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.     

The Arabidopsis cer26 mutant,like the cer2 mutant,is specifically affected in the very long chain fatty acid elongation process

Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl‐CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2‐like family: CER2, CER26 and CER26‐like . Expression pattern analysis showed that the three genes are differentially expressed in an organ‐ and tissue‐specific manner. Using individual T–DNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl‐CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity.     

Isolation and characterization of eceriferum (cer) mutants induced by T-DNA insertions in Arabidopsis thaliana

Thirteen Arabidopsis thaliana mutants with deviating epicuticular wax layers (i.e., cer mutants) were isolated by screening 13 000 transformed lines produced by the seed transformation method. After crossing the 13 mutants to some of the previously known cer mutant lines, 12 of our mutants mapped to 6 of the 21 known complementation groups (cer1 through cer4 as well as cer6 and cer10), while the other mutant corresponded to a previously unknown locus, cer21. Mutant phenotypes of 6 of the 13 mutant lines were caused by T-DNA insertions within cer genes. We also analyzed the chemical composition of the epicuticular wax layers of the cer mutants isolated in this study relative to that of Arabidopsis wild-type plants. Our results suggest that the five genes we tagged regulate different steps in wax biosynthesis, i.e., the decarbonylation of fatty aldehydes to alkanes, the elongation of hexacosanoic acid to octacosanoic acid, the reduction of fatty aldehydes to primary alcohols and the production of free aldehydes, while an insertion in the fifth gene causes an alteration in the chain length distribution of the different classes of wax compounds.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Composition of alkyl esters in the cuticular wax on inflorescence stems of Arabidopsis thaliana cer mutants

Wax biosynthetic pathways proceed via the elongation of 16:0 acyl-CoA to very long-chain fatty acids (VLCFA), and by further modifications that include reduction to primary alcohols and formation of alkyl esters. We have analyzed the alkyl esters in the stem wax of ten cer mutants of Arabidopsis thaliana together with the corresponding wild types. Alkyl esters with chain lengths between C(38) and C(52) were identified, and the levels of esters ranged from 0.15 microg cm(-2) in Wassilewskija (WS) to 1.20 microg cm(-2) in cer2. Esters with even numbers of carbons prevailed, with C(42), C(44) and C(46) favoured in the wild types, a predominance of C(42) in cer2 and cer6 mutants, and a relative shift towards C(46) in cer3 and cer23 mutants. The esters of all mutants and wild types were dominated by 16:0 acyl moieties, whereas the chain lengths of esterified alcohols were between C(20) and C(32). The alkyl chain-length distributions of the wild-type esters had a maximum for C(28) alcohol, similar to the free alcohols accompanying them in the wax mixtures. The esterified alcohols of cer2, cer6 and cer9 had largely increased levels of C(26) alcohol, closely matching the patterns of the corresponding free alcohols and, therefore, differing drastically from the corresponding wild type. In contrast, cer1, cer3, cer10, cer13 and cer22 showed ester alcohol patterns with increased levels of C(30), only partially following the shift in chain lengths of the free alcohols in stem wax. These results provide information on the composition of substrate pools and/or the specificity of the ester synthase involved in wax ester formation. We conclude that alcohol levels at the site of biosynthesis are mainly limiting the ester formation in the Arabidopsis wild-type epidermis.     

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.     

Leaf sheath cuticular waxes on bloomless and sparse-bloom mutants of Sorghum bicolor

Leaf sheath cuticular waxes on wild-type Sorghum bicolor were approximately 96% free fatty acids, with the C28 and C30 acids being 77 and 20% of these acids, respectively. Twelve mutants with markedly reduced wax load were characterized for chemical composition. In all of the 12 mutants, reduction in the amount of C28 and C30 acids accounted for essentially all of the reduction in total wax load relative to wildtype. The bm2 mutation caused a 99% reduction in total waxes. The bm4, bm5, bm6, bm7 and h10 mutations caused more than 91% reduction in total waxes, whereas the remaining six mutants, bm9, bm11, h7, h11, h12 and h13, caused between 35 and 78% reduction in total wax load. Relative to wild-type, bm4 caused a large increase in the absolute amount of C22, C24 and C26 acids, and reduction in the C28 and longer acids, suggesting that bm4 may suppress elongation of C26, acyl-CoA primarily. The h10 mutation increased the absolute amounts of the longest chain length acids, but reduced shorter acids, suggesting that h10 may suppress termination of acyl-CoA elongation. The bm6, bm9, bm11, h7, h11, h12 and h13 mutations increased the relative amounts, but not absolute amounts, of longer chain acids. Based on chemical composition alone, it is still uncertain which genes and their products were altered by these mutations. Nevertheless, these Sorghum cuticular wax mutants should provide a valuable resource for future studies to elucidate gene involvement in the biosynthesis of cuticular waxes, in particular, the very-long-chain fatty acids.     

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.     

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.     

Arabidopsis ECERIFERUM2 Is a Component of the Fatty Acid Elongation Machinery Required for Fatty Acid Extension to Exceptional Lengths

Primary aerial surfaces of land plants are coated by a lipidic cuticle, which forms a barrier against transpirational water loss and protects the plant from diverse stresses. Four enzymes of a fatty acid elongase complex are required for the synthesis of very-long-chain fatty acid (VLCFA) precursors of cuticular waxes. Fatty acid elongase substrate specificity is determined by a condensing enzyme that catalyzes the first reaction carried out by the complex. In Arabidopsis (Arabidopsis thaliana), characterized condensing enzymes involved in wax synthesis can only elongate VLCFAs up to 28 carbons (C28) in length, despite the predominance of C29 to C31 monomers in Arabidopsis stem wax. This suggests additional proteins are required for elongation beyond C28. The wax-deficient mutant eceriferum2 (cer2) lacks waxes longer than C28, implying that CER2, a putative BAHD acyltransferase, is required for C28 elongation. Here, we characterize the cer2 mutant and demonstrate that green fluorescent protein-tagged CER2 localizes to the endoplasmic reticulum, the site of VLCFA biosynthesis. We use site-directed mutagenesis to show that the classification of CER2 as a BAHD acyltransferase based on sequence homology does not fit with CER2 catalytic activity. Finally, we provide evidence for the function of CER2 in C28 elongation by an assay in yeast (Saccharomyces cerevisiae).Land plants have a lipidic cuticle that seals the outer surface of all of their primary aerial organs. Structurally, the cuticle consists of two components, cutin and cuticular waxes. Together these form a hydrophobic barrier that plays a critical role in plant survival by restricting nonstomatal water loss (Riederer and Schreiber, 2001). Cuticles also protect the plant from biotic and abiotic stresses, profoundly affect plant-insect interactions (Müller, 2006), prevent epidermal fusions (Sieber et al., 2000), and are involved in drought stress signaling (Wang et al., 2011).Cutin is a polymer of mainly midchain- and ω-hydroxy and -epoxy 16 carbon (C16) and C18 fatty acids, which are cross-linked in ester bonds directly or through a glycerol backbone (Pollard et al., 2008). Cuticular waxes are aliphatic monomers that are deposited within the cutin matrix as intracuticular wax, and on top of it as epicuticular wax film and crystals. Wax is a heterogeneous mixture of very-long-chain fatty acids (VLCFAs) and their alkane, aldehyde, alcohol, ketone, and ester derivatives, which typically range from C24 to C32 in length (Samuels et al., 2008). The composition of cuticular wax varies greatly among species and tissues, often providing physical and chemical properties to the plant surface that are advantageous in specific environments.Genetic analyses have revealed that a fatty acid elongase (FAE) complex is responsible for the synthesis of VLCFA wax precursors (Millar et al., 1999; Fiebig et al., 2000; Kunst and Samuels, 2009). FAE complexes are heterotetramers of independently transcribed, monofunctional proteins localized to the endoplasmic reticulum (ER). Together, they catalyze a series of four reactions to elongate long-chain acyl-CoAs or very-long-chain acyl-CoAs by sequential addition of two carbon units. The condensing enzyme, or β-ketoacyl-CoA synthase (KCS), catalyzes the first reaction in this sequence and is both rate limiting and specific for the chain length of acyl-CoA synthesized (Millar and Kunst, 1997). Two very dissimilar families of KCSs have been identified in Arabidopsis (Arabidopsis thaliana): a FAE1-type family homologous to the first such KCS enzyme discovered in association with seed oil biosynthesis (Kunst et al., 1992; James et al., 1995; Lassner et al., 1996), and an ELONGATION DEFECTIVE (ELO)-like family homologous to the yeast (Saccharomyces cerevisiae) ELO family responsible for sphingolipid synthesis (Dunn et al., 2004). To date, no function has been ascribed to Arabidopsis ELOs. Of the 21 FAE1-type KCS enzymes in Arabidopsis (Joubès et al., 2008), 11 have been shown by microarray analysis to be up-regulated in the stem epidermis (Suh et al., 2005). Only one of these, ECERIFERUM6 (CER6/KCS6/CUT1; Millar et al., 1999; Fiebig et al., 2000; Joubès et al., 2008), has a dominant role in the elongation of VLCFAs for cuticular wax synthesis, as CER6 suppression results in a dramatic reduction of all wax monomers longer than C24 (Millar et al., 1999). Heterologous expression of CER6 in yeast has demonstrated that the CER6 condensing enzyme can produce C28 VLCFAs (O. Rowland and L. Kunst, unpublished data). However, CER6 appears to be unable to produce VLCFAs longer than C28 in yeast; this presents a problem as the bulk of Arabidopsis stem wax is made up of C29 alkanes, secondary alcohols, and ketones derived from C30 VLCFAs. Mutant screens have not revealed any other KCS enzymes necessary for VLCFA elongation past C28 in Arabidopsis. Therefore, there may be other proteins unrelated to condensing enzymes that are required for acyl chain extension beyond C28 that remain unknown.The wax-deficient mutant cer2 shows a dramatic reduction in all stem waxes longer than C28 and increased accumulation of waxes C28 or shorter, suggesting that CER2 has a role in the final steps of VLCFA elongation. Surprisingly, the cer2 mutation has been mapped to At4g24510 (Negruk et al., 1996; Xia et al., 1996), a gene homologous to plant BAHD acyltransferases. However, the CER2 protein was reported to localize exclusively to the nucleus (Xia et al., 1997). This does not fit with CER2 annotation as a BAHD acyltransferase, as all characterized BAHD acyltransferases are soluble cytosolic enzymes (D’Auria, 2006).The objective of this work was to more precisely evaluate the role of CER2 in fatty acid elongation using a new CER2 allele, cer2-5 (Columbia-0 [Col-0] ecotype). We provide evidence that CER2 has a metabolic function specific to wax synthesis, and that the CER2 homolog CER2-LIKE1 has an analogous role in leaf wax synthesis. Despite the classification of CER2 as a BAHD acyltransferase based on sequence homology, we demonstrate that CER2 cannot share the catalytic mechanism that has been confirmed for other members of the BAHD family, and provide biochemical support for a function of CER2 in VLCFA elongation by an assay in yeast.     

ECERIFERUM2-LIKE Proteins Have Unique Biochemical and Physiological Functions in Very-Long-Chain Fatty Acid Elongation

The extension of very-long-chain fatty acids (VLCFAs) for the synthesis of specialized apoplastic lipids requires unique biochemical machinery. Condensing enzymes catalyze the first reaction in fatty acid elongation and determine the chain length of fatty acids accepted and produced by the fatty acid elongation complex. Although necessary for the elongation of all VLCFAs, known condensing enzymes cannot efficiently synthesize VLCFAs longer than 28 carbons, despite the prevalence of C28 to C34 acyl lipids in cuticular wax and the pollen coat. The eceriferum2 (cer2) mutant of Arabidopsis (Arabidopsis thaliana) was previously shown to have a specific deficiency in cuticular waxes longer than 28 carbons, and heterologous expression of CER2 in yeast (Saccharomyces cerevisiae) demonstrated that it can modify the acyl chain length produced by a condensing enzyme from 28 to 30 carbon atoms. Here, we report the physiological functions and biochemical specificities of the CER2 homologs CER2-LIKE1 and CER2-LIKE2 by mutant analysis and heterologous expression in yeast. We demonstrate that all three CER2-LIKEs function with the same small subset of condensing enzymes, and that they have different effects on the substrate specificity of the same condensing enzyme. Finally, we show that the changes in acyl chain length caused by each CER2-LIKE protein are of substantial importance for cuticle formation and pollen coat function.The extension of fatty acids to lengths greater than 28 carbons (C28) is an exceptional process in plant metabolism in that it requires unique biochemical machinery, and the elongation products are used for the synthesis of specialized plant metabolites. Derivatives of C30 to C34 fatty acids make up the bulk of plant cuticular wax, which coats all of a plant’s primary aerial surfaces. Cuticular wax serves as a barrier against transpirational water loss (Riederer and Schreiber, 2001) and protects the plant from both biotic (Eigenbrode, 1996) and abiotic (Grace and van Gardingen, 1996) stresses. C30 to C34 fatty acid-derived lipids are also components of the pollen coat, where they function in pollen hydration and germination on dry stigma (Elleman et al., 1992; Preuss et al., 1993).The core complex that elongates long-chain fatty acids (C16–C18) to very-long-chain fatty acids (VLCFAs; C20–C34) consists of four interacting proteins localized to the endoplasmic reticulum (ER). β-Keto-acyl-CoA synthases (KCSs), also known as condensing enzymes, catalyze the first reaction required for VLCFA elongation, condensing malonyl-CoA with an acyl-CoA (n) to produce a β-keto-acyl-CoA (n + 2). Condensation is both a specific and rate-limiting step in elongation (Millar and Kunst, 1997). Chain length specificity of KCSs is of particular importance because VLCFA length determines the downstream use of the fatty acid (for review, see Joubès et al., 2008; Haslam and Kunst, 2013a). There are two families of condensing enzymes in Arabidopsis (Arabidopsis thaliana). The ELONGATION-DEFECTIVE (ELO)-LIKE family is homologous to yeast (Saccharomyces cerevisiae) ELOs, and has putative functions in sphingolipid biosynthesis (Quist et al., 2009). Although our current understanding of plant ELO-LIKE physiological function and biochemical activity is limited, the mechanism of yeast Elo protein activity has been thoroughly investigated (Denic and Weissman, 2007). The FATTY ACID ELONGATION1 (FAE1)-type family is homologous to the first condensing enzyme identified in Arabidopsis, which is required for the synthesis of C20 to C22 VLCFAs in Arabidopsis oilseeds. Many of the 21 FAE1-type condensing enzymes of Arabidopsis have been characterized using reverse genetics and heterologous expression in yeast (Trenkamp et al., 2004; Blacklock and Jaworski, 2006; Paul et al., 2006; Tresch et al., 2012). This work has revealed the intriguing caveat that, although FAE1-type KCSs are involved in the synthesis of diverse downstream metabolites and use a broad range of acyl chain lengths, none are able to efficiently elongate VLCFAs beyond C28 (for review, see Haslam and Kunst, 2013a), which is essential for the production of cuticular wax components.Eceriferum2 (cer2) and glossy2 (gl2) mutants of Arabidopsis and Zea mays, respectively, are deficient in specific VLCFA-derived waxes longer than C28 (Bianchi et al., 1975; McNevin et al., 1993; Jenks et al., 1995). Both mutations were mapped to genes that do not resemble any component of the elongase complex (Tacke et al., 1995; Xia et al., 1996), but are homologous to the BAHD family of acyltransferases (St-Pierre et al., 1998). However, site-directed mutagenesis of conserved acyltransferase catalytic site amino acids in CER2 revealed that this motif is not required for CER2 function in cuticular wax synthesis (Haslam et al., 2012).CER6 is a condensing enzyme necessary for the accumulation of stem cuticular waxes in Arabidopsis, but when expressed in yeast, CER6 can only elongate VLCFAs to C28. When CER2 is expressed in yeast, it has no elongation activity. However, coexpression of CER2 and CER6 results in efficient production of C30 VLCFAs. Coexpression of CER2 with LfKCS45, a condensing enzyme from the crucifer Lesquerella fendleri that generates C28 and a small amount of C30 VLCFAs (Moon et al., 2004), does not alter product chain length (Haslam et al., 2012). Based on these observations, it was hypothesized that CER2 modifies the chain length specificity of the core elongase complex by interaction with specific KCS enzymes (Haslam et al., 2012).CER2 homologs are found in diverse flowering plant lineages, and many species have multiple CER2 homologs (Tuominen et al., 2011). A BLAST search of proteins from Arabidopsis identified two sequences with substantial similarity to CER2. {"type":"entrez-protein","attrs":{"text":"NP_193120","term_id":"15236357","term_text":"NP_193120"}}NP_193120 is 36% identical to CER2, and is encoded by the gene At4g13840. We named this gene CER2-LIKE1 (also known as CER26) (Pascal et al., 2013). {"type":"entrez-protein","attrs":{"text":"NP_566741","term_id":"18403923","term_text":"NP_566741"}}NP_566741 is 38% identical to CER2, and is encoded by the gene At3g23840. We named this gene CER2-LIKE2 (also named CER26-LIKE) (Pascal et al., 2013). Characterization of a cer2-like1 null mutant revealed a role for the CER2-LIKE1 protein in the elongation of leaf wax precursors beyond C30, analogous to the role of CER2 in C28 elongation in stems (Haslam et al., 2012; Pascal et al., 2013). cer2 cer2-like1 double mutants are deficient in the formation of wax components longer than C28 in both stems and leaves. As the cer2 single mutant has no leaf wax phenotype, the additive effect of these two mutations on leaf wax composition indicates that there is partial functional redundancy between the two genes.A comprehensive investigation of the biochemical and physiological functions of CER2-LIKE proteins is necessary. Beyond the value of knowing the specific roles of each homolog, such an investigation has potential to elucidate the nature of CER2-LIKE protein function. With this objective, we used our data to address the following questions: (1) Do CER2-LIKE proteins function with CER6 alone, or can they modify the activity of other FAE1-type condensing enzymes? (2) Do CER2-LIKE proteins have different effects on the substrate specificity of the same condensing enzyme, or is substrate specificity determined exclusively by the condensing enzyme? (3) What is the physiological relevance of the subtle changes in acyl lipid chain length that CER2-LIKE proteins induce?     

Disruptions of the Arabidopsis Enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis

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 enoyl-CoA 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.     

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).     

Cytological and biochemical analysis of COF1, an Arabidopsis mutant of an ABC transporter gene

In transposon-tagged lines of Arabidopsis we found two new mutants, cof1-1 and cof1-2 (cuticular defect and organ fusion), that show the phenotype of wilting when grown in soil, organ fusion of rosette leaves and infertility. Toluidine blue testing and scanning electron microscopy observation revealed that these mutants had cuticular defects in the stems and adult leaves, but not in cotyledones. Transmission electron microscopy observation revealed thinner cuticle layers in the mutants, and cuticular materials interspersed between the two fused epidermal layers were observed in the mutant rosette leaves. These two mutants had a transposon insertion in the coding regions of WBC11, which was classified as a member of ABC transporter genes in Arabidopsis. WBC11 showed high sequence similarity to CER5 (also called WBC12), which was involved in cuticular lipid export. Gas chromatographic analysis revealed that C29 alkane extracted from the stem surface of cof1 mutants was reduced whereas C29 ketone was accumulated, which was different from the case of cer5 mutants. While cer5 mutants had fairly normal morphology, cof1 mutants had pleiotropic phenotypes so that COF1/WBC11 could have important roles different from those of CER5/WBC12. We also found that C29 alkane was accumulated in the intracellular extract of cof1 mutants, suggesting a function for WBC11 in the direct transport of lipid molecules. Pollen observation showed that mutant pollen grains were irregularly shaped. The function of COF1/WBC11 in lipid transport for the construction of cuticle layers and pollen coats for normal organ formation is discussed.     

The CER3 wax biosynthetic gene from Arabidopsis thaliana is allelic to WAX2/YRE/FLP1

The cuticle coats the aerial organs of land plants and is composed of a cutin matrix embedded and overlayed with waxes. The Arabidopsis CER3 gene is important for cuticular wax biosynthesis and was reported to correspond to At5g02310 encoding an E3 ubiquitin ligase. Here, we demonstrate that CER3 is not At5g02310 and instead corresponds to WAX2/YRE/FLP1 (At5g57800), a gene of unknown function required for wax biosynthesis. CER3 protein has also been implicated in cutin production because strong cer3 alleles display organ fusions. Leaf cutin analysis of two cer3 alleles did not reveal significant differences in cutin load or composition, indicating that CER3 has no major role in leaf cutin formation.     

Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids

Whereas the physiological significance of microsomal fatty acid elongation is generally appreciated, its molecular nature is poorly understood. Here, we describe tissue-specific regulation of a novel mouse gene family encoding components implicated in the synthesis of very long chain fatty acids. The Ssc1 gene appears to be ubiquitously expressed, whereas Ssc2 and Cig30 show a restricted expression pattern. Their translation products are all integral membrane proteins with five putative transmembrane domains. By complementing the homologous yeast mutants, we found that Ssc1 could rescue normal sphingolipid synthesis in the sur4/elo3 mutant lacking the ability to synthesize cerotic acid (C(26:0)). Similarly, Cig30 reverted the phenotype of the fen1/elo2 mutant that has reduced levels of fatty acids in the C(20)-C(24) range. Further, we show that Ssc1 mRNA levels were markedly decreased in the brains of myelin-deficient mouse mutants known to have very low fatty acid chain elongation activity. Conversely, the dramatic induction of Cig30 expression during brown fat recruitment coincided with elevated elongation activity. Our results strongly implicate this new mammalian gene family in tissue-specific synthesis of very long chain fatty acids and sphingolipids.