Profiling candidate genes involved in wax biosynthesis in Arabidopsis thaliana by microarray analysis

Plant epidermal wax forms a hydrophobic layer covering aerial plant organs which constitutes a barrier against uncontrolled water loss and biotic stresses. Wax biosynthesis requires the coordinated activity of a large number of enzymes for the formation of saturated very-long-chain fatty acids and their further transformation in several aliphatic compounds. We found in the available database 282 candidate genes that may play a role in wax synthesis, regulation and transport. To identify the most interesting candidates, we measured the level of expression of 204 genes in the aerial parts of 15-day-old Arabidopsis seedlings by performing microarray experiments. We showed that only 25% of the putative candidates were expressed to significant levels in our samples, thus significantly reducing the number of genes which will be worth studying using reverse genetics to demonstrate their involvement in wax accumulation. We identified a beta-keto acyl-CoA synthase gene, At5g43760, which is co-regulated with the wax gene CER6 in a number of conditions and organs. By contrast, we showed that neither the fatty acyl-CoA reductase genes nor the wax synthase genes were expressed in 15-day-old leaves and stems, raising questions about the identity of the enzymes involved in the acyl-reduction pathway that accounts for 20% of the total wax amount.     

Cloning and characterization of CER2, an Arabidopsis gene that affects cuticular wax accumulation.

Cuticular waxes are complex mixtures of very long chain fatty acids and their derivatives that cover plant surfaces. Mutants of the ECERIFERUM2 (cer2) gene of Arabidopsis condition bright green stems and siliques, indicative of the relatively low abundance of the cuticular wax crystals that comprise the wax bloom on wild-type plants. We cloned the CER2 gene via chromosome walking. Three lines of evidence establish that the cloned sequence represents the CER2 gene: (1) this sequence is capable of complementing the cer2 mutant phenotype in transgenic plants; (2) the corresponding DNA sequence isolated from plants homozygous for the cer2-2 mutant allele contains a sequence polymorphism that generates a premature stop codon; and (3) the deduced CER2 protein sequence exhibits sequence similarity to that of a maize gene (glossy2) that also is involved in cuticular wax accumulation. The CER2 gene encodes a novel protein with a predicted mass of 47 kD. We studied the expression pattern of the CER2 gene by in situ hybridization and analysis of transgenic Arabidopsis plants carrying a CER2-beta-glucuronidase gene fusion that includes 1.0 kb immediately upstream of CER2 and 0.2 kb of CER2 coding sequences. These studies demonstrate that the CER2 gene is expressed in an organ- and tissue-specific manner; CER2 is expressed at high levels only in the epidermis of young siliques and stems. This finding is consistent with the visible phenotype associated with mutants of the CER2 gene. Hence, the 1.2-kb fragment of the CER2 gene used to construct the CER2-beta-glucuronidase gene fusion includes all of the genetic information required for the epidermis-specific accumulation of CER2 mRNA.     

RDR1 and SGS3, Components of RNA-Mediated Gene Silencing, Are Required for the Regulation of Cuticular Wax Biosynthesis in Developing Inflorescence Stems of Arabidopsis

The cuticle is a protective layer that coats the primary aerial surfaces of land plants and mediates plant interactions with the environment. It is synthesized by epidermal cells and is composed of a cutin polyester matrix that is embedded and covered with cuticular waxes. Recently, we have discovered a novel regulatory mechanism of cuticular wax biosynthesis that involves the ECERIFERUM7 (CER7) ribonuclease, a core subunit of the exosome. We hypothesized that at the onset of wax production, the CER7 ribonuclease degrades an mRNA specifying a repressor of CER3, a wax biosynthetic gene whose protein product is required for wax formation via the decarbonylation pathway. In the absence of this repressor, CER3 is expressed, leading to wax production. To identify the putative repressor of CER3 and to unravel the mechanism of CER7-mediated regulation of wax production, we performed a screen for suppressors of the cer7 mutant. Our screen resulted in the isolation of components of the RNA-silencing machinery, RNA-DEPENDENT RNA POLYMERASE1 and SUPPRESSOR OF GENE SILENCING3, implicating RNA silencing in the control of cuticular wax deposition during inflorescence stem development in Arabidopsis (Arabidopsis thaliana).     

Leaf Epicuticular Waxes of the Eceriferum Mutants in Arabidopsis

Wild-type Arabidopsis leaf epicuticular wax (EW) occurs as a smooth layer over the epidermal surface, whereas stem EW has a crystalline microstructure. Wild-type EW load was more than 10-fold lower on leaves than on stems. Compared with the EW on wild-type stems, EW on wild-type leaves had a much higher proportion of their total EW load in the form of alkanes and 1-alcohols; a large reduction in secondary alcohols, ketones, and esters; and a chain-length distribution for major EW classes that was skewed toward longer lengths. The eceriferum (cer) mutations often differentially affected leaf and stem EW chemical compositions. For example, the cer2 mutant EW phenotype was expressed on the stem but not on the leaf. Compared to wild type, the amount of primary alcohols on cer9 mutants was reduced on leaves but elevated on stems, whereas an opposite differential effect for primary alcohols was observed on cer16 leaves and stems. Putative functions for CER gene products are discussed. The CER4 and CER6 gene products may be involved in fatty aldehyde reduction and C26 fatty acylcoenzyme A elongation, respectively. CER1, CER8, CER9, and CER16 gene products may be involved in EW substrate transfer. The CER3 gene product may be involved in release of fatty acids from elongase complexes. CER2 gene product may have regulatory functions.     

Molecular characterization of the CER1 gene of arabidopsis involved in epicuticular wax biosynthesis and pollen fertility.

The aerial parts of plants are coated with an epicuticular wax layer, which is important as a first line of defense against external influences. In Arabidopsis, the ECERIFERUM (CER) genes effect different steps of the wax biosynthesis pathway. In this article, we describe the isolation of the CER1 gene, which encodes a novel protein involved in the conversion of long chain aldehydes to alkanes, a key step in was biosynthesis. CER1 was cloned after gene tagging with the heterologous maize transposable element system Enhancer-Inhibitor, also known as Suppressor-mutator. cer1 mutants display glossy green stems and fruits and are conditionally male sterile. The similarity of the CER1 protein with a group of integral membrane enzymes, which process highly hydrophobic molecules, points to a function of the CER1 protein as a decarbonylase.     

Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses

Land plant aerial organs are covered by a hydrophobic layer called the cuticle that serves as a waterproof barrier protecting plants against desiccation, ultraviolet radiation, and pathogens. Cuticle consists of a cutin matrix as well as cuticular waxes in which very-long-chain (VLC) alkanes are the major components, representing up to 70% of the total wax content in Arabidopsis (Arabidopsis thaliana) leaves. However, despite its major involvement in cuticle formation, the alkane-forming pathway is still largely unknown. To address this deficiency, we report here the characterization of the Arabidopsis ECERIFERUM1 (CER1) gene predicted to encode an enzyme involved in alkane biosynthesis. Analysis of CER1 expression showed that CER1 is specifically expressed in the epidermis of aerial organs and coexpressed with other genes of the alkane-forming pathway. Modification of CER1 expression in transgenic plants specifically affects VLC alkane biosynthesis: waxes of TDNA insertional mutant alleles are devoid of VLC alkanes and derivatives, whereas CER1 overexpression dramatically increases the production of the odd-carbon-numbered alkanes together with a substantial accumulation of iso-branched alkanes. We also showed that CER1 expression is induced by osmotic stresses and regulated by abscisic acid. Furthermore, CER1-overexpressing plants showed reduced cuticle permeability together with reduced soil water deficit susceptibility. However, CER1 overexpression increased susceptibility to bacterial and fungal pathogens. Taken together, these results demonstrate that CER1 controls alkane biosynthesis and is highly linked to responses to biotic and abiotic stresses.     

An ABC transporter gene of Arabidopsis thaliana, AtWBC11, is involved in cuticle development and prevention of organ fusion

Cuticle, including wax and cutin, is the barrier covering plant aerial organs and protecting the inner tissues. The Arabidopsis thaliana ATP-binding cassette (ABC) transporter CER5 (AtWBC12) has been identified as a wax exporter. In agreement with the latest report of another wax exporter, AtWBC11, here we show that atwbc11 mutants displayed organ fusions and stunted growth, and became vulnerable to chlorophyll leaching and toluidine blue staining. Chemical analysis showed that wax and cutin monomers were both reduced in the atwbc11 mutant. AtWBC11 was widely expressed in aerial organs. Interestingly, we found that the expression was light dependent, and the phytohormone ABA up-regulated AtWBC11 expression. We also found that while the AtWBC11 promoter had a broad pattern of activity, the expression was converted to epidermis specific when the reporter gene was fused to AtWBC11 cDNA. Furthermore, RNA blot analysis supported epidermis-specific expression of AtWBC11. Our results support that AtWBC11 is involved in cuticle development.     

The cytochrome P450 enzyme CYP96A15 is the midchain alkane hydroxylase responsible for formation of secondary alcohols and ketones in stem cuticular wax of Arabidopsis

Most aerial surfaces of plants are covered by cuticular wax that is synthesized in epidermal cells. The wax mixture on the inflorescence stems of Arabidopsis (Arabidopsis thaliana) is dominated by alkanes, secondary alcohols, and ketones, all thought to be formed sequentially in the decarbonylation pathway of wax biosynthesis. Here, we used a reverse-genetic approach to identify a cytochrome P450 enzyme (CYP96A15) involved in wax biosynthesis and characterized it as a midchain alkane hydroxylase (MAH1). Stem wax of T-DNA insertional mutant alleles was found to be devoid of secondary alcohols and ketones (mah1-1) or to contain much lower levels of these components (mah1-2 and mah1-3) than wild type. All mutant lines also had increased alkane amounts, partially or fully compensating for the loss of other compound classes. In spite of the chemical variation between mutant and wild-type waxes, there were no discernible differences in the epicuticular wax crystals on the stem surfaces. Mutant stem wax phenotypes could be partially rescued by expression of wild-type MAH1 under the control of the native promoter as well as the cauliflower mosaic virus 35S promoter. Cauliflower mosaic virus 35S-driven overexpression of MAH1 led to ectopic accumulation of secondary alcohols and ketones in Arabidopsis leaf wax, where only traces of these compounds are found in the wild type. The newly formed leaf alcohols and ketones had midchain functional groups on or next to the central carbon, thus matching those compounds in wild-type stem wax. Taken together, mutant analyses and ectopic expression of MAH1 in leaves suggest that this enzyme can catalyze the hydroxylation reaction leading from alkanes to secondary alcohols and possibly also a second hydroxylation leading to the corresponding ketones. MAH1 expression was largely restricted to the expanding regions of the inflorescence stems, specifically to the epidermal pavement cells, but not in trichomes and guard cells. MAH1-green fluorescent protein fusion proteins localized to the endoplasmic reticulum, providing evidence that both intermediate and final products of the decarbonylation pathway are generated in this subcellular compartment and must subsequently be delivered to the plasma membrane for export toward the cuticle.     

Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

Plant epidermal cells dedicate more than half of their lipid metabolism to the synthesis of cuticular lipids, which seal and protect the plant shoot. The cuticle is made up of a cutin polymer and waxes, diverse hydrophobic compounds including very-long-chain fatty acids and their derivatives. How such hydrophobic compounds are exported to the cuticle, especially through the hydrophilic plant cell wall, is not known. By performing a reverse genetic screen, we have identified LTPG, a glycosylphosphatidylinositol-anchored lipid transfer protein that is highly expressed in the epidermis during cuticle biosynthesis in Arabidopsis thaliana inflorescence stems. Mutant plant lines with decreased LTPG expression had reduced wax load on the stem surface, showing that LTPG is involved either directly or indirectly in cuticular lipid deposition. In vitro 2-p-toluidinonaphthalene-6-sulfonate assays showed that recombinant LTPG has the capacity to bind to this lipid probe. LTPG was primarily localized to the plasma membrane on all faces of stem epidermal cells in the growing regions of inflorescence stems where wax is actively secreted. These data suggest that LTPG may function as a component of the cuticular lipid export machinery.     

Alterations in CER6, a gene identical to CUT1, differentially affect long-chain lipid content on the surface of pollen and stems

Very long chain lipids contribute to the hydrophobic cuticle on the surface of all land plants and are an essential component of the extracellular pollen coat in the Brassicaceae. Mutations in Arabidopsis CER genes eliminate very long chain lipids from the cuticle surface and, in some cases, from the pollen coat. In Arabidopsis, the loss of pollen coat lipids can disrupt interactions with the stigma, inhibiting pollen hydration and causing sterility. We have positionally cloned CER6 and demonstrate that a wild-type copy complements the cer6-2 defect. In addition, we have identified a fertile, intragenic suppressor, cer6-2R, that partially restores pollen coat lipids but does not rescue the stem wax defect, suggesting an intriguing difference in the requirements for CER6 activity on stems and the pollen coat. Importantly, analysis of this suppressor demonstrates that low amounts of very long chain lipids are sufficient for pollen hydration and germination. The predicted CER6 amino acid sequence resembles that of fatty acid-condensing enzymes, consistent with its role in the production of epicuticular and pollen coat lipids >28 carbons long. DNA sequence analysis revealed the nature of the cer6-1, cer6-2, and cer6-2R mutations, and segregation analysis showed that CER6 is identical to CUT1, a cDNA previously mapped to a different chromosome arm. Instead, we have determined that a new gene, CER60, with a high degree of nucleotide and amino acid similarity to CER6, resides at the original CUT1 locus.     

The YORE-YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis

We have identified a new Arabidopsis mutant, yore-yore (yre), which has small trichomes and glossy stems. Adhesion between epidermal cells was observed in the organs of the yre shoot. The cloned YRE had high homology to plant genes involved in epicuticular wax synthesis, such as ECERIFERUM1 (CER1) and maize GLOSSY1. The phenotype of transgenic plants harboring double-stranded RNA interference (dsRNAi) YRE was quite similar to that of the yre mutant. The amount of epicuticular wax extracted from leaves and stems of yre-1 was approximately one-sixth of that from the wild type. YRE promoter::GUS and in situ hybridization revealed that YRE was specifically expressed in cells of the L1 layer of the shoot apical meristem and young leaves, stems, siliques, and lateral root primordia. Strong expression was detected in developing trichomes. The trichome structure of cer1 was normal, whereas that of the yre cer1 double mutant was heavily deformed, indicating that epicuticular wax is required for normal growth of trichomes. Double mutants of yre and trichome-morphology mutants, glabra2 (gl2) and transparent testa glabra1 (ttg1), showed that the phenotype of the trichome structure was additive, suggesting that the wax-requiring pathway is distinct from the trichome development pathway controlled by GL2 and TTG1.