We describe a green fluorescent protein (GFP)-based assay for investigating membrane traffic on the secretory pathway in plants. Expression of AtRab1b(N121I), predicted to be a dominant inhibitory mutant of the Arabidopsis Rab GTPase AtRab1b, resulted in accumulation of a secreted GFP marker in an intracellular reticulate compartment reminiscent of the endoplasmic reticulum. This accumulation was alleviated by coexpressing wild-type AtRab1b but not AtRab8c. When a Golgi-targeted and N-glycosylated variant of GFP was coexpressed with AtRab1b(N121I), the variant also accumulated in a reticulate network and an endoglycosidase H-sensitive population appeared. Unexpectedly, expression of AtRab1b(N121I), but not of the wild-type AtRab1b, resulted in a reduction or cessation of vectorial Golgi movement, an effect that was reversed by coexpression of the wild type. We conclude that AtRab1b function is required for transport from the endoplasmic reticulum to the Golgi apparatus and suggest that this process may be coupled to the control of Golgi movement.
A waxy protective cuticle coats all primary aerial plant tissues. Its synthesis requires extensive export of lipids from epidermal cells to the plant surface. Arabidopsis cer5 mutants had reduced stem cuticular wax loads and accumulated sheetlike inclusions in the cytoplasm of wax-secreting cells. These inclusions represented abnormal deposits of cuticular wax and resembled inclusions found in a human disorder caused by a defective peroxisomal adenosine triphosphate binding cassette (ABC) transporter. We found that the CER5 gene encodes an ABC transporter localized in the plasma membrane of epidermal cells and conclude that it is required for wax export to the cuticle.
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 verylong-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 b-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.
Wax esters are neutral lipids composed of aliphatic alcohols and acids, with both moieties usually long-chain (C 16 and C 18 ) or very-long-chain (C 20 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.
In the absence of cell migration, plant architecture is largely determined by the direction and extent of cell expansion during development. In this report, we show that very-long-chain fatty acid (VLCFA) synthesis plays an essential role in cell expansion. The Arabidopsis thaliana eceriferum10 (cer10) mutants exhibit severe morphological abnormalities and reduced size of aerial organs. These mutants are disrupted in the At3g55360 gene, previously identified as a gene coding for enoylCoA reductase (ECR), an enzyme required for VLCFA synthesis. The absence of ECR activity results in a reduction of cuticular wax load and affects VLCFA composition of seed triacylglycerols and sphingolipids, demonstrating in planta that ECR is involved in all VLCFA elongation reactions in Arabidopsis. Epidermal and seed-specific silencing of ECR activity resulted in a reduction of cuticular wax load and the VLCFA content of seed triacylglycerols, respectively, with no effects on plant morphogenesis, suggesting that the developmental phenotypes arise from abnormal sphingolipid composition. Cellular analysis revealed aberrant endocytic membrane traffic and defective cell expansion underlying the morphological defects of cer10 mutants.
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