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First published online September 15, 2006; 10.1104/pp.106.086785 Plant Physiology 142:866-877 (2006) © 2006 American Society of Plant Biologists CER4 Encodes an Alcohol-Forming Fatty Acyl-Coenzyme A Reductase Involved in Cuticular Wax Production in Arabidopsis1,[W]Department of Botany (O.R., H.Z., S.R.H., P.L., R.J., L.K.) and Department of Chemistry (R.J.), University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
A waxy cuticle that serves as a protective barrier against uncontrolled water loss and environmental damage coats the aerial surfaces of land plants. It is composed of a cutin polymer matrix and waxes. Cuticular waxes are complex mixtures of very-long-chain fatty acids and their derivatives. We report here the molecular cloning and characterization of CER4, a wax biosynthetic gene from Arabidopsis (Arabidopsis thaliana). Arabidopsis cer4 mutants exhibit major decreases in stem primary alcohols and wax esters, and slightly elevated levels of aldehydes, alkanes, secondary alcohols, and ketones. This phenotype suggested that CER4 encoded an alcohol-forming fatty acyl-coenzyme A reductase (FAR). We identified eight FAR-like genes in Arabidopsis that are highly related to an alcohol-forming FAR expressed in seeds of jojoba (Simmondsia chinensis). Molecular characterization of CER4 alleles and genomic complementation revealed that one of these eight genes, At4g33790, encoded the FAR required for cuticular wax production. Expression of CER4 cDNA in yeast (Saccharomyces cerevisiae) resulted in the accumulation of C24:0 and C26:0 primary alcohols. Fully functional green fluorescent protein-tagged CER4 protein was localized to the endoplasmic reticulum in yeast cells by confocal microscopy. Analysis of gene expression by reverse transcription-PCR indicated that CER4 was expressed in leaves, stems, flowers, siliques, and roots. Expression of a  -glucuronidase reporter gene driven by the CER4 promoter in transgenic plants was detected in epidermal cells of leaves and stems, consistent with a dedicated role for CER4 in cuticular wax biosynthesis. CER4 was also expressed in all cell types in the elongation zone of young roots. These data indicate that CER4 is an alcohol-forming FAR that has specificity for very-long-chain fatty acids and is responsible for the synthesis of primary alcohols in the epidermal cells of aerial tissues and in roots.
A continuous lipophilic layer called the cuticle covers most of the aerial surfaces of vascular plants. The cuticle is synthesized by epidermal cells and is one of their distinctive features. The main function of the cuticle is to serve as a waterproof barrier. It restricts nonstomatal water loss and repels rainwater, thus minimizing the deposition of dust, pollen, and spores (Kerstiens, 1996  ; Riederer and Schreiber, 2001). The cuticle also protects underlying tissues from excess levels of UV radiation, mechanical damage, as well as bacterial and fungal pathogens (Jenks et al., 2002) and insects (Eigenbrode and Espelie, 1995). Furthermore, the cuticle has a critical role in plant development by preventing the fusion of cell walls from adjacent organs (Sieber et al., 2000).
The cuticle consists primarily of cutin and waxes. Cutin, the main structural component of the cuticle, is composed of C16 and C18 hydroxy and epoxy fatty acid monomers (Heredia, 2003
Cuticular waxes are complex mixtures of lipids, mostly composed of very-long-chain aliphatic molecules, including primary and secondary alcohols, aldehydes, alkanes, ketones, and esters that are derived from saturated very-long-chain fatty acids (VLCFAs). Each lipid class can be present as a series of characteristic chain lengths (e.g. C24, C26, C28, C30) or one chain length may predominate. Wax load and composition varies considerably between different plant species (Post-Beittenmiller, 1996
The first step in wax biosynthesis is the elongation of saturated C16 and C18 fatty acyl-CoAs, produced in the plastid, to generate VLCFA wax precursors between 20 and 34 carbons in length. Fatty acid elongation is catalyzed by an endoplasmic reticulum (ER)-associated, multienzyme system referred to as the fatty acid elongase (von Wettstein-Knowles, 1982
Even though major wax biosynthetic steps have been defined, our knowledge of the enzymes involved and the nature of the biochemical reactions that they catalyze is limited. Only a small number of genes cloned from Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) encode biosynthetic enzymes of known function. For example, CER6, KCS1, GL8A, GL8B, and CER10 are components of the fatty acid elongases required to generate VLCFA wax precursors (Xu et al., 1997
It is notable that, among the wax-related genes cloned thus far, none specify an enzyme that catalyzes a reaction following fatty acid elongation. Therefore, the most detailed information on the acyl-reduction pathway currently comes from early reports on the biochemistry of plant cuticular wax formation and from recent investigations characterizing related genes involved in the formation of VLCFA primary alcohols and alkyl esters in other organisms and tissues. The biochemistry of wax alcohol formation was first examined in Brassica oleracea and led to the proposal that this is a two-step process carried out by two separate enzymes, an NADH-dependent fatty acyl reductase (FAR) that reduces the fatty acyl-CoAs to free aldehydes and an NADPH-dependent aldehyde reductase that converts the aldehydes to primary alcohols (Kolattukudy, 1971
For Arabidopsis, corresponding FARs involved in cuticular wax biosynthesis have not been investigated and it is currently unknown whether the acyl-reduction pathway proceeds in one or two enzymatic steps from acyl-CoA precursors to primary alcohols. Earlier studies (Hannoufa et al., 1993 The goal of this work was to clone and characterize the gene disrupted in Arabidopsis cer4 mutants to investigate whether the CER4 protein (1) is indeed a FAR; (2) can form aldehyde and/or primary alcohols; (3) has chain length specificity for very-long-chain fatty acyl substrates; and (4) is responsible for wax alcohol formation in all organs of the plant; and (5) to determine its subcellular location.
Molecular Identification of the CER4 Gene
A query of the Arabidopsis genome database with the deduced amino acid sequence of the jojoba FAR using BLAST search programs revealed a group of eight sequences in the Arabidopsis genome that were highly related to the jojoba FAR (28%–54% amino acid sequence identity) over their entire length (Fig. 1
). One of these sequences is MS2 (At3g11980), the gene that encodes a tapetum-specific protein essential for pollen fertility (Aarts et al., 1997
Sequencing of the At4g33790 genomic clone revealed an error in the deposited genomic sequence (GenBank accession no. NC_003075) through comparisons to deposited full-length cDNA sequences (GenBank accession nos. AY057657 and AY070065; Yamada et al., 2003 ). This error was an extra nucleotide causing a frame shift in the predicted coding sequence. The corrected At4g33790 gene consists of 10 exons (Fig. 2A
) and the entire exon-intron region is 3,567 bp in length. The full-length cDNA sequence contains an open reading frame of 1,482 bp encoding a 493-amino acid protein. PCR amplification of the At4g33790 gene in the cer4-1 mutant indicated the presence of a deletion and/or rearrangement of at least 3,350 bp spanning 500 bp of the promoter region, the 5'-untranslated region, and at least 2,800 bp downstream of the translation start. A T-DNA insertion is present in the fourth intron of At4g33790 (at nucleotide 1,960 relative to the start codon) in the cer4-2 mutant. The finding that two independently isolated mutant lines with the same glossy stem wax phenotype both had changes in the same gene indicated that CER4 is indeed encoded by At4g33790.
To confirm the identity of the CER4 gene, we also obtained two SALK T-DNA insertion lines, SALK_038693 (cer4-3) and SALK_000575 (cer4-4), containing T-DNA insertions in the fifth exon (nucleotide 2,225) and fourth intron (nucleotide 1,755), respectively, of the At4g33790 gene from the Arabidopsis Biological Resource Center (ABRC). Homozygotes of cer4-3 and cer4-4 exhibited glossy stem phenotypes similar to the previously characterized cer4-1 and cer4-2 lines. Finally, the extent of cer4 gene disruption was examined by semiquantitative reverse transcription (RT)-PCR. Very low levels of CER4 mRNA were detected in the cer4-2, cer4-3, and cer4-4 lines, whereas no transcript was found in cer4-1 (Fig. 2B). Taken together, these results demonstrate that CER4 is identical to the FAR-like gene At4g33790.
The total wax loads on stems of all three wild-type lines of Arabidopsis were found to be very similar under the growth conditions used here, ranging from 20 µg/cm2 for Ws to 23 µg/cm2 for Ler and Columbia-0 (Col-0; Table I ). The total wax coverage on inflorescence stems of the four mutant lines did not differ from respective wild types. Furthermore, both the absolute amounts and the relative proportions of the fatty acids, aldehydes, alkanes, secondary alcohols, and ketones did not differ between the wax mixtures of the wild-type lines and between the cer4 lines and the corresponding wild types (Table I; Fig. 3 ). The only compound classes affected by CER4 mutations were the primary alcohols and the alkyl esters, reduced from wild-type levels of 2 to 3 µg/cm2 and 1 to 4 µg/cm2, respectively, to only trace amounts in the mutant lines (Tables I and II ).
A close examination of the chain length patterns within compound classes revealed that most of the individual wax compounds were unchanged in cer4-1. The most drastic effect was found for the C24, C26, and C28 primary alcohols, all three compounds being reduced to trace amounts in the stem wax of all four mutant lines. In contrast, C30 primary alcohol still accumulated in the cer4-1 cuticle, reaching approximately 16% of wild-type levels (Fig. 3). Similar chain length patterns were found for the primary alcohols in cer4-2 and cer4-3, whereas cer4-4 showed a drastic reduction of C24 and C26 alcohols, and partially reduced amounts of C28 and C30 alcohols (data not shown). The alkyl esters of the wild-type lines showed chain lengths ranging from C38 to C54, with a strong predominance of even-numbered homologs and a maximum for C44 (Table II). In contrast to this, the mutant lines cer4-1, cer4-2, and cer4-3 had largely reduced levels of even-numbered esters, but not of odd-numbered compounds. The remaining even-numbered esters showed a bimodal distribution with maxima at C46 and at C50 to C54. The ester fraction was dominated by the C45 ester, which, according to mass spectrometry (MS) data, consisted mainly of C16:0 acid esterified with a C29 alcohol. Because this is the predominant chain length of the secondary alcohols in Arabidopsis stem wax, it seems very likely that this residual ester contains the secondary alcohols nonacosan-14-ol and -15-ol. These alcohols were not further analyzed in the cer4 mutant esters because secondary alcohols are formed on the decarbonylation pathway without involvement of FAR enzymes and the acyl reduction pathway. The even-numbered alkyl moieties of all the esters were quantified using MS data and summarized according to the alcohol chain lengths present. Whereas Col-0 wild-type esters were dominated by C26 alkyl chains, the mutant lines cer4-1, cer4-2, and cer4-3 were characterized by drastically decreased amounts of this alcohol. Instead, they had notably increased levels of C22 and C30 alcohols in their stem wax esters, together with varying levels of C24 and C28 alcohols. The wax of cer4-4 showed an ester composition intermediate between wild-type and the other three mutant lines. Even though the total stem wax loads of the cer4 plants were similar to wild-type plants, the stems of all four mutant lines appeared glossy. This indicated that the epicuticular wax crystals on the cer4 stems were affected by the mutations. Because these crystals had not been reported for cer4 plants before, we examined the density and shape of wax crystals on stems of cer4-1 plants by scanning electron microscopy (SEM; Fig. 4 ). A dense array of epicuticular wax crystals consisting of vertical rods, tubes, longitudinal bundles of rodlets, and horizontal, reticulate platelets cover the stem surfaces of wild-type Arabidopsis (Fig. 4, A and B). Conversely, the stem surfaces of cer4-1 plants are almost devoid of wax crystals and instead have a thick film of wax with a relatively smooth surface (Fig. 4, C and D), accounting for the change in surface light reflectance.
Both the wax chemical and the surface micromorphological phenotypes could be rescued by complementation with the At4g33790 genomic sequence (Fig. 3; Table I), providing further evidence that we have identified the CER4 gene.
To verify that CER4 is an alcohol-forming FAR involved in the production of very-long-chain primary alcohols, we expressed the 1,482-bp coding region of the CER4 gene in Saccharomyces under the control of the yeast (Saccharomyces cerevisiae) GAL1 promoter. Under inducing conditions, cells transformed with an empty-vector control accumulated C16:0, C16:1, C18:0, C18:1, and C26:0 fatty acids, but no fatty alcohols could be detected (Fig. 5A ). Conversely, yeast cells expressing CER4 produced two novel compounds. They were identified as primary alcohols C24:0-OH and C26:0-OH (Fig. 5B), based on their gas chromatography (GC) behavior and MS characteristics. The CER4-expressing cells also consistently accumulated greater levels of C26:0 fatty acid compared to the control yeast. No primary alcohols derived from saturated and monounsaturated C16 and C18 fatty acids could be detected.
The subcellular localization of the CER4 FAR was carried out in yeast expressing CER4 cDNA N-terminally tagged with green fluorescent protein (GFP) under the control of the GAL1 promoter. The fusion of the GFP epitope to the CER4 protein did not affect the FAR activity of the modified enzyme in yeast (data not shown) and allowed us to visualize it by confocal microscopy. This experiment revealed a fluorescence pattern typical of the yeast ER (Fig. 6A , arrow). Counterstaining of yeast cells with hexyl rhodamine B, a dye that can stain the ER, outlined the same cellular domains (Fig. 6, C–E), confirming that CER4 resides in the ER in yeast.
Characterization of the Predicted CER4 Protein
The CER4 transcript encodes a polypeptide of 493 amino acids with an estimated molecular mass of 56.0 kD. The predicted CER4 protein sequence is 54% identical with the NADPH-dependent membrane-associated alcohol-forming FAR from jojoba embryos (Metz et al., 2000
The jojoba FAR is thought to be an integral membrane protein containing two membrane-spanning domains between residues 309 to 329 and between residues 378 to 398. CER4 has a similar number of hydrophobic residues in these two predicted segments (Fig. 7). However, hydropathy plots and other sequence analysis programs do not reveal strong transmembrane domains anywhere in the CER4 protein sequence. It thus remains uncertain whether these segments are true transmembrane regions in the FAR enzymes. The deduced CER4 amino acid sequence also lacks the typical NAD(P)H binding motif or Rossmann fold: GXGXX(G/A) (Wierenga et al., 1986 ). Aarts et al. (1997) speculated that MS2 binds NAD(P)H through a related nucleotide-binding sequence (I/V/F)X(I/L/V)TGXTGFL(G/A) and CER4 contains this motif between residues 19 and 29 (Fig. 7).
Semiquantitative RT-PCR was used to investigate the transcription profile of the CER4 gene in various whole tissues (Fig. 8 ). Aerial tissue samples were derived from 6-week-old Arabidopsis plants and the root tissue sample from 14-d-old seedlings. CER4 was expressed in all tissues, but to varying levels. Highest transcript accumulation was detected in open flowers and roots. Modest transcript levels were observed in stems, closed flowers, and siliques; low, but consistently detectable, CER4 transcripts were observed in rosette and cauline leaves.
To analyze the cell-type expression pattern of CER4 in detail, the 2.1 kb of genomic sequence immediately upstream of the CER4 coding region was fused to the -glucuronidase (GUS) reporter gene (CER4pro:GUS) and the construct was used to transform Arabidopsis. Tissue samples from 20 independent transgenic lines were stained for GUS activity, with four of these lines being characterized in detail. Consistent with RT-PCR analysis, reporter gene expression was observed in both shoots and roots (Fig. 9
). In the stem, CER4 was expressed along the entire length exclusively in the epidermal cells (Fig. 9, A and B). We confirmed the epidermis-specific expression of CER4 in the inflorescence by in situ hybridization (Fig. 9C) and observed that CER4 expression was confined to the region below the shoot apical and floral meristems.
In rosette leaves, CER4pro:GUS was strongly expressed in the trichomes, which are specialized epidermal cells (Fig. 9E). This very restricted expression pattern in leaves may explain the low levels of the CER4 transcript present in whole-leaf tissues because the transcript would be diluted by the surrounding pavement cells and underlying mesophyll cells. Expression of CER4 in cauline leaves was more widespread, being detected in trichomes, pavement cells, and the vasculature, and was especially high at the base of the leaf (Fig. 9D). However, this was not reflected by higher levels of total expression detected by RT-PCR using whole-tissue samples from cauline leaves (Fig. 8). In flowers, CER4pro-directed GUS activity was detected in the petals, the filaments of the stamens, and the carpel (Fig. 9F). In situ hybridization of the CER4 transcript verified the expression in the carpel and showed that this expression was epidermis specific (Fig. 9G). In siliques, CER4pro:GUS activity was present throughout the outer layer of the silique, but no activity was observed in the seeds (Fig. 9H). Unexpectedly, we also detected CER4 expression in the central and lateral roots of 14-d-old seedlings (Fig. 9I), but not in the root tip (Fig. 9J). CER4 expression was strongest in the elongation zone behind the root tip and appeared to diminish in the older sections of the differentiation zone (Fig. 9I). In the elongation zone, GUS activity was detected in all cell types (Fig. 9K), although GUS staining appeared more pronounced in the epidermal cells compared to interior cell types.
Primary alcohols account for 10% to 15% of the total wax load on wild-type Arabidopsis stems and 15% to 25% on Arabidopsis leaves either as free alcohols or in the form of wax esters. Mutations in CER4 virtually abolish the formation of primary alcohols in both plant organs, indicating that CER4 is the key enzyme catalyzing their biosynthesis. In this study, we have used the cer4 mutants to clone the CER4 (At4g33790) gene and characterize the functions of CER4 in wax production.
The CER4 (At4g33790) gene encodes a protein with an estimated molecular mass of 56 kD, similar to values reported for the jojoba and pea FAR enzymes (Vioque and Kolattukudy, 1997
The CER4 substrate specificity revealed in yeast is in good agreement with the established wax profiles of cer4 mutants, which lack C24 to C28 free alcohols as reported here and previously (Hannoufa et al., 1993 Interestingly, despite the massive reduction in primary alcohol and alkyl ester levels, cer4 mutants have almost wild-type stem wax loads (Table I) and yet their stems are strongly glossy. The SEM survey of the cer4 stem surface showed that they are covered with a relatively smooth film and are virtually devoid of wax crystals, resulting in a glossy stem appearance. These data indicate that primary alcohols and/or wax esters are required for epicuticular wax crystal formation. It is unlikely, however, that the wax crystals themselves are formed from these two wax components alone because alkanes, secondary alcohols, and ketones make up the majority of the Arabidopsis stem wax load (approximately 75%) and are expected to make the major contribution to crystal form. It thus remains unclear how the lack of acyl-reduction products prevents wax crystal formation.
The CER4 transcript was detected exclusively in the epidermal cells of stems. Expression of CER4 in the epidermal layer of the shoot was anticipated based on the phenotype of cer4 mutants, which lack primary alcohols in cuticular wax deposited on the stem surface. This suggests that the primary function of CER4 is in cuticular wax formation, at least in stems. CER4 was also expressed in the epidermal cells of leaves, but was confined to trichome cell types in rosette leaves. This trichome-specific pattern of expression has been observed for CER2 (Xia et al., 1997
Examination of the active GFP-tagged CER4 in yeast by confocal microscopy revealed that this FAR resides in the ER. ER localization of an alcohol-forming FAR was already suggested for the jojoba embryo enzyme, which was membrane bound and associated with the ER membrane fraction (Metz et al., 2000 In summary, we have cloned the CER4 gene disrupted in the cer4 wax-deficient mutants of Arabidopsis and demonstrate that it encodes an ER-localized FAR. CER4 is specifically involved in the production of C24 to C28 very-long-chain primary alcohols, one of the major cuticular wax components found on Arabidopsis shoots.
Plant Material and Growth Conditions
SALK T-DNA insertional lines, SALK_038693 and SALK_000575 (Col-0 ecotype), and cer4-1 (Ler ecotype) mutant seeds were obtained from the ABRC (www.arabidopsis.org). cer4-2 (Ws ecotype) was a gift from Dr. Bertrand Lemieux (York University). Seeds were stratified for 3 to 4 d at 4°C, and were then germinated on AT-agar plates (Somerville and Ogren, 1982
Wax load was determined on 6-week-old Arabidopsis (Arabidopsis thaliana) plants. Stems, leaves, and siliques were immersed in chloroform for 30 s to remove epi- and intracuticular waxes. After extraction, wax samples were evaporated to dryness under a stream of nitrogen, dissolved in 50 µL of N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS; Pierce), and derivatized at 80°C for 90 min. Samples were then analyzed by gas-liquid chromatography as described in Pighin et al. (2004) For wax ester analyses, stem cuticular wax mixtures of Arabidopsis wild-type and cer4 mutant lines were separated by thin-layer chromatography on silica gel using 1,1,1-trichloroethane as the mobile phase. Compound classes were localized by staining with primuline and UV light, removed from the plates, eluted with CHCl3, filtered, concentrated in a stream of N2, and stored at 4°C. The fraction containing alkyl esters (Rf 0.13) was subjected to detailed GC analyses. A GC-flame ionization detector was employed to quantify the relative amounts of ester homologs, whereas GC-MS was used to determine the isomer composition for each ester chain length based on acyl fragments RCOOH2+.
Segments from the apical 1 cm of the stem were mounted onto SEM stubs, allowed to dry, and then coated with gold particles in a SEMPrep2 sputter coater (Nanotech). The coated samples were viewed with a Hitachi S4700 field emission SEM using an accelerating voltage of 1 kV and a working distance of 12 mm.
Protein sequences were aligned with the ClustalW 1.83 program (Thompson et al., 1997
A 6,138-bp DNA fragment containing the coding region of At4g33790 was amplified by PCR from genomic DNA isolated from wild-type Ler plants. This fragment contained 2,160 bp of sequence 5' of the ATG start codon and 438 bp of sequence 3' of the stop codon. The primers used for amplification were At4g33790-PromEcoRI (5'-GAGGAATTCTTTCCTTGTAGCCGCCTTTA-3') and At4g33790-TermXbaI (5'-GAGTCTAGAAACTTTACATGGGGGCAATG-3'). To minimize PCR-induced errors, amplification was carried out using the Expand high-fidelity PCR system (Roche Diagnostics). The amplified DNA fragment was digested with EcoRI and XbaI and cloned into the corresponding sites of pRD400 (Datla et al., 1992
We first isolated full-length CER4 cDNA for expression in yeast (Saccharomyces cerevisiae). Total RNA was extracted from wild-type Col-0 plants using TRIzol Reagent (Invitrogen) according to manufacturer's protocol. First-strand cDNA synthesis was carried out using 1 µg of total RNA, oligo(dT)18, and SuperScript II reverse transcriptase (Invitrogen). To amplify CER4 cDNA, PCR was performed using the Expand high-fidelity PCR system (Roche) with 1 µL of cDNA. Primers used for amplification were CER4-F5BamHI (5'-GAGGGATCCATGTCGACAGAAATGGAGGTC-3') and CER4-R7SacI (5'-CACGAGCTCTTAGAAGACATACTTAAGCAGC-3'). The amplified DNA fragment was digested with BamHI and SacI and cloned into the corresponding sites of pBluescript II SK+ to generate pBS/CER4. Sequencing of an individual clone confirmed that there were no errors in the CER4 cDNA. pBS/CER4 was digested with EcoRI and Ecl136II and cloned between the EcoRI and EcoRV restriction sites of the yeast expression vector p423-GAL1 (Mumberg et al., 1994
p423-GAL1/CER4 and an empty-vector p423-GAL1 (control) were transformed into yeast strain W3031A (MATa ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 can1-100) according to Gietz and Woods (2002)
To generate GFP fused in frame with the coding region of CER4, pBS/CER4 was digested with BamHI and SacI and subcloned into the corresponding sites of the binary vector pVKH18-GFPN (Zheng et al., 2005
Aerial tissues used for RNA extractions were harvested from 6-week-old Arabidopsis plants. Whole-root tissue was harvested from 14-d-old seedlings grown on AT-agar plates under continuous light conditions. With the exception of silique tissue, total RNA was extracted using a guanidine-HCl and phenol-chloroform extraction procedure (Logemann et al., 1987 For analysis of the transcription profile of CER4 by RT-PCR, RNA was reverse transcribed using 1 µg of total RNA template, oligo(dT)18, and SuperScript II reverse transcriptase (Invitrogen). One microliter of a 1:1 diluted RT reaction was used as template in a 20-µL reaction with gene-specific primers CER4-F5BamHI (above) and CER4-R3EcoRI (5'-CACGAATTCCCCTCAGTCCAACCAGGAAA-3') designed to amplify a 851-bp cDNA fragment of CER4. To examine the extent of gene disruption in each of four cer4 alleles, gene-specific primers TSH450 (5'-CTTCTTCTGTGATCTTGATGC-3') and TSH451 (5'-TAGAAGACATACTTAAGCAGCC-3') that can amplify a 578-bp cDNA fragment of CER4 were used. The glyceraldehyde-3-P dehydrogenase (GAPC) constitutive control was amplified using primers GAPC-p1 (5'-TCAGACTCGAGAAAGCTGCTAC-3') and GAPC-p2 (5'-GATCAAGTCGACCACACGG-3'), which amplifies a 245-bp cDNA fragment.
A DNA fragment containing 2,147 bp of sequence immediately upstream of the ATG start codon of CER4 was amplified by PCR from Arabidopsis bacterial artificial chromosome T16L1 (AL031394) using the Expand high-fidelity system (Roche Diagnostics). The primers used for amplification were CER4-PROMSalIfor (5'-GAGGTCGACTTTCCTTGTAGCCGCCTTTA-3') and CER4-PROMXbaIrev (5'-GAGTCTAGAGTATATACGTTTGAGTGAGAGA-3'). The amplified product was digested with SalI and XbaI and cloned between the corresponding sites of pBluescript II SK+ to generate pBS/CER4pro. Sequencing of an individual clone confirmed that there were no errors in the CER4 promoter. pBS/CER4pro was digested with SalI and XbaI and cloned between the corresponding sites of pBI101 (CLONTECH) to generate a transcriptional fusion of the CER4 promoter with the GUS reporter gene. This construct was introduced into wild-type Col-0 plants by Agrobacterium-mediated transformation as described above.
GUS staining solution was composed of 50 mM sodium phosphate, pH 7.0, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.1% (v/v) Triton X-100, and 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-
In situ hybridization of Arabidopsis inflorescences (tissue fixation, sectioning, hybridization, signal detection, and probe synthesis) was carried out as described previously (Samach et al., 1997
The following materials are available in the online version of this article.
We thank the ABRC at Ohio State University for the cer4-1 mutant, Bertrand Lemieux for providing the cer4-2 mutant, and the Salk Institute for Genomic Analysis Laboratory for providing sequence-indexed Arabidopsis T-DNA insertion mutants cer4-3 (SALK_038693) and cer4-4 (SALK_000575). We thank Helena Friesen for the p423GAL1 vector. We are grateful to the Bioimaging Facility at the University of British Columbia for providing microscopy support. Received July 15, 2006; accepted September 1, 2006; published September 15, 2006.
1 This work was supported by a grant from the National Sciences and Engineering Research Council of Canada. 
2 Present address: Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6. 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. www.plantphysiol.org/cgi/doi/10.1104/pp.106.086785 * Corresponding author; e-mail kunst{at}interchange.ubc.ca; fax 604–822–6089.
Aarts MG, Hodge R, Kalantidis K, Florack D, Wilson ZA, Mulligan BJ, Stiekema WJ, Scott R, Pereira A (1997) The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes. Plant J 12: 615–623[CrossRef][ISI][Medline] Aarts MGM, Keijzer CJ, Stiekema WJ, Pereira A (1995) Molecular characterization of the CER1 gene of Arabidopsis involved in epicuticular wax biosynthesis and pollen fertility. Plant Cell 7: 2115–2127[Abstract] Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A (2004) The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell 16: 2463–2480 Birnbaum K, Shasha DE, Wang JY, Jung JW, Lambert GM, Galbraith DW, Benfey PN (2003) A gene expression map of the Arabidopsis root. Science 302: 1956–1960 Broun P, Poindexter P, Osborne E, Jiang C-Z, Riechmann JL (2004) WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc Natl Acad Sci USA 101: 4706–4711 Cheesbrough TM, Kolattukudy PE (1984) Alkane biosynthesis by decarbonylation of aldehydes catalyzed by a particulate preparation from Pisum sativum. Proc Natl Acad Sci USA 81: 6613–6617 Chen X, Goodwin SM, Boroff VL, Liu X, Jenks MA (2003) Cloning and characterization of the WAX2 gene of Arabidopsis involved in cuticle membrane and wax production. Plant Cell 15: 1170–1185 Cheng JB, Russell DW (2004) Mammalian wax biosynthesis. I. Identification of two fatty acyl-Coenzyme A reductases with different substrate specificities and tissue distributions. J Biol Chem 279: 37789–37797 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743[CrossRef][ISI][Medline] Datla RS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller W (1992) Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 122: 383–384[CrossRef][ISI][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][ISI][Medline] Downing WL, Mauxion F, Fauvarque MO, Reviron MP, de Vienne D, Vartanian N, Giraudat J (1992) A Brassica napus transcript encoding a protein related to the Kunitz protease inhibitor family accumulates upon water stress in leaves, not in seeds. Plant J 2: 685–693[CrossRef][ISI][Medline] Eigenbrode SD, Espelie KE (1995) Effects of plant epicuticular lipids on insect herbivores. Annu Rev Entomol 40: 171–194[CrossRef][ISI] 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 Gietz RD, Woods RA (2002) Transformation of yeast by the lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350: 87–96[CrossRef][ISI][Medline] Hannoufa A, McNevin J, Lemieux B (1993) Epicuticular waxes of eceriferum mutants of Arabidopsis thaliana. Phytochemistry 33: 851–855[CrossRef] Hannoufa A, Negruk V, Eisner G, Lemieux B (1996) The CER3 gene of Arabidopsis thaliana is expressed in leaves, stems, roots, flowers and apical meristems. Plant J 10: 459–467[CrossRef][ISI][Medline] Hansen JD, Pyee J, Xia Y, Wen TJ, Robertson DS, Kolattukudy PE, Nikolau BJ, Schnable PS (1997) The glossy1 locus of maize and an epidermis-specific cDNA from Kleinia odora define a class of receptor-like proteins required for the normal accumulation of cuticular waxes. Plant Physiol 113: 1091–1100[Abstract] Heredia A (2003) Biophysical and biochemical characteristics of cutin, a plant barrier biopolymer. Biochim Biophys Acta 1620: 1–7[Medline] Jenks MA, Eigenbrode SD, Lemieux B (2002) Cuticular waxes of Arabidopsis. In CR Somerville, EM Meyerowitz, eds, The Arabidopsis Book. American Society of Plant Biologists, Rockville, MD Jenks MA, Tuttle HA, Eigenbrode SD, Feldmann KA (1995) Leaf epicuticular waxes of the eceriferum mutants in Arabidopsis. Plant Physiol 108: 369–377[Abstract] Kerstiens G (1996) Signalling across the divide: a wider perspective of cuticular structure-function relationships. Trends Plant Sci 1: 125–129[CrossRef][ISI] Kolattukudy PE (1971) Enzymatic synthesis of fatty alcohols in Brassica oleracea. Arch Biochem Biophys 142: 701–709[CrossRef][ISI][Medline] Kolattukudy PE (1996) Biosynthetic pathways of cutin and waxes, their sensitivity to environmental stresses. In G Kersteins, ed, Plant Cuticles, An Integrated Functional Approach. BIOS Scientific Publishers, Oxford, pp 83–108 Koornneef M, Hanhart CJ, Thiel F (1989) A genetic and phenotypic description of eceriferum (cer) mutants in Arabidopsis thaliana. J Hered 80: 118–122 Krolikowski KA, Victor JL, Wagler TN, Lolle SJ, Pruitt RE (2003) Isolation and characterization of the Arabidopsis organ fusion gene HOTHEAD. Plant J 35: 501–511[CrossRef][ISI][Medline] Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res 42: 51–80[CrossRef][ISI][Medline] 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][ISI][Medline] Kurdyukov S, Faust A, Nawrath C, Bar S, Voisin D, Efremova N, Franke R, Schreiber L, Saedler H, Metraux JP, et al (2006a) The epidermis-specific extracellular BODYGUARD controls cuticle development and morphogenesis in Arabidopsis. Plant Cell 18: 321–339 Kurdyukov S, Faust A, Trenkamp S, Bar S, Franke R, Efremova N, Tietjen K, Schreiber L, Saedler H, Yephremov A (2006b) Genetic and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long-chain alpha-,omega-dicarboxylic fatty acids and formation of extracellular matrix. Planta 224: 315–329[CrossRef][ISI][Medline] Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem 163: 16–20[CrossRef][ISI][Medline] McNevin JP, Woodward W, Hannoufa A, Feldmann K, Lemieux B (1993) Isolation and characterization of eceriferum (cer) mutants induced by T-DNA insertions in Arabidopsis thaliana. Genome 36: 610–618[Medline] Metz JG, Pollard MR, Anderson L, Hayes TR, Lassner MW (2000) Purification of a jojoba embryo fatty acyl-Coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed. Plant Physiol 122: 635–644 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 Moose SP, Sisco PH (1996) Glossy15, an APETALA2-like gene from maize that regulates leaf epidermal cell identity. Genes Dev 10: 3018–3027 Moto K, Yoshiga T, Yamamoto M, Takahashi S, Okano K, Ando T, Nakata T, Matsumoto S (2003) Pheromone gland-specific fatty-acyl reductase of the silkmoth, Bombyx mori. Proc Natl Acad Sci USA 100: 9156–9161 |
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