PerspectivePlant Biology

A Plant ABC Transporter Takes the Lotus Seat

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Science  22 Oct 2004:
Vol. 306, Issue 5696, pp. 622-625
DOI: 10.1126/science.1105227

When plants moved from water to land 450 million years ago, they needed to develop a sealed surface to protect themselves against water loss in the “dry” air environment. To solve this problem, plants invented an epicuticular wax layer that covers the entire surface of the plant that is exposed to air. This protective wax cuticle also serves a multitude of other functions. Its elaborate micro- and nanostructure prevents water and other particles from sticking to the surface of leaves, keeping them clean and so enhancing their ability to trap light for photosynthesis. Adhering water droplets and other particles are washed away in a self-cleaning process called the lotus effect (1). The wax layer also filters out damaging ultraviolet rays, prevents volatile chemicals and pollutants from sticking to leaves and stems, and protects plants against attack by microbes and herbivores.

The plant cuticle is composed of a mixture of cutins and polysaccharides, an intracuticular wax layer, and an epicuticular surface layer of wax crystals (see the figure). The wax layer is formed from wax precursor molecules—very long chain fatty acids (VLCFAs) and their derivatives—that are synthesized by plant epidermal cells. But how is such an elaborate structure constructed on the surface of plants? How do the highly hydrophobic wax precursor molecules get to the construction site outside of the plant cell? And what were the evolutionary steps that led to this innovation? On page 702 of this issue, Pighin et al. (2) provide crucial information on the mechanism by which wax precursor molecules are exported to the plant surface. They show that plant cells use an ABC (ATP-binding cassette) transporter protein similar to the ABC transporters found in mammalian cells for this purpose.

Waxing its own surface.

Different models for the transport of wax precursor molecules (VLCFAs) from their site of synthesis in the endoplasmic reticulum (ER) to their site of deposition on the outer surface of plant cells. VLCFAs (orange) synthesized by ER-localized elongases (pink) may be transported to the cell surface by several routes. (1) They may be picked up by fatty-acid binding proteins (FABPs; green moons) and transported to the ABC transporter CER5 (pale blue) localized in the plasma membrane. CER5 may actively expel VLCFAs into the cell wall space in an ATP-dependent process. (1a) In a variation on this model, VLCFAs could be transported through a side port of CER5 into the upper leaflet of the plasma membrane bilayer. (2) Alternatively, the CER5 transporter may act as a flippase, flipping VLCFAs from the inner to the outer leaflet of the plasma membrane. (1b) In all cases, extracellular lipid-transfer proteins (dark pink moons) will be required to facilitate transport of VLCFAs to destinations outside the cell. Current data (2) are also compatible with vesicular transport of VLCFAs in either (3) oleosome bodies covered by oleosin-like proteins (purple) or (4) uncoated vesicles that contain the VLCFAs in lipid rafts. Available CER5 localization data do not exclude the possibility that CER5 loads intracellular vesicles with VLCFAs by direct transport or through a flippase mechanism.

CREDIT: ILLUSTRATION ADAPTED BY KATHARINE SUTLIFF/SCIENCE

Traditionally, VLCFAs were thought to be exported by a vesicular pathway from their site of synthesis in the endoplasmic reticulum to their destination at the plant surface (see the figure). Sequestration in vesicles would keep these potentially harmful wax precursor molecules in a hydrophobic compartment inside the cell in the same way as plant triglycerides are stored in oleosin-coated vesicular oil bodies (3, 4). Given the difficulty in analyzing the export of wax precursors biochemically, Pighin et al. chose a genetic approach. They exploited a large collection of Arabidopsis mutants with visibly altered wax cuticles on the surface of their inflorescence stems. In these so-called cer or eceriferum (not wax-carrying) mutants (5), VLCFA biosynthesis is affected. Through careful characterization of the cer5 Arabidopsis mutant, Pighin et al. identified an interesting candidate protein for VLCFA export. They show that this CER5 protein is an ABC transporter expressed in plant epidermal cells that, when defective, results in reduced wax on the surface of the cer5 plant stem. Fluorescence imaging revealed that CER5 resides close to or at the plasma membrane of the plant cell. Intriguingly, the total VLCFA content in epidermal cells from mutant and wild-type Arabidopsis is comparable. This can be explained by the intracellular accumulation of VLCFAs in trilamellar inclusions in the mutant plant cells. The inclusions might be modified vesicles that contain large amounts of wax precursor molecules that are either destined for export or need to be stored in a separate compartment to protect cell membranes from accumulating too many VLCFAs.

Members of the ABC transporter family transport a wide variety of substrates including hydrophilic molecules, drugs, and lipids across the membranes of mammalian cells. Some cancer cells are able to survive despite treatment with multiple antitumor drugs because they are induced to express multidrug resistance (MDR) ABC transporters that pump out the drugs faster than they can accumulate inside the cancer cells. Transporters of this kind also export hydrophobic substrates such as platelet-activating factor (PAF), glycerophospholipids, and sphingolipids. The predicted transport activity of the plant CER5 ABC transporter resembles that of three other ABC lipid transporters: MsbA, a recently crystallized bacterial protein involved in export of lipophilic molecules (6); TDG1, which transports fatty acids across the chloroplast envelope (7); and ALDP, an ABC transporter that is mutated in the neurological disease adrenoleukodystrophy. ALDP and its plant counterpart PXA1/comatose (8) are found in peroxisomes, and both are crucial for VLCFA degradation inside these organelles. Mammalian cells expressing the mutant ALDP transporter also exhibit the trilamellar inclusions seen in cer5 mutant plant cells (9).

A seemingly simple hypothesis is that CER5, a half-size ABC transporter, forms a homo- or heterodimeric pore through which VLCFAs are actively transported across the plasma membrane (see the figure). This hypothesis seems to rule out a vesicular pathway of export. Given the near-insolubility of the substrate and the difficulty in setting up an export assay for the CER5 transporter, it is not easy to directly determine transport activity and substrate specificity. But the identification of CER5 does not absolutely rule out a vesicular pathway. Because fluorescence imaging has limited resolution, it remains possible that CER5 is localized in a subapical compartment involved in secretion. Another possibility is that CER5 acts as a “flippase” (6), flipping VLCFAs from the inner to the outer leaflet of the plant cell plasma membrane. Whether bacterial MsbA acts as a pore-like transporter or as a flippase also remains a matter of debate (10, 11). CER5, like MsbA, may have a side port that permits lipids to enter or exit the pore.

No matter how the lipid transporters work, the low solubility of the VLCFAs implicates fatty acid-binding proteins in all of these models. Such proteins would be analogous to serum albumin, which binds fatty acids in serum, and to other fatty acid-binding proteins of the mammalian cell cytosol. These proteins need to be identified to clarify further the export pathway for wax precursors in plants. An orphan gene in the Arabidopsis genome encoding protein At5g58070 and extracellular lipid-transfer proteins (LTPs) are solid candidates. Independent of the actual transport mechanism, the identification of CER5 sheds light on wax secretion in plants and may help to elucidate how the elaborate micro- and nanostructure of the wax layer is constructed. How did land plants invent wax secretion? The genomes of living land plants contain more than 100 ABC transporter genes (12). Because transporters seem to be sloppy with respect to their substrate specificity (13, 14), it is feasible that when plants crept out of the water, they turned a member of the ABC transporter family into a lipid exporter by ensuring that it became localized to a different cellular compartment. Perhaps this is an example of an evolutionary principle in which sloppiness is transformed into flexibility.

Obviously, there is more work to be done to identify other components of the lipid export machinery. We need to define the exact export pathway and its components. The remaining Arabidopsis cer mutants provide an outstanding resource with which to fill in the gaps to obtain a more complete picture. Given that the reduced-wax phenotype of the cer5 mutant is restricted to stems, the transporters involved in wax deposition on leaves and pollen will need to be identified. A comparative analysis of fatty acid transport in bacteria, plants, and animals, although likely to reveal variations as well as commonalities, will cross-fertilize research in these respective fields. Such an analysis will help to answer crucial questions, including whether the fatty acid substrates are free or bound and how the trilamellar inclusions form. The new insight provided by Pighin and colleagues into the ABC lipid transporter of plants has implications beyond understanding the lotus effect—given the multifunctional role of the wax cuticle, the new findings will be a boon to agriculture.

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