Very-long-chain Fatty Acids Biosynthesis Research Articles

Molecular and Functional Characterization of Elovl4 Genes in Sparus aurata and Solea senegalensis Pointing to a Critical Role in Very Long-Chain (>C24) Fatty Acid Synthesis during Early Neural Development of Fish.

Very long-chain fatty acids (VLC-FA) play critical roles in neural tissues during the early development of vertebrates. However, studies on VLC-FA in fish are scarce. The biosynthesis of VLC-FA is mediated by elongation of very long-chain fatty acid 4 (Elovl4) proteins and, consequently, the complement and activity of these enzymes determines the capacity that a given species has for satisfying its physiological demands, in particular for the correct development of neurophysiological functions. The present study aimed to characterize and localize the expression of elovl4 genes from Sparus aurata and Solea senegalensis, as well as to determine the function of their encoded proteins. The results confirmed that both fish possess two distinct elovl4 genes, named elovl4a and elovl4b. Functional assays demonstrated that both Elovl4 isoforms had the capability to elongate long-chain (C20–24), both saturated (SFA) and polyunsaturated (PUFA), fatty acid precursors to VLC-FA. In spite of their overlapping activity, Elovl4a was more active in VLC-SFA elongation, while Elovl4b had a preponderant elongation activity towards n-3 PUFA substrates, particularly in S. aurata, being additionally the only isoform that is capable of elongating docosahexaenoic acid (DHA). A preferential expression of elovl4 genes was measured in neural tissues, being elovl4a and elovl4b mRNAs mostly found in brain and eyes, respectively.

Genome-Wide Identification of Peanut KCS Genes Reveals That AhKCS1 and AhKCS28 Are Involved in Regulating VLCFA Contents in Seeds

The peanut (Arachis hypogaea L.) is an important oilseed crop worldwide. Compared to other common edible vegetable oils, peanut oil contains a higher content of saturated fatty acids (SFAs), approximately 20–40% of which are very long chain fatty acids (VLCFAs). To understand the basis for this oil profile, we interrogated genes for peanut β-ketoacyl-CoA synthase (KCS), which is known to be a key enzyme in VLCFA biosynthesis. A total of 30 AhKCS genes were identified in the assembled genome of the peanut. Based on transcriptome data, nine AhKCS genes with high expression levels in developing seeds were cloned and expressed in yeast. All these AhKCSs could produce VLCFAs but result in different profiles, indicating that the AhKCSs catalyzed fatty acid elongation with different substrate specificities. Expression level analysis of these nine AhKCS genes was performed in developing seeds from six peanut germplasm lines with different VLCFA contents. Among these genes, the expression levels of AhKCS1 or AhKCS28 were, 4–10-fold higher than that of any other AhKCS. However, only the expression levels of AhKCS1 and AhKCS28 were significantly and positively correlated with the VLCFA content, suggesting that AhKCS1 and AhKCS28 were involved in the regulation of VLCFA content in the peanut seed. Further subcellular localization analysis indicated that AhKCS1 and AhKCS28 were located at the endoplasmic reticulum (ER). Overexpression of AhKCS1 or AhKCS28 in Arabidopsis increased the contents of VLCFAs in the seed, especially for very long chain saturated fatty acids (VLCSFAs). Taken together, this study suggests that AhKCS1 and AhKCS28 could be key genes in regulating VLCFA biosynthesis in the seed, which could be applied to improve the health-promoting and nutritional qualities of the peanut.

Genome-wide mining and comparative analysis of fatty acid elongase gene family in Brassica napus and its progenitors

Very long chain fatty acids (VLCFAs) that are structural components of cell membrane lipid, cuticular waxes and seed oil, play crucial roles in plant growth, development and stress response. Fatty acid elongases (FAEs) comprising KCS and ELO, are key enzymes for VLCFA biosynthesis in plants. Although reference genomes of Brassica napus and its parental speices both have been sequenced, whole-genome analysis of FAE gene family in these Brassica speices is not reported. Here, 58, 33 and 30 KCS genes were identified in B. napus, B. rapa and B. oleracea genomes, respectively, whereas 14, 6 and 8 members were obtained for ELO genes. These KCS genes were unevenly located in 37 chromosomes and 3 scaffolds of 3 Brassica species, while these ELO genes were mapped to 19 chromosomes. The KCS and ELO proteins were divided into 8 and 4 subclasses, respectively. Gene structure and protein motifs remained highly conserved in each KCS or ELO subclass. Most promoters of KCS and ELO genes harbored various plant growth-, phytohormone-, and stress response-related cis-acting elements. 20 SSR loci existed in the KCS and ELO genes/promoters. The whole-genome duplication and segmental duplication mainly contributed to expansion of KCS and ELO genes in these genomes. Transcriptome analysis showed that KCS and ELO genes in 3 Brassica species were expressed in various tissues/organs with different levels, whereas 1 BnELO gene and 6 BnKCS genes might be pathogen-responsive genes. The qRT-PCR assay showed that BnKCS22 and BnELO04 responded to various phytohormone treatments and abiotic stresses. This work lays the foundation for further function identification of KCS and ELO genes in B. napus and its progenitors.

GFP Scaffold-Based Engineering for the Production of Unbranched Very Long Chain Fatty Acids in Escherichia coli With Oleic Acid and Cerulenin Supplementation.

Currently, very long chain fatty acids (VLCFAs) for oleochemical, pharmaceutical, cosmetic, or food applications are extracted from plant or marine organism resources, which is associated with a negative environmental impact. Therefore, there is an industrial demand to develop sustainable, microbial resources. Due to its ease of genetic modification and well-characterized metabolism, Escherichia coli has established itself as a model organism to study and tailor microbial fatty acid biosynthesis using a concerted genetic engineering approach. In this study, we systematically implemented a plant-derived (Arabidopsis thaliana) enzymatic cascade in Escherichia coli to enable unbranched VLCFA biosynthesis. The four Arabidopsis thaliana membrane-bound VLCFA enzymes were expressed using a synthetic expression cassette. To facilitate enzyme solubilization and interaction of the synthetic VLCFA synthase complex, we applied a self-assembly GFP scaffold. In order to initiate VLCFA biosynthesis, external oleic acid and cerulenin were supplemented to cultures. In this context, we detected the generation of arachidic (20:0), cis-11-eicosenoic (20:1) and cis-13-eicosenoic acid (20:1).

AKR2A participates in the regulation of cotton fibre development by modulating biosynthesis of very-long-chain fatty acids.

SummaryThe biosynthesis of very‐long‐chain fatty acids (VLCFAs) and their transport are required for fibre development. However, whether other regulatory factors are involved in this process is unknown. We report here that overexpression of an Arabidopsis gene ankyrin repeat‐containing protein 2A (AKR2A) in cotton promotes fibre elongation. RNA‐Seq analysis was employed to elucidate the mechanisms of AKR2A in regulating cotton fibre development. The VLCFA content and the ratio of VLCFAs to short‐chain fatty acids increased in AKR2A transgenic lines. In addition, AKR2A promotes fibre elongation by regulating ethylene and synergizing with the accumulation of auxin and hydrogen peroxide. Analysis of RNA‐Seq data indicates that AKR2A up‐regulates transcript levels of genes involved in VLCFAs’ biosynthesis, ethylene biosynthesis, auxin and hydrogen peroxide signalling, cell wall and cytoskeletal organization. Furthermore, AKR2A interacted with KCS1 in Arabidopsis both in vitro and in vivo. Moreover, the VLCFA content and the ratio of VLCFAs to short‐chain fatty acids increased significantly in seeds of AKR2A‐overexpressing lines and AKR2A/KCS1 co‐overexpressing lines, while AKR2A mutants are the opposite trend. Our results uncover a novel cotton fibre growth mechanism by which the critical regulator AKR2A promotes fibre development via activating hormone signalling cascade by mediating VLCFA biosynthesis. This study provides a potential candidate gene for improving fibre yield and quality through genetic engineering.

Very Long-Chain Fatty Acids in Composition of Plant Membrane Lipids

Literary data on very long-chain fatty acids (VLCFAs) that are present in polar lipids of the plant cell membranes are discussed. Large amounts of VLCFA are found in polar lipids of some cellular organelles as well as in nonextractable lipids from diverse plant objects, where the influence of surface lipids on the relative content of these FAs is excluded. In some plants, the VLCFA fraction in membrane lipids increases under several kinds of stress. Amounts and diversity of VLCFAs are lower in flowering plants as compared with the representatives of more ancient taxons—gymnosperms, ferns, and marine algae. Presence of VLCFAs in the composition of annular lipids of the cell membranes is assumed. Biosynthesis of VLCFAs, enzymes involved in the process, and encoding genes are discussed.

Very-long-chain fatty acid diversity in nine Heliophila seed oils

Heliophila is a genus of approximately 90 recognized plant species endemic to southern Africa and considered the most morphologically diverse genus in the family Brassicaceae. To begin to characterize chemical diversity in the genus, seed oil content and fatty acid composition were determined for 9 species, with representatives from the three major recognized clades. All species produced very-long-chain fatty acids (VLCFAs) as major components of the seed triacylglycerol (TAG) oil, with considerable diversity of fatty acid chain length. The 22-carbon (C22) monounsaturated fatty acid erucic acid (cis-13-docosenoic acid) was the dominant VLCFA in the oil of Heliophila coronopifolia at 47.6% of total seed fatty acids whereas the unusual C26 fatty acid ximenic acid (cis-17-hexacosenoic acid) dominated the VLCFA profile of oil from Heliophila scoparia at 35.8%. Heliophila africana oil was identified as a potential novel source of the nutraceutical fatty acid, nervonic acid (cis-15-tetracosenoic acid). Analysis of the oil of 3 species by sn-1,3 regioselective lipase digestion demonstrated the virtual exclusion of VLCFAs from the sn-2 position of TAG. Examination of the oil from H. scoparia by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry revealed the presence of high molecular weight TAGs and widespread distribution of VLCFAs among the TAG molecular species. Heliophila species have significant potential as sources of novel seed oil enriched in VLCFAs and as model species to study the mechanisms and evolution of VLCFA biosynthesis in plants.

Genome-wide identification and analysis of elongase of very long chain fatty acid genes in the silkworm, Bombyx mori.

Very long chain fatty acids (VLCFAs), such as sphingolipids, are components of cellular lipids, which are essential for cell proliferation. Mutations in the genes that encode proteins participating in VLCFA biosynthesis may cause inherited diseases, such as macular degeneration. Elongases of very long chain fatty acid (ELOVL) are enzymes that are involved in the biosynthesis of VLCFAs. Here, a total of 13 ELOVL genes, distributed across three chromosomes, were identified in the silkworm genome; all the ELOVL members contain a distinct ELO domain and a conserved HXXHH motif. Phylogenetic reconstruction was performed to analyze the evolutionary relationships among different species and to predict gene functions. The 13 ELOVL genes were assigned to the ELOVL3/6, ELOVL1/7, and ELOVL4 clades. Microarray and semiquantitative PCR analyses indicated that these genes are differentially expressed among various tissues, in turn suggesting functional divergence in the growth and development of each tissue. Further investigation showed that the expression level of the BGIBMGA000424 gene is significantly negatively correlated with the cocoon-shell weight among different silkworm strains. Taken together, the present study is the first comprehensive analysis of ELOVL genes in silkworm, and the results may serve as a foundation for further analysis of the physiological functions of ELOVL genes in silkworm.

Wax Crystal-Sparse Leaf 4, encoding a β-ketoacyl-coenzyme A synthase 6, is involved in rice cuticular wax accumulation.

WSL4 encodes a KCS6 protein which is required for cuticular wax accumulation in rice. Very long chain fatty acids (VLCFAs) are essential precursors for cuticular wax biosynthesis. VLCFA biosynthesis occurs in the endoplasmic reticulum and requires the fatty acid elongase (FAE) complex. The β-ketoacyl-coenzyme A synthase (KCS) catalyzes the first step of FAE-mediated VLCFA elongation. Here we characterized the Wax Crystal-Sparse Leaf 4 (WSL4) gene involved in leaf cuticular wax accumulation in rice. The wsl4 mutant displayed a pleiotropic phenotype including dwarfism, less tiller numbers and reduced surface wax load. Map-based cloning and nucleotide sequencing results revealed that wsl4 carried a single nucleotide substitution in the second exon of a putative KCS6 gene, encoding one subunit of the FAE complex for VLCFAs. Genetic complementation confirmed that the mutation in WSL4 was responsible for the phenotype of wsl4. WSL4 was constitutively expressed in various rice tissues and localized in the endoplasmic reticulum. Both WSL4-RNAi transgenic lines and WSL4 knocked-out mutants exhibited wax-deficient phenotypes similar to the wsl4 mutant. These data indicate that WSL4 is required for cuticular wax accumulation in rice.

A β-Ketoacyl-CoA Synthase Is Involved in Rice Leaf Cuticular Wax Synthesis and Requires a CER2-LIKE Protein as a Cofactor.

Cuticular waxes are complex mixtures of very-long-chain fatty acids (VLCFAs) and their derivatives, forming a natural barrier on aerial surfaces of terrestrial plants against biotic and abiotic stresses. In VLCFA biosynthesis, β-ketoacyl-CoA synthase (KCS) is the key enzyme, catalyzing the first reaction in fatty acid elongation and determining substrate specificity. We isolated a rice (Oryza sativa) wax crystal-sparse leaf 4 (WSL4) gene using a map-based cloning strategy. WSL4 is predicted to encode a KCS, a homolog of Arabidopsis (Arabidopsis thaliana) CER6. Complementation of the mutant wsl4-1 with WSL4 genomic DNA rescued the cuticular wax-deficient phenotype, confirming the function of WSL4 The load of wax components longer than 30 carbons (C30) and C28 were reduced markedly in wsl4-1 and wsl4-2 mutants, respectively. Overexpression of WSL4 increased the cuticular wax load in rice leaves. We further isolated a cofactor of WSL4, OsCER2, a homolog of Arabidopsis CER2, by coimmunoprecipitation and confirmed their physical interaction by split-ubiquitin yeast two-hybrid experiments. Expression of WSL4 alone in elo3 yeast cells resulted in increased C24 but did not produce VLCFAs of greater length, whereas expressing OsCER2 alone showed no effect. Coexpression of WSL4 and OsCER2 in elo3 yeast cells yielded fatty acids up to C30. OsCER2 with a mutated HxxxD motif (H172E, D176A, and D176H) interrupted its interaction with WSL4 and failed to elongate VLCFAs past C24 when expressed with WSL4 in elo3 yeast cells. These results demonstrated that WSL4 was involved in VLCFA elongation beyond C22 and that elongation beyond C24 required the participation of OsCER2.

Dual Fatty Acid Elongase Complex Interactions in Arabidopsis.

Very long chain fatty acids (VLCFAs) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase 3 hydroxyacyl-CoA dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFAs elongation but not the plant pas2-1 defects. PTPLA interacted with elongase subunits in the Endoplasmic Reticulum (ER) and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function increased VLCFA levels, an effect that was dependent on the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted to the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes based on PAS2 or PTPLA dehydratase that are functionally interacting.

Wax crystal-sparse leaf 3 encoding a β-ketoacyl-CoA reductase is involved in cuticular wax biosynthesis in rice.

WSL3 encodes β-ketoacyl-CoA reductase (KCR) in rice, in a similar way to YBR159w in yeast, and is essential for VLCFA biosynthesis and leaf wax accumulation. Cuticular waxes on plant surfaces limit non-stomatal water loss, protect plants against deposits of dust and impose a physical barrier to pathogen infection. We identified a wax-deficient mutant of rice, wax crystal-sparse leaf 3 (wsl3), which exhibits a pleiotropic phenotype that includes reduced epicuticular wax crystals on the leaf surface and altered wax composition. Map-based cloning demonstrated that defects in the mutant were caused by two adjacent single-nucleotide changes in a gene encoding β-ketoacyl-CoA reductase (KCR) that catalyzes the second step of the fatty acid elongation reaction. The identity of WSL3 was further confirmed by genetic complementation. Transient assays of fluorescent protein-tagged WSL3 in tobacco protoplasts showed that WSL3 localizes to the endoplasmic reticulum, the compartment of fatty acid elongation in cells. Quantitative PCR and histochemical staining indicated that WSL3 is universally expressed in tissues. RNA interference of WSL3 caused a phenotype that mimicked the wsl3 mutant. Very long-chain fatty acids (VLCFAs) 20:0 and 22:0, or 20:1Δ(11) and 22:1Δ(13), were detected when WSL3 and Arabidopsis fatty acid elongation 1 (FAE1) were co-expressed in a yeast ybr159wΔ mutant strain. Our results indicated that WSL3 affects rice cuticular wax production by participating in VLCFA elongation.

Very-long-chain fatty acids restrict regeneration capacity by confining pericycle competence for callus formation in Arabidopsis

The already differentiated organs in plants have a remarkable capacity to regenerate new individuals under culture conditions. Plant in vitro regeneration practically starts with the induction of a pluripotent cell mass, the callus, from detached organs on auxin-rich callus-inducing medium (CIM), which is generally required for subsequent regeneration of new bodies. Recent studies show that CIM-induced callus formation occurs from the pericycle or pericycle-like cells through a root developmental pathway, whereas the signals involved in governing callus-forming capacity of pericycle cells remain unknown. Here we report that very-long-chain fatty acids (VLCFAs) play a critical role in confining the pericycle competence for callus formation and thus the regeneration capacity of Arabidopsis By genetic screening, we identified the callus formation-related 1 (cfr1) mutant, which bypasses the inhibition of callus-forming capacity in roots by solitary-root (slr/iaa14). We show that CFR1 encodes 3-ketoacyl-CoA synthase 1 (KCS1), which catalyzes a rate-limiting step of VLCFA biosynthesis. Our biochemical and genetic analyses demonstrate that VLCFAs restrict the pericycle competence for callus formation, at least in part, by regulating the transcription of Aberrant Lateral Root Formation 4 (ALF4). Moreover, we provide evidence that VLCFAs act as cell layer signals to mediate the pericycle competence for callus formation. Taken together, our results identify VLCFAs or their derivatives as the confining signals for mediating the pericycle competence for callus formation and thus the regeneration capacity of plant organs.

Combinatorial Effects of Fatty Acid Elongase Enzymes on Nervonic Acid Production in Camelina sativa.

Very long chain fatty acids (VLCFAs) with chain lengths of 20 carbons and longer provide feedstocks for various applications; therefore, improvement of VLCFA contents in seeds has become an important goal for oilseed enhancement. VLCFA biosynthesis is controlled by a multi-enzyme protein complex referred to as fatty acid elongase, which is composed of β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD) and enoyl reductase (ECR). KCS has been identified as the rate-limiting enzyme, but little is known about the involvement of other three enzymes in VLCFA production. Here, the combinatorial effects of fatty acid elongase enzymes on VLCFA production were assessed by evaluating the changes in nervonic acid content. A KCS gene from Lunaria annua (LaKCS) and the other three elongase genes from Arabidopsis thaliana were used for the assessment. Five seed-specific expressing constructs, including LaKCS alone, LaKCS with AtKCR, LaKCS with AtHCD, LaKCS with AtECR, and LaKCS with AtKCR and AtHCD, were transformed into Camelina sativa. The nervonic acid content in seed oil increased from null in wild type camelina to 6-12% in LaKCS-expressing lines. However, compared with that from the LaKCS-expressing lines, nervonic acid content in mature seeds from the co-expressing lines with one or two extra elongase genes did not show further increases. Nervonic acid content from LaKCS, AtKCR and AtHCD co-expressing line was significantly higher than that in LaKCS-expressing line during early seed development stage, while the ultimate nervonic acid content was not significantly altered. The results from this study thus provide useful information for future engineering of oilseed crops for higher VLCFA production.

Do seed VLCFAs trigger spongy tissue formation in Alphonso mango by inducing germination?

Spongy tissue is a physiological disorder in Alphonso mango caused by the inception of germination-associated events during fruit maturation on the tree, rendering the fruit inedible. Inter-fruit competition during active fruit growth is a major contributing factor for the disorder which leads to reduced fat content in spongy tissue affected fruits. This study was, therefore, carried out to determine the possible association between seed fats and ST formation. The study of the fat content during fruit growth showed that it increased gradually from 40 percent fruit maturity. At 70 percent maturity, however, there was a sudden increase of fat content of whole fruit, leading to acute competition and resulting in differential allocation of resources among developing fruits. As a result, the seed in spongy-tissue-affected mature ripe fruit showed a marked drop in the levels of fats and the two very long chain fatty acids (VLCFAs), tetracosanoic acid and hexacosanoic acid together with an increase of linolenic acid and a fall in oleic acid contents, which are known to be key determinants for the initiation of pre-germination events in seed. Subsequently, a rise in the level of cytokinin and gibberellins in ST seed associated with a fall in abscisic acid level clearly signalled the onset of germination. Concurrently, a significant reduction in the ratio of linolenic acid/linoleic acid in pulp led to the loss of membrane integrity, cell death and the eventual formation of spongy tissue. Based on the above, it is concluded that a significant reduction in the biosynthesis of VLCFAs in seeds during fruit growth might trigger pre-germination events followed by a cascade of biochemical changes in the pulp, leading to lipid peroxidation and membrane injury in pulp culminating in ST development. Thus, this study presents crucial experimental evidence to highlight the critical role played by VLCFAs in inducing ST formation in Alphonso mango during the pre-harvest phase of fruit growth.

PAG1, a cotton brassinosteroid catabolism gene, modulates fiber elongation.

Cotton (Gossypium hirsutum) is the major source of natural textile fibers. Brassinosteroids (BRs) play crucial roles in regulating fiber development. The molecular mechanisms of BRs in regulating fiber elongation, however, are poorly understood. pagoda1 (pag1) was identified via an activation tagging genetic screen and characterized by genome walking and brassinolide (BL) supplementation. RNA-Seq analysis was employed to elucidate the mechanisms of PAG1 in regulating fiber development. pag1 exhibited dwarfism and reduced fiber length due to significant inhibition of cell elongation and expansion. BL treatment rescued its growth and fiber elongation. PAG1 encodes a homolog of Arabidopsis CYP734A1 that inactivates BRs via C-26 hydroxylation. RNA-Seq analyses showed that the constitutive expression of PAG1 downregulated the expression of genes involved in very-long-chain fatty acids (VLCFA) biosynthesis, ethylene-mediated signaling, response to cadmium, cell wall development, cytoskeleton organization and cell growth. Our results demonstrate that PAG1 plays crucial roles in regulating fiber development via controlling the level of endogenous bioactive BRs, which may affect ethylene signaling cascade by mediating VLCFA. Therefore, BR may be a critical regulator of fiber elongation, a role which may in turn be linked to effects on VLCFA biosynthesis, ethylene and cadmium signaling, cell wall- and cytoskeleton-related gene expression.

Arabidopsis 3-ketoacyl-coenzyme a synthase9 is involved in the synthesis of tetracosanoic acids as precursors of cuticular waxes, suberins, sphingolipids, and phospholipids.

Very-long-chain fatty acids (VLCFAs) with chain lengths from 20 to 34 carbons are involved in diverse biological functions such as membrane constituents, a surface barrier, and seed storage compounds. The first step in VLCFA biosynthesis is the condensation of two carbons to an acyl-coenzyme A, which is catalyzed by 3-ketoacyl-coenzyme A synthase (KCS). In this study, amino acid sequence homology and the messenger RNA expression patterns of 21 Arabidopsis (Arabidopsis thaliana) KCSs were compared. The in planta role of the KCS9 gene, showing higher expression in stem epidermal peels than in stems, was further investigated. The KCS9 gene was ubiquitously expressed in various organs and tissues, including roots, leaves, and stems, including epidermis, silique walls, sepals, the upper portion of the styles, and seed coats, but not in developing embryos. The fluorescent signals of the KCS9::enhanced yellow fluorescent protein construct were merged with those of BrFAD2::monomeric red fluorescent protein, which is an endoplasmic reticulum marker in tobacco (Nicotiana benthamiana) epidermal cells. The kcs9 knockout mutants exhibited a significant reduction in C24 VLCFAs but an accumulation of C20 and C22 VLCFAs in the analysis of membrane and surface lipids. The mutant phenotypes were rescued by the expression of KCS9 under the control of the cauliflower mosaic virus 35S promoter. Taken together, these data demonstrate that KCS9 is involved in the elongation of C22 to C24 fatty acids, which are essential precursors for the biosynthesis of cuticular waxes, aliphatic suberins, and membrane lipids, including sphingolipids and phospholipids. Finally, possible roles of unidentified KCSs are discussed by combining genetic study results and gene expression data from multiple Arabidopsis KCSs.

Natural Variation in Seed Very Long Chain Fatty Acid Content Is Controlled by a New Isoform of KCS18 in Arabidopsis thaliana

Oil from oleaginous seeds is mainly composed of triacylglycerols. Very long chain fatty acids (VLCFAs) are major constituents of triacylglycerols in many seed oils and represent valuable feedstock for industrial purposes. To identify genetic factors governing natural variability in VLCFA biosynthesis, a quantitative trait loci (QTL) analysis using a recombinant inbred line population derived from a cross between accessions Bay-0 and Shahdara was performed in Arabidopsis thaliana. Two fatty acid chain length ratio (CLR) QTL were identified, with one major locus, CLR.2, accounting for 77% of the observed phenotypic variation. A fine mapping and candidate gene approach showed that a key enzyme of the fatty acid elongation pathway, the β-ketoacyl-CoA synthase 18 (KCS18), was responsible for the CLR.2 QTL detected between Bay-0 and Shahdara. Association genetics and heterologous expression in yeast cells identified a single point mutation associated with an alteration of KCS18 activity, uncovering the molecular bases for the modulation of VLCFA content in these two natural populations of Arabidopsis. Identification of this kcs18 mutant with altered activity opens new perspectives for the modulation of oil composition in crop plants.

Biochemical Characterization of Sinorhizobium meliloti Mutants Reveals Gene Products Involved in the Biosynthesis of the Unusual Lipid A Very Long-chain Fatty Acid

Sinorhizobium meliloti forms a symbiosis with the legume alfalfa, whereby it differentiates into a nitrogen-fixing bacteroid. The lipid A species of S. meliloti are modified with very long-chain fatty acids (VLCFAs), which play a central role in bacteroid development. A six-gene cluster was hypothesized to be essential for the biosynthesis of VLCFA-modified lipid A. Previously, two cluster gene products, AcpXL and LpxXL, were found to be essential for S. meliloti lipid A VLCFA biosynthesis. In this paper, we show that the remaining four cluster genes are all involved in lipid A VLCFA biosynthesis. Therefore, we have identified novel gene products involved in the biosynthesis of these unusual lipid modifications. By physiological characterization of the cluster mutant strains, we demonstrate the importance of this gene cluster in the legume symbiosis and for growth in the absence of salt. Bacterial LPS species modified with VLCFAs are substantially less immunogenic than Escherichia coli LPS species, which lack VLCFAs. However, we show that the VLCFA modifications do not suppress the immunogenicity of S. meliloti LPS or affect the ability of S. meliloti to induce fluorescent plant defense molecules within the legume. Because VLCFA-modified lipids are produced by other rhizobia and mammalian pathogens, these findings will also be important in understanding the function and biosynthesis of these unusual fatty acids in diverse bacterial species.

Biosynthesis of very long-chain fatty acids (C>24) in Atlantic salmon: Cloning, functional characterisation, and tissue distribution of an Elovl4 elongase

The elongases of very long-chain fatty acids (Elovl) account for the rate-limiting condensation step of the elongation process in fatty acid (FA) biosynthesis in vertebrates. One member of the Elovl family, Elovl4, has been regarded as a critical enzyme in vertebrates in the production of the so-called very long-chain fatty acids (VLC-FA), a group of compounds that has been scarcely explored in fish. Here we report on the cloning of a novel Elovl4-like elongase from Atlantic salmon (Salmo salar). The salmon Elovl4 cDNA codes for a putative protein containing 306 amino acids. Heterologous expression in yeast demonstrated that salmon Elovl4 efficiently elongated saturated FAs up to 36:0, with 24:0 and 26:0 appearing as preferred substrates. Additionally, salmon Elovl4 effectively converted C20 and C22 polyunsaturated fatty acids to elongated polyenoic products up to C36. Tissue distribution showed that Elovl4 mRNA transcripts are abundant in eye, brain and testes, suggesting that, as described in mammals, these tissues are important metabolic sites for the biosynthesis of VLC-FA. Our results are discussed in comparison with the functional analyses observed in Elovl4 proteins from other vertebrates, and also other Elovl proteins investigated previously in Atlantic salmon.