This enzyme resides in the ER and uses long-chain and very-long-chain primary alcohols with C16 fatty acid for wax ester production.
Plant Material and Growth Conditions T-DNA insertion mutant lines SALK_067714 and SALK_118165 were obtained from the ABRC (www.arabidopsis.org). Seeds were stratified for 3 to 4 d at 4°C and plants were grown on Sunshine Mix 5 (Sun Gro Horticulture) under continuous white fluorescent light (80–100 µE m–2 s–1). The temperature was set at 20°C to 22°C.
Total RNA was extracted from each sample by using the RNeasy mini kit (Qiagen) and treated with RNase-free DNase (Promega). Total RNA (2.5 µg) was used for reverse transcriptase reactions. First-strand cDNA synthesis was performed using the SuperScript III platinum two-step qRT-PCR kit with SYBR green (Invitrogen). Glyceraldehyde-3-P dehydrogenase C subunit (GAPC) was used as a reference. Primers for WSD1 (forward 5'-CTGATGTTTGATCAGGGTTATG-3' and reverse 5'-TGCCAACTGTTCATAAAACACC-3') and primers for GAPC (forward 5'-ACTCGAGAAAGCTGCTAC-3' and reverse 5'-ATTCGTTGTCGTACCATG-3') were used for real-time PCR. The real-time PCR reaction included an initial 5-min denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 20 s, 55°C for 10 s, 72°C for 10 s, and a final 5-min extension at 72°C. The quantitative real-time PCR was performed using a Bio-Rad miniOpticon real-time PCR detection system (Bio-Rad) according to the manufacturer's directions. RT-PCR was carried out in an MJ Research PTC 200 thermocycler with WSD1 primers (forward 5'-ATGAAAGCGGAAAAAGTTATGG-3' and reverse 5'-TATTTCCCCTTGTGTGGCAGA-3') and GAPC primers (forward 5'-ACTCGAGAAAGCTGCTAC-3' and reverse 5'-ATTCGTTGTCGTACCATG-3') using the following amplification conditions: initial denaturation 94°C for 5 min, followed by 28 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s, and a final 5-min extension at 72°C.
Cauline leaves, flowers, and siliques were removed from the inflorescence stems and cuticular waxes extracted by immersing the stems two times for 30 s into chloroform at room temperature. Both chloroform extracts were combined and C24 alkane was added as internal standard. The solvent was removed under a gentle stream of nitrogen and the wax mixtures were treated with bis-N,N-(trimethylsilyl)trifluoroacetamide (Sigma) in pyridine for 1 h at 70°C to transform all hydroxyl-containing compounds into the corresponding trimethylsilyl derivatives. The wax composition was determined with capillary GC (5890N; Agilent) and a mass spectrometric detector (5973N; Agilent). GC was carried out with temperature-programmed on-column injection at 53°C, oven 2 min at 50°C, raised by 40°C min–1 to 200°C, held for 2 min at 200°C, raised by 3°C min–1 to 320°C, and held for 30 min at 320°C, and He carrier gas inlet pressure was programmed for constant flow of 1.4 mL min–1. The quantitative analyses of wax mixtures were carried out by capillary GC with flame ionization detector (FID) under the same GC conditions as above, but with H2 carrier gas at a constant flow of 2 mL min–1. Wax loads were determined by comparing GC-FID peak areas against internal standard and dividing by the surface area determined for the corresponding sample. Stem surface areas were calculated by measuring the projected stem areas in photographs and multiplying by In TLC analysis, wax mixtures from stems of wild-type, mutant, and transgenic plants were separated on silica gel 60 (EMD Chemicals) using hexane-diethylether-acetic acid (90:7.5:1 [v/v/v]) and visualized under UV light after spraying with primuline (Sigma).
To determine the fatty acid composition of the seed oil, fatty acid methyl esters were prepared by refluxing the seed samples in 2 mL of 1 N methanolic HCl for 90 min at 80°C. After cooling, 2 mL of 0.9% NaCl solution and 150 µL of hexane were added and the mixture was vortexed vigorously. The fatty acid methyl esters in the hexane phase were analyzed by GC-liquid chromatography as described previously (Kunst et al., 1992
For the GUS activity assays, the 1,978-bp fragment immediately upstream of the WSD1 coding region was amplified by PCR from genomic Arabidopsis DNA using forward 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTAATAAATTTCCCTCACCATCC-3' and reverse 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTTTTCCGCTTTCATAGTCAG-3' primers. The amplified fragment was introduced into a donor vector pDONR221 (Invitrogen), resulting in pDONR221-WSD1 promoter constructs, and then the WSD1 promoter was cloned into a binary vector pGWB3 (Nakagawa et al., 2007
To generate an N-terminal YFP fusion to WSD1, the WSD1 coding region was amplified from wild-type cDNA using gene-specific primers (forward 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAAAGCGGAAAAAGTTATGG-3' and reverse 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTATTTCCCCTTGTGTGGCAGA-3'). The PCR product was cloned into the pEARLEYGATE104 vector (Earley et al., 2006
The tissues collected from transgenic plants harboring the WSD1 promoter:GUS construct were incubated in GUS assay buffer as described previously (Jefferson, 1987
Tissues from 10-d-old transgenic seedlings harboring the 35S:YFP:WSD1 were immersed in 1.6 mM hexyl rhodamine B solution (Molecular Probes) for 10 to 30 min, then mounted in distilled water and observed in a Zeiss LSM 5 Pascal confocal laser-scanning microscope (Carl Zeiss; http://www.zeiss.com). YFP fluorescence was detected using excitation of 514 nm with a 535- to 580-nm band-pass emission filter. Hexyl rhodamine B was excited with a 543-nm argon ion laser line with a 600- to 650-nm band-pass emission filter. All confocal images obtained were processed with LSM 5 Image Browser (Carl Zeiss) and Adobe Photoshop 5.0 software.
Tissue fixation, sectioning, hybridization, signal detection, and probe synthesis were performed as previously described (Hooker et al., 2002
For expression in E. coli, full-length WSD1 cDNA was cloned into the expression vector pGEX4T2 (GE Healthcare) using BamHI and XhoI restriction sites. The resulting plasmid pGEX4T2:WSD1 was transformed into E. coli BL21 (DE3). Recombinant E. coli was grown in Luria-Bertani medium for 6 h at 37°C in the presence of 1 mM isopropyl-β-D-thiogalactopyranoside. Cells were then harvested, washed, resuspended in 125 mM sodium phosphate buffer (pH 7.4), and disrupted by glass beads.
For expression in yeast (Saccharomyces cerevisiae), full-length WSD1 cDNA was amplified by PCR using forward and reverse primers 5'-AGAGGATCCATGAAAGCGGAAAAAGTTATGG-3' and 5'-AGACTCGAGTCAAACTTCCGTTTTGTGAAA-3', respectively. The PCR product was cloned into the BamHI-XhoI-restricted vector pESC-URA (Stratagene) collinear with the Gal-inducible GAL1 promoter. The generated construct was transformed into yeast strain H1246 defective in storage lipid accumulation (Sandager et al., 2002
Enzyme activities were determined according to the method reported by Kalscheuer et al. (2004)
For extraction of total lipids, yeast cells were harvested by centrifugation and 2 mL of methanol were added to each sample. After 5 min, 4 mL of chloroform and 1 mL of 0.9% NaCl were added, the two-phase system was mixed by vortexing, and the chloroform phase transferred to a new tube and concentrated under a stream of nitrogen. Wax esters were purified by preparative TLC and analyzed by GC-MS and GC-FID as described above. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number NP_568547.
The following materials are available in the online version of this article.
We thank the Genomic Analysis Laboratory of the Salk Institute for providing sequence indexed Arabidopsis T-DNA insertion mutants, Tsuyoshi Nakagawa for the pGWB3 vector, and Keith Earley for the pEARLEYGATE104 vector. Received May 23, 2008; accepted July 9, 2008; published July 11, 2008.
1 This work was supported by the Natural Sciences and Engineering Research Council of Canada (Special Research Opportunity grant to R.J., L.S., and L.K.). Additional funding was provided by the Canadian Foundation for Innovation and the Canada Research Chairs Program (to R.J.). 
2 This work is part of ICON, a European Community Seventh Framework Programme.
3 Present address: Department of Biological Sciences, University of Manitoba, Winnipeg Manitoba, Canada R3T 2N2.
4 Present address: Department of Biology, McGill University, Montreal, Quebec, Canada H3A 1B1. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ljerka Kunst (kunst{at}interchange.ubc.ca).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.123471 * Corresponding author; e-mail kunst{at}interchange.ubc.ca.
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, et al (2003) Genome-wide insertional mutagenesis of Arabidopsis. Science 301: 653–657 Barthlott W, Neinhuis C (1997) Purity of the sacred lotus, or escape from contamination in biological surfaces. Planta 202: 1–8[CrossRef][Web of Science] Benzioni A, Forti M (1989) Jojoba. In G Robbelen, RK Downey, A Ashri, eds, Oil Crops of the World. McGraw-Hill Publishing Company, New York, pp 448–461 Bird D, Beisson F, Brigham A, Shin J, Greer S, Jetter R, Kunst L, Wu X, Yephremov A, Samuels L (2007) Characterization of Arabidopsis WBC11/ABCG11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion. Plant J 52: 485–498[CrossRef][Medline] Boevink P, Cruz S, Hawes C, Harris N, Oparka KJ (1996) Virus-mediated delivery of the green fluorescent protein to the endoplasmic reticulum of plant cells. Plant J 10: 935–941[CrossRef][Web of Science] Carver TLW, Gurr SJ (2006) Filamentous fungi on plant surfaces. In M Riederer, C Müller, eds, Annual Plant Reviews 23: Biology of the Plant Cuticle. Blackwell, Oxford, pp 368–397 Cheng JB, Russell DW (2004) Mammalian wax biosynthesis. 2. Expression cloning of wax synthase cDNAs encoding a member of the acyltransferase enzyme family. J Biol Chem 279: 37798–37807 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis. Plant J 16: 735–743[CrossRef][Web of Science][Medline] Dietrich CR, Perera MA, D Yandeau-Nelson M, Meeley RB, Nikolau BJ, Schnable PS (2005) Characterization of two GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development. Plant J 42: 844–861[CrossRef][Web of Science][Medline] Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS (2006) Gateway-compatible vectors for plant functional genomics and proteomics. Plant J 45: 616–629[CrossRef][Web of Science][Medline] Eigenbrode SD, Espelie KE (1995) Effects of plant epicuticular lipids on insect herbivores. Annu Rev Entomol 40: 171–194[CrossRef][Web of Science] Fiebig A, Mayfield JA, Miley NL, Chau S, Fischer RL, Preuss D (2000) Alterations in CER6, a gene identical to CUT1, differentially affect long-chain lipid content on the surface of pollen and stems. Plant Cell 12: 2001–2008 Greer S, Wen M, Bird D, Wu X, Samuels AL, Kunst L, Jetter R (2007) The cytochrome P450 enzyme CYP96A15 is the midchain alkane hydroxylase responsible for formation of secondary alcohols and ketones in stem cuticular wax of Arabidopsis. Plant Physiol 145: 653–667 Hepworth SR, Zhang Y, McKim S, Li X, Haughn GW (2005) BLADE-ON-PETIOLE–dependent signaling controls leaf and floral patterning in Arabidopsis. Plant Cell 17: 1434–1448 Heredia A (2003) Biophysical and biochemical characteristics of cutin, a plant barrier biopolymer. Biochim Biophys Acta 1620: 1–7[Medline] Hooker TS, Millar AA, Kunst L (2002) Significance of the expression of the CER6 condensing enzyme for cuticular wax production in Arabidopsis. Plant Physiol 129: 1568–1580 Ishige T, Tani A, Sakai Y, Kato N (2003) Wax ester production by bacteria. Curr Opin Microbiol 6: 244–250[CrossRef][Web of Science][Medline] Jefferson RA (1987) Assaying chimeric gene in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405[CrossRef] Jenks MA, Tuttle HA, Eigenbrode SD, Feldmann KA (1995) Leaf epicuticular waxes of the eceriferum mutants in Arabidopsis. Plant Physiol 108: 369–377[Abstract] Jetter R, Kunst L, Samuels AL (2006) Composition of plant cuticular waxes. In M Riederer, C Müller, eds, Annual Plant Reviews 23: Biology of the Plant Cuticle. Blackwell, Oxford, pp 145–181 Kalscheuer R, Luftmann H, Steinbüchel A (2004) Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase. Appl Environ Microbiol 70: 7119–7125 Kalscheuer R, Steinbüchel A (2003) A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278: 8075–8082 King A, Nam JW, Han J, Hilliard J, Jaworski JG (2007) Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes wax-esters. Planta 226: 381–394[Medline] Kolattukudy PE (1976) Chemistry and Biochemistry of Natural Waxes. Elsevier, Amsterdam Krauss P, Markstädter C, Riederer M (1997) Attenuation of UV radiation by plant cuticles from woody species. Plant Cell Environ 20: 1079–1085[CrossRef] Kunst L, Jetter R, Samuels AL (2006) Biosynthesis and transport of plant cuticular waxes. In M Riederer, C Müller, eds, Annual Plant Reviews 23: Biology of the Plant Cuticle. Blackwell, Oxford, pp 182–215 Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res 42: 51–80[CrossRef][Web of Science][Medline] Kunst L, Taylor DC, Underhill EW (1992) Fatty acid elongation in developing seeds of Arabidopsis. Plant Physiol Biochem 30: 425–434[Web of Science] Kurata T, Kawabata-Awai C, Sakuradani E, Shimizu S, Okada K, Wada T (2003) The YORE-YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis. Plant J 36: 55–66[CrossRef][Web of Science][Medline] Lai C, Kunst L, Jetter R (2007) Composition of alkyl esters in the cuticular wax on inflorescence stems of Arabidopsis cer mutants. Plant J 50: 189–196[CrossRef][Web of Science][Medline] Lardizabal KD, Metz JG, Sakamoto T, Hutton WC, Pollard MR, Lassner MW (2000) Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic Arabidopsis. Plant Physiol 122: 645–656 Lessire R, Juguelin H, Moreau P, Cassagne C (1985) Elongation of acyl-CoAs by microsomes from etiolated leek seedlings. Phytochemistry 24: 1187–1192[CrossRef][Web of Science] Leveau JHJ (2006) Microbial communities in the phyllosphere. In M Riederer, C Müller, eds, Annual Plant Reviews 23: Biology of the Plant Cuticle. Blackwell, Oxford, pp 334–367 Lolle SJ, Hsu W, Pruitt RE (1998) Genetic analysis of organ fusion in Arabidopsis. Genetics 149: 607–619 Millar AA, Clemens S, Zachgo S, Giblin EM, Taylor DC, Kunst L (1999) CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant Cell 11: 825–838 Müller C (2006) Plant-insect interactions on cuticular surfaces. In M Riederer, C Müller, eds, Annual Plant Reviews 23: Biology of the Plant Cuticle. Blackwell, Oxford, pp 398–422 Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K, Matsuoka K, Jinbo T, Kimura T (2007) Development of the series of gateway binary vectors, pGWB, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104: 34–41[CrossRef][Web of Science][Medline] Pfündel EE, Agati G, Cerovic ZG (2006) Optical properties of plant surfaces. In M Riederer, C Müller, eds, Annual Plant Reviews 23: Biology of the Plant Cuticle. Blackwell, Oxford, pp 216–249 Pighin JA, Zheng H, Balakshin LJ, Goodman IP, Western TL, Jetter R, Kunst L, Samuels AL (2004) Plant cuticular lipid export requires an ABC transporter. Science 306: 702–704 Riederer M, Schreiber L (2001) Protecting against water loss: analysis of the barrier properties of plant cuticles. J Exp Bot 52: 2023–2032 Rowland O, Zheng H, Hepworth SR, Lam P, Jetter R, Kunst L (2006) CER4 encodes an alcohol-forming fatty acyl-Coenzyme A reductase involved in cuticular wax production in Arabidopsis. Plant Physiol 142: 866–877 Samuels L, Kunst L, Jetter R (2008) Sealing plant surfaces: cuticular wax formation by epidermal cells. Annu Rev Plant Biol 59: 683–707[CrossRef][Medline] Sandager L, Gustavsson MH, Ståhl U, Dahlqvist A, Wiberg E, Banas A, Lenman M, Ronne H, Stymne S (2002) Storage lipid synthesis is non-essential in yeast. J Biol Chem 277: 6478–6482 Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Scholkopf B, Weigel D, Lohmann JU (2005) A gene expression map of Arabidopsis development. Nat Genet 37: 501–506[CrossRef][Web of Science][Medline] Sieber P, Schorderet M, Ryser U, Buchala A, Kolattukudy P, Metraux JP, Nawrath C (2000) Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure and properties of the cuticle and postgenital organ fusions. Plant Cell 12: 721–737 Solovchenko A, Merzlyak M (2003) Optical properties and contribution of cuticle to UV protection in plants: experiments with apple fruit. Photochem Photobiol Sci 2: 861–866[CrossRef][Web of Science][Medline] Stöveken T, Kalscheuer R, Malkus U, Reichelt R, Steinbüchel A (2005) The wax ester synthase/acyl Coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase. J Bacteriol 187: 1369–1376 Suh MC, Samuels AL, Jetter R, Kunst L, Pollard M, Ohlrogge J, Beisson F (2005) Cuticular lipid composition, surface structure, and gene expression in Arabidopsis stem epidermis. Plant Physiol 139: 1649–1665 Wältermann M, Stöveken T, Steinbüchel A (2006) Key enzymes for biosynthesis of neutral lipid storage compounds in prokaryotes: properties, function and occurrence of wax ester synthases/acyl-CoA: diacylglycerol acyltransferases. Biochimie 89: 230–242[CrossRef][Web of Science][Medline] Xia YJ, Nikolau BJ, Schnable PS (1997) Developmental and hormonal regulation of the Arabidopsis CER2 gene that codes for a nuclear-localized protein required for the normal accumulation of cuticular waxes. Plant Physiol 115: 925–937[Abstract] Xu XJ, Dietrich CR, Delledonne M, Xia YJ, Wen TJ, Robertson DS, Nikolau BJ, Schnable PS (1997) Sequence analysis of the cloned glossy8 gene of maize suggests that it may code for a β-ketoacyl reductase required for the biosynthesis of cuticular waxes. Plant Physiol 115: 501–510[Abstract] Zheng H, Rowland O, Kunst L (2005) Disruptions of the Arabidopsis enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis. Plant Cell 17: 1467–1481
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C12) in the petunia (Petunia hybrida) petal (King et al., 2007
.