Plant Cuticular Lipid Export Requires an ABC Transporter

All primary aerial organs of land plants are covered with a waxy cuticle that is essential for their protection and interaction with the environment. The cuticle is composed of very-long-chain fatty acids and their derivatives, collectively termed cuticular wax, embedded within and encasing the cutin matrix (1). Cuticle synthesis requires extensive transport of lipids out of the epidermal cells to the plant surface. The mechanism of export of the cuticular lipids is unknown.

To identify mutants defective in lipid transport to the cuticle, we examined a collection of Arabidopsis thaliana eceriferum (or cer) lines for changes in wax-secreting epidermal cells by transmission electron microscopy (TEM). Cer mutants have a glossy, bright green stem phenotype because of a reduction or altered composition of cuticular wax (2). TEM study (3) of the stem epidermis of the cer5 mutant revealed an unusual cellular phenotype. Similar to the wild type, cer5 cells were entirely filled with a central vacuole with the cytoplasm present in a thin rim around the edge of the cell (Fig. 1A), but they also contained large protrusions of cytoplasm into the vacuole (Fig. 1B). Within these protrusions, loose bundles of linear inclusions, distinct from the endoplasmic reticulum, Golgi, and cytoskeletal elements, were found (Fig. 1C). These inclusions were found only in the epidermis; they were never observed in other cell types. Morphologically similar trilamellar inclusions had been described in the cells of patients with X-linked adrenoleukodystrophy (ALD), a neurodegenerative disease caused by a defect in an ABC transporter involved in transport of saturated very-long-chain fatty acids into the peroxisome for β-oxidation (4).

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The cer5 stem epidermis was further examined by cryo–scanning electron microscopy (SEM) (Fig. 1D) (3). The stem surface was sparsely covered with epicuticular wax crystals, consistent with reports that the wax load on cer5 stems is merely reduced, not eliminated (5). The cer5 epidermal cells contained large sheetlike structures, which corresponded in size and arrangement with the rod-shaped inclusion profiles seen in TEM sections (Fig. 1, E and F). Nile red staining and examination by light microscopy demonstrated that these inclusions were lipidic in nature (fig. S1).

Morphological similarities between the cer5 inclusions and those found in ALD cells raised the question of whether both structures had similar composition. Because the cer5 inclusions could not be prepared selectively, we inferred their composition from comparisons between isolated epidermal cells with and without inclusions (3). The total fatty acid profiles of cer5 and wild-type epidermal peels did not differ significantly. Thus, it is unlikely that the cer5 inclusions consist of fatty acids, distinguishing them from the corresponding structures in ALD cells.

When cuticular wax components were quantified (3), wild-type plants showed a wax load of 0.24 μg/mm², whereas the mutant had only 0.11 μg/mm² of wax (Fig. 2A). The amounts of all wax components (e.g., alkanes, ketones, and primary and secondary alcohols) on the cer5 surface were significantly reduced (Fig. 2B). In contrast, the amounts of total epidermal wax (surface plus intracellular) extracted from isolated epidermal peels of wild type (0.31 μg/mm²) and cer5 (0.28 μg/mm²), did not differ significantly. Thus, wax biosynthesis was not compromised in cer5, but wax components were retained within epidermal cells.

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To determine the molecular basis of the cer5 defect, we isolated the CER5 gene by using a combination of positional cloning and insertional mutagenesis (3). Complementation of the cer5 mutant with the wild-type CER5 gene (At1g51500) rescued the wax-deficient phenotype (Fig. 2A). Thus, CER5 is the At1g51500 gene encoding an ABC transporter. During cloning, we identified an additional allele of CER5 in the Salk transfer-DNA (T-DNA) insertional mutation collection (Salk 036776) (Fig. 2A). We designated the original mutant allele cer5-1 and the Salk 036776 allele cer5-2. Sequencing of the At1g51500 gene in cer5-1 identified a point mutation that, in the predicted CER5 protein, would cause the replacement of a glycine with an aspartate within the consensus ABC C motif (6) (fig. S2). The T-DNA insertion in the cer5-2 allele was located in an exon encoding the region after the predicted fifth transmembrane domain of the CER5 protein. We examined CER5 transcript levels in the cer5 mutants to determine the extent of gene disruption in each line. Whereas the abundance of the CER5 transcript in cer5-1 was comparable to the wild type, no transcript could be detected in cer5-2, indicating that it is a transcriptional knockout (fig. S3).

Analysis of the predicted CER5 protein sequence revealed the presence of the characteristic ABC transporter domains near the N terminus, including the Walker A and B boxes and C motif for nucleotide binding and six transmembrane domains (TMD) near the C terminus (fig. S2). When compared with prototype ABC transporters, which have the TMD near the N terminus followed by the ABC domain, the ABC-TMD orientation found in CER5 would be considered a reverse arrangement. Known ABC transporters consist of two (ABC-TMD) units (7) either within one polypeptide or as two “half-transporters” making up homo- or heterodimers. CER5 sequence predicts that it would encode a half-transporter, so presumably CER5 would require dimerization to function.

The CER5 sequence has been designated WBC12, a member of the white-brown complex subfamily, in an analysis of the 129 putative ABC transporters of the Arabidopsis genome (8). This is the largest subfamily of ABC transporters in Arabidopsis, and, although some have been cloned (9), CER5 is the only member of the subfamily that has been characterized functionally. Two other putative Arabidopsis ABC transporters have high similarity to CER5, At3g21090 (WBC 15) and At1g51460 (WBC 13) (10). Furthermore, there is similarity to two human ABC transporters from the WBC/ABCG subfamily: breast cancer resistance protein and a placental ABCG2, which are localized to the plasma membrane (11) and believed to function in lipid and xenobiotic export (12). The simplest hypothesis is that CER5, like other WBC subfamily members, acts as a primary transporter of lipids. However, it cannot be ruled out that it acts indirectly by regulating the activities of other transporters.

CER5 was expressed exclusively in the epidermal cells, as shown by GUS activity assays in plants transformed with the CER5 promoter::GUS construct (Fig. 3A) (3). CER5 transcript was found in all examined plant organs, including stems, leaves, siliques, flowers, and roots (fig. S4). This was unexpected, because the cer5 phenotype is only apparent in stems or detectable by gas chromatography in stems and leaves (85% of wild-type wax load) (5). It suggests that additional transporters must be involved in delivering wax components to the cuticle in other tissues.

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To investigate the subcellular localization of CER5 in Arabidopsis, we introduced a GFP-CER5 (where GFP indicates green fluorescent protein) construct driven by the native CER5 promoter into cer5-1 plants (3). The wild-type phenotype was restored in 42 of 43 transgenic plants expressing the GFP-CER5 fusion protein, indicating that the protein was fully functional. The GFP-tagged CER5 was localized in the plasma membrane of epidermal cells (Fig. 3B). When the cell wall was stained with propidium iodide, the GFP-CER5 plasma membrane fluorescence was clearly separated by the red propidium iodide signal (Fig. 3B, inset).

We identified the plasma membrane-localized ABC transporter, CER5, involved in wax export to the plant cuticle. CER5 must be an important component of the export machinery in the Arabidopsis stem, because disruption of this transporter results in striking accumulations of wax inside the epidermal cells. The absence of a detectable phenotype in tissues other than the stem and leaf and accumulation of residual surface wax on the stem of cer5-2 knockout line suggest that additional wax export mechanisms must exist in plants. Chemical analysis of the mutant wax demonstrated that CER5, like many ABC transporters, has broad substrate specificity and is capable of transporting a variety of wax substrates. We conclude that in plants, as in other eukaryotes, proteins of the WBC/ABCG subfamily are key components of lipid transport systems.