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First published online April 22, 2005; 10.1104/pp.104.058164 Plant Physiology 138:478-489 (2005) © 2005 American Society of Plant Biologists Cloning and Characterization of GLOSSY1, a Maize Gene Involved in Cuticle Membrane and Wax Production1,[w]Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo, 24126 Bergamo, Italy (M.S., H.H., M.M.); and Max-Planck Institut für Züchtungsforschung, D–50829 Cologne, Germany (E.S., R.V., F.S.)
The cuticle covering the aerial organs of land plants plays a protective role against several biotic and abiotic stresses and, in addition, participates in a variety of plant-insect interactions. Here, we describe the molecular cloning and characterization of the maize (Zea mays) GLOSSY1 (GL1) gene, a component of the pathway leading to cuticular wax biosynthesis in seedling leaves. The genomic and cDNA sequences we isolated differ significantly in length and in most of the coding region from those previously identified. The predicted GL1 protein includes three histidine-rich domains, the landmark of a family of membrane-bound desaturases/hydroxylases, including fatty acid-modifying enzymes. GL1 expression is not restricted to the juvenile developmental stage of the maize plant, pointing to a broader function of the gene product than anticipated on the basis of the mutant phenotype. Indeed, in addition to affecting cuticular wax biosynthesis, gl1 mutations have a pleiotropic effect on epidermis development, altering trichome size and impairing cutin structure. Of the many wax biosynthetic genes identified so far, only a few from Arabidopsis (Arabidopsis thaliana) were found to be essential for normal cutin formation. Among these is WAX2, which shares 62% identity with GL1 at the protein level. In wax2-defective plants, cutin alterations induce postgenital organ fusion. This trait is not displayed by gl1 mutants, suggesting a different role of the maize and Arabidopsis cuticle in plant development.
The cuticle forms the outermost layer of the above-ground parts of most plants. The physical and chemical properties of this structure support vital functions such as prevention of nonstomatal water loss, protection against UV irradiation, and reduction of deposition of dust, pollen, and air pollutants. In addition, it plays a critical role in plant defense against bacterial and fungal pathogens and participates in a variety of plant-insect interactions (Post-Beittenmiller, 1996  ).
The cuticle is synthesized by the epidermal cells and consists of an outer layer of epicuticular waxes overlaying the cuticle membrane, which is composed of a network of interesterified hydroxy and epoxy-hydroxy fatty acids of mainly 16 and 18 atoms in length (cutin) interspersed by intracuticular waxes (Walton, 1990
Although advances have been made in the understanding of the biosynthesis of specific cutin and wax constituents, many questions pertaining to the organization and regulation of the concerned biochemical pathways remain unanswered. The availability of mutants deficient in cuticular wax accumulation in a variety of species and the isolation of the corresponding genes reveal helpful information to elucidate cuticular wax biosynthesis and to characterize molecular aspects of regulatory control (Kunst and Samuels, 2003
In maize (Zea mays), at least 18 loci (the GLOSSY or GL loci) have been found to affect the quantity and/or the composition of cuticular waxes on the surface of seedling leaves (Neuffer et al., 1997
Over the past years, various maize GLOSSY genes involved in cuticular wax production have been cloned (Moose and Sisco, 1994
Mutation at the GL1 locus causes dramatic alterations in the amount, composition, and crystallization patterns of juvenile cuticular waxes (Bianchi et al., 1985
In an effort to characterize the Gl1 gene, we performed transposon-tagging experiments with the Enhancer/Suppressor mutation (En/Spm) element, which led to the tagging of the GL1 locus (Maddaloni et al., 1990
Isolation of Transposon-Tagged Alleles of the GL1 Locus
From the cross outlined in "Materials and Methods," nine glossy seedlings with revertant nonglossy sectors were identified out of approximately 90,000 F1 seedlings. The new alleles were designated gl1-m1 through gl1-m9. Genetic analyses of the new mutable alleles are described by Maddaloni et al. (1990) Plants carrying the gl1-m5 allele were tested for the functional presence in their genome of the transposable element En/Spm. Plants heterozygous for the gl1-m5 allele and for the stable recessive reference allele gl1-ref were crossed with the En/Spm tester strains described in "Materials and Methods." Three independent test crosses were performed. F1 plants of each test cross were selfed, and the resulting ears were scored for F2 kernels displaying reversions of the a1-m1 or a1-m(r) tester allele. All three groups of F1 plants gave rise to segregating and nonsegregating ears, with a percentage of the former near to 75%, suggesting the presence, in the gl1-m5 progenitor, of two unlinked copies of an active En/Spm element. F2 kernels of both groups of ears were grown to seedlings, and these were scored for leaf variegation due to the gl1-m5 allele. The results of these experiments are summarized in Table I. Approximately one-half of the ears that segregated for variegated kernels also segregated gl1-m5 variegated seedlings, while ears without variegated seeds did not originate variegated seedlings. This result indicates that the gl1-m5 allele is likely to be caused by the insertion of an autonomous active En/Spm element.
Phenotypic Analysis of gl1 Mutants
Morphology of epicuticular waxes of wild-type and gl1 plants was previously examined with scanning electron microscopy (SEM) by Lorenzoni and Salamini (1975)
The wax phenotypes of a GL1 wild-type allele, of the recessive gl1-ref allele and of the unstable gl1-m5 allele are shown in Figure 1, A to C, as revealed by visual and microscopic inspections. The gl1-m5 allele shows clear somatic instability that is visible as sectors of wild-type tissue in a mutant background (Fig. 1D). The revertant sectors can cover small or large parts of a leaf (up to one-half) or are restricted to single epidermal cells. This finding shows that GL1, like other GL genes, acts cell autonomously during juvenile leaf development (Moose and Sisco, 1994
Epidermal cells of maize leaves are arranged in cell rows that extend longitudinally parallel with the veins and show a gradient of cell differentiation from the leaf base to the leaf tip. Selected cell rows are enriched with stomata, others with trichomes, while still others are devoid of both types of specialized epidermal cells. gl1 mutant trichomes are smaller and more closely spaced in comparison to the wild type. In the apical region of fully developed juvenile leaves, their size is about one-half that of the wild type; on the leaf margin, mean distance between mutant and wild-type trichomes is 124 ± 14 µm and 171 ± 28 µm, respectively (Fig. 1, E and F). Stomata distribution on gl1 leaves does not differ from the wild type. Ultrastuctural analysis of the leaf cuticle with transmission electron microscopy (TEM) indicates that the wild-type cuticle membrane appears to be divided into an outermost translucent layer (the cuticle proper) and an innermost opaque layer (the reticulated cuticular layer; Fig. 1G). In the gl1 mutant, cuticle membrane thickness is clearly reduced by about 50% and the cuticle proper appears almost absent (Fig. 1H). No differences in permeability to chlorophyll were detected in gl1 leaves compared to the wild type, as determined with extraction in 80% ethanol, with or without prior removal of cuticular waxes (data not shown). gl1 plants do not show reduction in pollen fertility, probably because waxes affected by the mutation are those found on juvenile leaves.
The internal EcoRI/BamHI fragment of the En/Spm element was used as a hybridization probe in DNA gel-blot analyses of families segregating for gl1-m5. An 8.3-kb HindIII fragment that cosegregated with the gl1-m5 mutant phenotype was identified. Representative homozygous (lanes 4 and 5) and heterozygous (lanes 6 and 7) gl1-m5 plants with the 8.3-kb En/Spm hybridizing fragment are shown in Figure 2A. Evidence that the HindIII fragment represents an En/Spm insertion in the GL1 gene came from the absence of this fragment in plants homozygous for germinal reverted alleles derived from gl1-m5 (Fig. 2A, lanes 2 and 3). No other En/Spm hybridizing fragments from gl1-m5 plants were consistently found to be missing from these derivatives.
A size-fractionated subgenomic library of HindIII fragments from heterozygous gl1-m5 plants was constructed in the  EMBL3 vector, and the 8.3-kb HindIII fragment was isolated in the clone -09 using the EcoRI/BamHI probe of the En/Spm element. The restriction map of the cloned HindIII fragment indicated the presence of an En/Spm element flanked by non-En/Spm sequences. A restriction fragment (0.95-kb HindIII-XhoI) carried by the non-En/Spm sequence was used as a hybridization probe to the same DNA gel blot shown in Figure 2A. The resulting hybridization pattern is shown in Figure 2B. Homozygous gl1-m5 plants, which exhibit an unstable phenotype in the presence of the autonomous En/Spm, showed the expected 8.3-kb HindIII fragment and a low-intensity 6.0-kb fragment (lanes 4 and 5). The 6.0-kb fragment was correlated with the generation of somatic revertant sectors from gl1-m5 and thus represented the original progenitor allele. Proof that the 0.95-kb HindIII-XhoI fragment represents part of the GL1 gene came from comparing the two independent germinal revertant derivatives of gl1-m5 (lanes 2 and 3) with their mutable siblings (lanes 4–7). The homozygous revertant plants contained only a 6.0-kb HindIII hybridizing fragment, whereas their mutable siblings heterozygous for the gl1-m5 and gl1-ref alleles, contained the 8.3- and 6.0-kb fragments (lanes 6 and 7). The size difference between the restriction fragment representing gl1-m5 and its somatic and germinal revertant derivatives was consistent with the En/Spm insertion observed within the cloned 8.3-kb HindIII fragment (Fig. 2B).
Although these results indicated that
The HindIII-XhoI fragment derived from clone Computer-aided analysis of the genomic sequence obtained identified the putative exons encompassing the GL1 transcript. A database search for proteins homologous to the deduced GL1 polypeptide bolstered the postulated mRNA sequence. On the basis of these data, two primers were designed to isolate the full-length coding sequence of GL1 by reverse transcription (RT)-PCR. A single 2,056-bp fragment, including a 1,866-nucleotide-long open reading frame (ORF), was amplified from RNA extracted from wild-type seedling leaves of the inbred Wf9. From a partial cDNA clone isolated from a seedling cDNA library, we deduced that the GL1 transcript contains a 240-bp-long untranslated region (UTR) at its 3' end (data not shown). The 5' UTR was previously found to be 185 bp long. Taken together, these data suggest an approximate length of 2,291 bp for the GL1 transcript. An in-frame stop codon was present 87 bp upstream of the ATG start codon of the main ORF. No alternative translation start sites were present, indicating that the amplified fragment included the complete coding region. Putative CAAT- and TATA-box motifs were found in the promoter sequence 200 and 146 bp upstream of the ATG start codon, respectively, while a putative polyadenylation site was present 312 bp downstream of the translation stop codon. Alignment of cDNA and genomic sequences revealed the presence of eight noncontiguous stretches of homology, interspersed with seven intron sequences ranging in size from 95 to 2,025 bp (Fig. 3). The deduced coding sequence displays three single base differences with respect to the genomic sequence considered. This discrepancy is due to polymorphism between the two strains used for the isolation of genomic and cDNA clones, as confirmed by sequence analysis of the corresponding genomic regions of the Wf9 inbred line (data not shown).
Mapping of En/Spm Insertion Sites
The available unstable gl1 alleles were examined by Southern analysis using different fragments of the GL1 gene as probes. All unstable alleles were found to be caused by independent insertions of members of the En/Spm transposable element family. The approximate insertion site and the orientation of the elements with respect to the GL1 gene were determined for all seven unstable alleles. In four cases (gl1-m1, 2, 5, and 8), the transposable element was inserted distal to the HindIII restriction site present in the fourth intron of the GL1 locus, which has been used previously to delimit the end of the GL1 gene (Fig. 3; Hansen et al., 1997 The identification of the transposable elements present at the different unstable gl1 alleles and the location of the insertion point in the known sequence of the GL1 gene made possible a PCR-mediated amplification of specific fragments spanning the 5' and 3' junctions between the gl1 and terminal transposable element sequences. GL1- and En/Spm-specific primers were employed in PCR amplification reactions (see Supplemental Tables II and III) generating amplified fragments of the expected lengths. All amplification products were sequenced to determine the precise insertion point of the transposable element present in each of the unstable gl1 alleles (Fig. 3). The seven En/Spm insertions are all placed within the genomic region encompassed by the cDNA sequence. In all cases, the characteristic target site duplication of three nucleotides for En/Spm was observed (data not shown).
Conceptual translation of the 1,866-nucleotide-long ORF present in the GL1 cDNA sequence gave rise to a putative polypeptide of 621 amino acids with an apparent molecular mass of 69.6 kD and a pI of 9.89. Hydropathy analysis predicted the presence of several transmembrane domains in the N-terminal region of the GL1 polypeptide, as well as of a hydrophilic C-terminal domain. Furthermore, a tripartite His-rich motif characteristic of a family of membrane-bound desaturases/hydroxylases was present in the N-terminal part.
Compared to our cDNA, the sequence previously identified as the GL1 transcript by Hansen et al. (1997)
A database search for proteins homologous to GL1 with the TBLASTX algorithm revealed several sequences exhibiting high levels of similarity with the query sequence used. In particular, a putative polypeptide of 619 amino acids encoded by a cDNA from rice (Oryza sativa; AK060786) showed 84% identity over its entire coding sequence. Furthermore, significant homologies, with a 67% identity score, were found with the products of two other rice cDNAs (AK066569 and AK070469), with the WAX2 locus of Arabidopsis encoding a protein involved in cuticle synthesis (62% identity), and a partial polypeptide (L33792) derived from Senecio odorus (55% identity). The alignment of the deduced GL1 amino acid sequence and deduced protein sequences exhibiting high similarity scores is depicted in Figure 4. The highest degree of homology consistently regards the C-terminal part of the deduced proteins. A comparison of the deduced GL1 protein sequence and the product of the Arabidopsis ECERIFERUM1 (CER1) locus, a putative aldehyde decarbonylase active in the cuticular wax biosynthesis pathway, reveals an overall identity of 35%. This similarity score was significantly lower than the degree of similarity encountered between the putative GL1 and Arabidopsis WAX2 proteins (62%). Since previous results attributed to the maize GL1 locus a role as an Arabidopsis CER1 ortholog, we investigated amino acid sequence similarities among a restricted group of GL1 homolog sequences by means of phylogenetic analysis (Fig. 5). These analyses suggested the presence of two groups of protein sequences, the former containing the CER1 protein as a founder sequence, the latter including the WAX2 sequence. Interestingly, the GL1 sequence showed a high level of homology with the members of the WAX2 group, while a second maize sequence (GenBank AY104752) was located within the CER1 group with which it shares 55% amino acid identity. Thus, phylogenetic analysis indicated that Gl1-related sequences can be divided into two subgroups, each comprising genes from at least three species: maize, rice, and Arabidopsis (Fig. 5).
GL1 Transcription Analysis The 3' end of the GL1 cDNA was used as a probe in northern-blot experiments performed with total RNA extracted from different tissues of wild-type plants and from leaf tissue homozygous for the gl1-ref allele. As shown in Figure 6A, the RNA extracted from wild-type seedlings showed a transcript with an estimated size of 2,300 residues, in accordance with the expected length of the GL1 mRNA (lane 1). The accumulation of this RNA was dramatically reduced in the gl1-ref mutant (lane 2) and was completely blocked in the root where, instead, a transcript of greater size was detected (lane 3). GL1 expression was evident also in adult leaves (lane 4) and in floral organs (silks and anthers; lanes 5 and 6, respectively), suggesting that GL1 activity was not restricted to the juvenile developmental phase of the maize plant. The same pattern of hybridization was observed using the complete Gl1 cDNA as a probe (data not shown). To check the amount of RNA loading, the filters were stripped and reprobed with a maize cytosolic GAPDH clone (Fig. 6B). The GL1 transcript was further studied by RT-PCR analysis using the samples described above (Fig. 6C). The use of forward and reverse GL1 primers allowed the amplification of a fragment of the expected size from RNA samples obtained from wild-type seedlings (lane 2) and, at low abundance, from gl1-ref mutant leaf (lane 3), mature leaf (lane 5), and anther tissue (lane 7). PCR amplification with primers against cytosolic GAPDH was used to verify the integrity of the samples (Fig. 6D).
As can be seen in Figure 6C, from the root (lane 3) and silk (lane 5) extracts no RNA amplification was obtained by RT-PCR. In this respect, we identified an incomplete Gl1-related clone by screening a silk cDNA library using Gl1 as a probe (H. Hartings, R. Velasco, and M. Motto, unpublished data). This silk cDNA shows 78% identity with Gl1; northern experiments performed with the same samples as those in Figure 6A give a similar hybridization pattern but with a higher intensity in the silk extract (see Supplemental Fig. 7). This was taken as evidence that a Gl1-related gene is expressed mainly in the silk tissue and gives rise to an mRNA cross-hybridizing to the Gl1 probe. As concerns the band in lane 3 of Figure 6A, this might be either a root-specific transcript related in sequence to Gl1 or an unspliced version of Gl1 not amplified by RT-PCR with the conditions used. However, using different combinations of Gl1-specific primers aimed at identifying the presence of intron sequences in the Gl1 transcript, we had no indication of the occurrence of an unspliced version of the Gl1 mRNA in the root extract (data not shown). Accordingly, this band is likely to be the result of unspecific cross-hybridization.
Molecular Cloning and Characterization of the GL1 Locus
To obtain molecular insights into the nature of the genetic lesion that gives rise to the gl1 phenotype, a collection of unstable gl1 mutations induced by autonomous elements of the En/Spm family was generated (Maddaloni et al., 1990
Together, our results indicate that we have cloned the GL1 genomic sequence and the complete coding region of its major transcript. Hansen et al. (1997)
The putative protein encoded by GL1 is 621 amino acids long and is related in length and sequence to those coded by a number of loci from different plant species. These polypeptide sequences display several predicted transmembrane domains in the N-terminal region and a globular domain in the C-terminal part. A common feature shared by these proteins is the presence of eight conserved His motifs in the tripartite domain H-X2–4-H, H-X2–3-H-H, (H/Q)-X2–3-H-H, which form a di-iron-binding site essential for catalytic activity in a large family of integral membrane enzymes, such as acyl desaturases, alkyl-hydroxylases, epoxydases, acetylenases, methyl oxidases, ketolases, and decarbonylases, activities found in prokaryotes and eukaryotes (Shanklin and Cahoon, 1998
Mutations of GL1, CER1, and WAX2 cause dramatic alterations in composition and crystallization patterns of cuticular waxes (Lorenzoni and Salamini, 1975
CER1 was suggested to be an aldehyde decarbonylase because the mutant shows an increase in aldehydes and a reduction of the products of aldehyde decarbonylation, namely, alkane, secondary alcohols, and ketones (Aarts et al., 1995 In wax2 mutants, total wax load is diminished by about 80% because of the reduced accumulation of all the prevalent wax constituents, including aldehydes, with the exception of C30 primary alcohols, which are increased on wax2 stems.
Wax load on gl1 juvenile leaves is reduced by 73% compared to the wild type due to a decreased accumulation of both aldehydes and primary alcohols, while the amount of esters does not change. The mutation has a pronounced effect on the synthesis of long-chain wax compounds (C32), whereas those with shorter acyl chains are less affected or even increased (Bianchi et al., 1977
The Arabidopsis WAX2/YRE gene described by Chen et al. (2003)
Alternatively, different roles on plant development may be ascribed to maize and Arabidopsis cuticles. In addition to wax2, the abnormal leaf shape1 (ale1) and lacerata (lcr) Arabidopsis mutants are altered in cuticle membrane morphology and display postgenital organ fusion (Tanaka et al., 2001
A similar correlation is not observed in maize and other monocots. Maize mutants with adhesion-competent epidermal cells include crinkly4 (cr4) and adherent1 (ad1). The cr4 mutation has a broad effect on epidermal cell morphology, not restricted to the cuticular layer (Becraft et al., 1996
As far as GL1 function is concerned, analysis of the mutant phenotype indicates that GL1, like the other GLOSSY genes, is an essential component of the juvenile wax layer biosynthetic route (Bianchi et al., 1985
From the expression profile experiments, it can be argued that the regulation of the expression of the characterized GLOSSY genes is more complex than predicted only on the basis of seedling phenotypes. GL8 turns out to be expressed in different organs of the adult plant, including the roots, although to a lesser extent than in seedling leaves (Xu et al., 1997
Plant Material
The origin and maintenance of the wx-m7 transposon stock, the gl1-ref allele used in this study, and the recovery of the gl1-mutable strains have been described previously (Maddaloni et al., 1990
The wx-m7 stock also contains copies of active autonomous En/Spm elements (Michel et al., 1995
Maize (Zea mays) DNA was isolated from leaves of flowering plants or from seedlings as described (Michel et al., 1995 Southern analysis was performed to map the position of the En element in seven unstable gl1 alleles. For this purpose, genomic DNA from homozygous mutant plants was digested with the restriction enzymes BamHI, HindIII, ApaI, SstI, SpeI, and KpnI. Blots were probed with four PCR-derived fragments of the GL1 allele covering regions (base positions are indicated relative to the translation start site) from –2,302 to –648 (probe I), from +1,213 to +2,048 (probe II), from +2,426 to +3,581 (probe III), and from +4,131 to +4,745 (probe IV).
HindIII-digested genomic fragments from 7 to 11 kb from plants carrying the gl1-m5 allele were cloned into the Recombinant clones carrying a wild-type GL1 allele were recovered from a maize BAC library derived from DNA extracted from the inbred F2, kindly provided by Dr. Keith Edwards (University of Bristol, UK).
Total RNA was isolated from the following organs and tissues of the inbred Wf9: second and third leaf of wild-type and gl1-ref mutant seedlings, wild-type roots of 1-week-old seedlings, wild-type adult leaf (top leaf, surrounding the tassel), wild-type silks, and wild-type anthers (both immature and pollen-shedding, from the same tassel). Extractions were performed using TRizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For northern experiments, 20 µg of total RNA samples were fractionated on denaturing gels, capillary blotted onto nylon membranes (Hybond N+; Amersham, Little Chalfont, UK), and hybridized at 45°C in Ultrahyb solution (Ambion, Austin, TX). Filters were washed at 55°C in 2x SSC/0.1% SDS (15 min), in 1x SSC/0.1%SDS (30 min), and in 0.1x SSC/0.1% SDS (30 min). RNA markers from 0.2 to 10 kb (Sigma-Aldrich, St. Louis) were used as size standards. Probes were the full-length GL1 cDNA and the 400-bp 3' end, including the complete 3' UTR, of a partial GL1 clone isolated from a seedling cDNA library. To check the amount of RNA loading, filters were rehybridized with a probe derived from maize cytosolic GAPDH cDNA. For RT-PCR, 5 µg of total RNA were reverse transcribed using SuperScriptII reverse transcriptase (Invitrogen) according to manufacturer's instructions. One-twentieth of the final reaction product was amplified by PCR with the following primers: forward, 5'-ATCGAATTCACGTACGGCACAGTTGCTAGC-3'; reverse, 5'-CGCTCTAGACCACCAATTCACACTCGACG-3'. The forward primer annealed to the region of the GL1 cDNA starting 70 bp upstream of the ATG start codon, while the reverse primer annealed to the region starting 101 bp downstream of the stop codon. To avoid formation of secondary structures, PCR reactions were performed in the presence of 10% DMSO (final concentration). The 5' end of the forward and reverse primers included, respectively, EcoRI and XbaI restriction sites (indicated in italics in the above sequences), which were used to subclone the GL1 cDNA from leaves of the inbred Wf9 into the pBluescriptSKII vector (Stratagene, La Jolla, CA) prior to sequencing. Five independent clones were sequenced on both strands to determine the sequence of the GL1 cDNA.
DNA sequencing was carried out with an automatic sequencer (CEQ 8000; Beckman-Coulter, Fullerton, CA). Genomic and cDNA sequences were determined on both strands.
SEM was used to study adaxial surfaces of primary leaves of gl1 mutant and wild-type plants grown for 2 to 3 weeks in a phytochamber at 26°C/19°C (day/night) and 40% humidity with a 16/8-h light/dark rhythm, and a light intensity of 1,900 µE m–2 s–1. Segments of the middle part of the leaf blade were fixed to a specimen holder by tissue tek and shock frozen with liquid nitrogen within a high vacuum cryo preparation stage. Samples were transferred under vacuum to a cryo preparation chamber where they were sputter coated with gold and examined on the cold stage of a Zeiss DSM 940 SEM (Carl Zeiss NTS GmbH, Oberkochen, Germany). For TEM investigation of the cuticle, small pieces (2–3 mm2) of primary leaf blades were fixed for 2 h at room temperature in 2.5% (v/v) glutaraldehyde and 2% (v/v) formaldehyde in 0.05 M phosphate buffer (PB), pH 6.8. After washing in PB, samples were postfixed for 1 h in 2% (v/v) osmium tetroxide in PB, washed again, and dehydrated through a graded series of ethanol. Samples were then infiltrated with LR White resin (Plano, Marburg, Germany) and polymerized for 48 h at 60°C. Ultrathin cross-sections were prepared and mounted on carbon-coated Formvar copper grids (200 mesh; Plano). After staining with 2% uranyl acetate for 2 h, sections were inspected with a Zeiss EM 10 TEM (Carl Zeiss NTS GmbH).
Multiple DNA and protein sequence alignments were performed using ClustalW (Thompson et al., 1994 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY505017 and AY505498.
We thank Dr. Keith Edwards, University of Bristol, UK, for providing a recombinant BAC clone carrying a wild-type GL1 gene. Received December 15, 2004; returned for revision February 9, 2005; accepted February 9, 2005.
1 This work was supported by grants from the Ministero delle Politiche Agricole e Forestali, Rome. 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.058164. * Corresponding author; e-mail motto{at}iscbg.it; fax 39–035–31–60–54.
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Xu X, Dietrich CR, Lessire R, Nikolau BJ, Schnable PS (2002) The endoplasmic reticulum-associated maize GL8 protein is a component of the acyl-coenzyme A elongase involved in the production of cuticular waxes. Plant Physiol 128: 924–934 This article has been cited by other articles:
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ABSTRACT
RESULTS
-ketoacyl reductase of the fatty acid elongase complex involved in wax production. GL2 is apparently involved in acyl chain elongation from C30 to C32. However, comparisons of the predicted GL2 sequence with those in protein databases revealed no similarities to any known fatty acyl synthases, as may be expected for a component of the acyl elongation pathway (Tacke et al., 1995
-hydroxylation in development. Proc Natl Acad Sci USA 98: 9694–9699
