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?     

Focus: Personalized Medicine: Simultaneously Detection of 50 Mutations at 20 Sites in the BRAF and RAS Genes by Multiplexed Single-Nucleotide Primer Extension Assay Using Fine-Needle Aspirates of Thyroid Nodules

Fine-needle aspiration (FNA) is commonly used for primary evaluation of thyroid nodules. Twenty to 30 percent of thyroid nodules remain indeterminate after FNA evaluation. Studies show the BRAF p.V600E to be highly specific for papillary thyroid carcinoma (PTC), while RAS mutations carry up to 88 percent positive predictive value for malignancy. We developed a two-tube multiplexed PCR assay followed by single-nucleotide primer extension assay for simultaneous detection of 50 mutations in the BRAF (p.V600E, p.K601E/Q) and RAS genes (KRAS and NRAS codons 12, 13, 19, 61 and HRAS 61) using FNA smears of thyroid nodules. Forty-two FNAs and 27 paired formalin-fixed, paraffin-embedded (FFPE) tissues were tested. All BRAF p.V600E-positive FNA smears (five) carried a final diagnosis of PTC on resection. RAS mutations were found in benign as well as malignant lesions. Ninety-two percent concordance was observed between FNA and FFPE tissues. In conclusion, our assay is sensitive and reliable for simultaneous detection of multiple BRAF/RAS mutations in FNA smears of thyroid nodules.     

Weight–length relationships of six batoids in the Ecuadorian Pacific

Weight–length relationships (WLR) were estimated for six batoids, namely: Urotrygon chilensis, Narcine entemedor, Rhinobatos leucorhynchus, Rhinobatos planiceps, Rhinobatos prahli and Urobatis tumbesensis captured in the Ecuadorian Pacific. Data were collected between October 2013 and August 2014 in two artisanal fishing ports. In addition, this represents the first WLR estimations for five of the species.     

Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR–Cas9-mediated mutagenesis

Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5′ upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus. Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.