Very-long-chain Fatty Acids Synthesis Research Articles

PaMYB11 promotes suberin deposition in Norway spruce embryogenic tissue during cryopreservation: A novel resistance mechanism against osmosis.

The osmotic resistance mechanism has been extensively studied in whole plants or plant tissues. However, little is known about it in embryogenic tissue (ET) which is widely used in plant-based biotechnological systems. Suberin, a cell wall aliphatic and aromatic heteropolymer, plays a critical role in plant cells against osmosis stress. The suberin regulatory biosynthesis has rarely been studied in gymnosperms. Here, PaMYB11, a subgroup 11 R2R3-MYB transcription factor, plays a key role in the osmotic resistance of Norway spruce (Picea abies) ETs during cryoprotectant pretreatment. Thus, RNA-seq, histological, and analytical chemical analyses are performed on the stable transformations of PaMYB11-OE and PaMYB11-SRDX in Norway spruce ETs. DAP-seq, Y1H, and LUC are further combined to explore the PaMYB11 targets. Activation of PaMYB11 is necessary and sufficient for suberin lamellae deposition on Norway spruce embryogenic cell walls, which plays a decisive role in ET survival under osmotic stress. Transcriptome analysis shows that PaMYB11 enhances suberin lamellae monomer synthesis by promoting very long-chain fatty acid (VLCFA) synthesis. PaPOP, PaADH1, and PaTET8L, the first two (PaADH1 and PaPOP, included) involved in VLCFA synthesis, are proved to be the direct targets of PaMYB11. Our study identified a novel osmotic response directed by PaMYB11 in Norway spruce ET, which provides a new understanding of the resistance mechanism against osmosis in gymnosperms.

PASTICCINO2 interacting with Golgi Anti-Apoptotic Proteins resists endoplasmic reticulum stress dependent on very long-chain fatty acids.

The endoplasmic reticulum (ER) is crucial for maintaining cell homeostasis because it is the primary site for synthesizing secreted and transmembrane proteins and lipids. The unfolded protein response (UPR) is activated to restore ER homeostasis under ER stress. However, the relationship between lipids and the ER stress response in plants is not well understood. Arabidopsis Golgi anti-apoptotic proteins (GAAPs) are involved in resisting ER stress. To elucidate the function of GAAPs, PASTICCINO2 (PAS2), involved in very long-chain fatty acid (VLCFA) synthesis, was found to interact with GAAPs and IRE1. Single pas2 and gaap1/gaap2pas2 double mutants exhibited increased seedling damage and impaired UPR response under chronic ER stress. Site mutation combined with genetic analysis revealed that the role of PAS2 in resisting ER stress depended on its VLCFA synthesis domain. VLCFA contents were upregulated under ER stress, which required GAAPs. Exogenous VLCFAs partially restored the defect in UPR upregulation caused by PAS2 or GAAP mutations under chronic ER stress. These findings demonstrate that the association of PAS2 with GAAPs confers plant resistance to ER stress by regulating VLCFA synthesis and the UPR. This provides a basis for further studies on the connection between lipids and cell fate decisions under stress.

3-ketoacyl-CoA synthase 19 contributes to the biosynthesis of seed lipids and cuticular wax in Arabidopsis and abiotic stress tolerance.

Very-long-chain fatty acids (VLCFAs) are essential precursors for plant membrane lipids, cuticular waxes, suberin, and storage oils. Integral to the fatty acid elongase (FAE) complex, 3-ketoacyl-CoA synthases (KCSs) function as crucial enzymes in the VLCFA pathway, determining the chain length of VLCFA. This study explores the in-planta role of the KCS19 gene. KCS19 is predominantly expressed in leaves and stem epidermis, sepals, styles, early silique walls, beaks, pedicels, and mature embryos. Localized in the endoplasmic reticulum, KCS19 interacts with other FAE proteins. kcs19 knockout mutants displayed reduced total wax and wax crystals, particularly alkanes, while KCS19 overexpression increased these components and wax crystals. Moreover, the cuticle permeability was higher for the kcs19 mutants compared to the wild type, rendering them more susceptible to drought and salt stress, whereas KCS19 overexpression enhanced drought and salt tolerance. Disrupting KCS19 increased C18 species and decreased C20 and longer species in seed fatty acids, indicating its role in elongating C18 to C20 VLCFAs, potentially up to C24 for seed storage lipids. Collectively, KCS19-mediated VLCFA synthesis is required for cuticular wax biosynthesis and seed storage lipids, impacting plant responses to abiotic stress.

Acetyl-CoA Carboxylase1 Influences ECERIFERUM2 Activity to Mediate the Synthesis of Very-Long-Chain Fatty Acid Past C28.

Cuticular wax is a protective layer on the aerial surfaces of land plants. In Arabidopsis (Arabidopsis thaliana), cuticular wax is mainly constituted of compounds derived from very-long-chain fatty acids (VLCFAs) with chain lengths longer than C28. CER2-LIKE (ECERIFERUM2-LIKE) proteins interact with CER6/KCS6 (ECERIFERUM6/β-Ketoacyl-CoA Synthase6), the key enzyme of the fatty acid elongase complex, to modify its substrate specificity for VLCFA elongation past C28. However, the molecular regulatory mechanism of CER2-LIKE proteins remains unclear. Arabidopsis eceriferum19 (cer19) mutants display wax-deficient stems caused by loss of waxes longer than C28, indicating that CER19 may participate in the CER2-LIKE-mediated VLCFA elongation past C28. Using positional cloning and genetic complementation, we showed that CER19 encodes Acetyl-CoA Carboxylase1 (ACC1), which catalyzes the synthesis of malonyl-CoA, the essential substrate for the CER6/KCS6-mediated condensation reaction in VLCFA synthesis. We demonstrated that ACC1 physically interacts with CER2-LIKE proteins via split-ubiquitin yeast two-hybrid (SUY2H) and firefly luciferase complementation imaging (LCI) analysis. Additionally, heterologous expression in yeast and genetic analysis in Arabidopsis revealed that ACC1 affects CER2 activity to influence VLCFA elongation past C28. These findings imply that CER2-LIKE proteins might function as a link between ACC1 and CER6/KCS6 and subsequently enhance CER6/KCS6 binding to malonyl-CoA for further utilization in VLCFA elongation past C28. This information deepens our understanding of the complex mechanism of cuticular wax biosynthesis.

Differential gene expression and potential regulatory network of fatty acid biosynthesis during fruit and leaf development in yellowhorn (Xanthoceras sorbifolium), an oil-producing tree with significant deployment values.

Xanthoceras sorbifolium (yellowhorn) is a woody oil plant with super stress resistance and excellent oil characteristics. The yellowhorn oil can be used as biofuel and edible oil with high nutritional and medicinal value. However, genetic studies on yellowhorn are just in the beginning, and fundamental biological questions regarding its very long-chain fatty acid (VLCFA) biosynthesis pathway remain largely unknown. In this study, we reconstructed the VLCFA biosynthesis pathway and annotated 137 genes encoding relevant enzymes. We identified four oleosin genes that package triacylglycerols (TAGs) and are specifically expressed in fruits, likely playing key roles in yellowhorn oil production. Especially, by examining time-ordered gene co-expression network (TO-GCN) constructed from fruit and leaf developments, we identified key enzymatic genes and potential regulatory transcription factors involved in VLCFA synthesis. In fruits, we further inferred a hierarchical regulatory network with MYB-related (XS03G0296800) and B3 (XS02G0057600) transcription factors as top-tier regulators, providing clues into factors controlling carbon flux into fatty acids. Our results offer new insights into key genes and transcriptional regulators governing fatty acid production in yellowhorn, laying the foundation for efforts to optimize oil content and fatty acid composition. Moreover, the gene expression patterns and putative regulatory relationships identified here will inform metabolic engineering and molecular breeding approaches tailored to meet biofuel and bioproduct demands.

The impact of the GLOSSY2 and GLOSSY2-LIKE BAHD-proteins in affecting the product profile of the maize fatty acid elongase.

The maize glossy2 and glossy2-like genes are homologs, which encode proteins that belong to the BAHD family of acyltransferases. In planta genetic studies have demonstrated that these genes may be involved in the elongation of very long chain fatty acids (VLCFAs) that are precursors of the cuticular wax fraction of the plant cuticle. VLCFAs are synthesized by a fatty acyl-CoA elongase complex (FAE) that consists of four component enzymes. Previously, we functionally identified the maize FAE component enzymes by their ability to complement haploid Saccharomyces cerevisiae strains that carry lethal deletion alleles for each FAE component enzyme. In this study we used these complemented haploid strains and wild-type diploid strains to evaluate whether the co-expression of either GLOSSY2 or GLOSSY2-LIKE with individual maize FAE component enzymes affects the VLCFA product-profile of the FAE system. Wild-type diploid strains produced VLCFAs of up to 28-carbon chain length. Co-expression of GLOSSY2 or GLOSSY2-LIKE with a combination of maize 3-ketoacyl-CoA synthases stimulated the synthesis of longer VLCFAs, up to 30-carbon chain lengths. However, such results could not be recapitulated when these co-expression experiments were conducted in the yeast haploid mutant strains that lacked individual components of the endogenous FAE system. Specifically, lethal yeast mutant strains that are genetically complemented by the expression of maize FAE-component enzymes produce VLCFAs that range between 20- and 26-carbon chain lengths. However, expressing either GLOSSY2 or GLOSSY2-LIKE in these complemented strains does not enable the synthesis of longer chain VLCFAs. These results indicate that the apparent stimulatory role of GLOSSY2 or GLOSSY2-LIKE to enable the synthesis of longer chain VLCFAs in diploid yeast cells may be associated with mixing plant enzyme components with the endogenous FAE complex.

Very-long-chain fatty acids are crucial to neuronal polarity by providing sphingolipids to lipid rafts

Fatty acids have long been considered essential to brain development; however, the involvement of their synthesis in nervous system formation is unclear. We generate mice with knockout of GPSN2, an enzyme for synthesis of very-long-chain fatty acids (VLCFAs) and investigate the effects. Both GPSN2-/- and GPSN2+/- mice show abnormal neuronal networks as a result of impaired neuronal polarity determination. Lipidomics of GPSN2-/- embryos reveal that ceramide synthesis is specifically inhibited depending on FA length; namely, VLCFA-containing ceramide is reduced. We demonstrate that lipid rafts are highly enriched in growth cones and that GPSN2+/- neurons lose gangliosides in their membranes. Application of C24:0 ceramide, but not C16:0 ceramide or C24:0 phosphatidylcholine, to GPSN2+/- neurons rescues both neuronal polarity determination and lipid-raft density in the growth cone. Taken together, our results indicate that VLCFA synthesis contributes to physiological neuronal development in brain network formation, in particular neuronal polarity determination through the formation of lipid rafts.

Tetracosanoic acids produced by 3-ketoacyl-CoA synthase 17 are required for synthesizing seed coat suberin in Arabidopsis.

Very long-chain fatty acids (VLCFAs) are precursors for the synthesis of membrane lipids, cuticular waxes, suberins, and storage oils in plants. 3-Ketoacyl CoA synthase (KCS) catalyzes the condensation of C2 units from malonyl-CoA to acyl-CoA, the first rate-limiting step in VLCFA synthesis. In this study, we revealed that Arabidopsis KCS17 catalyzes the elongation of C22-C24 VLCFAs required for synthesizing seed coat suberin. Histochemical analysis of Arabidopsis plants expressing GUS (β-glucuronidase) under the control of the KCS17 promoter revealed predominant GUS expression in seed coats, petals, stigma, and developing pollen. The expression of KCS17:eYFP (enhanced yellow fluorescent protein) driven by the KCS17 promoter was observed in the outer integument1 of Arabidopsis seed coats. The KCS17:eYFP signal was detected in the endoplasmic reticulum of tobacco epidermal cells. The levels of C22 VLCFAs and their derivatives, primary alcohols, α,ω-alkane diols, ω-hydroxy fatty acids, and α,ω-dicarboxylic acids increased by ~2-fold, but those of C24 VLCFAs, ω-hydroxy fatty acids, and α,ω-dicarboxylic acids were reduced by half in kcs17-1 and kcs17-2 seed coats relative to the wild type (WT). The seed coat of kcs17 displayed decreased autofluorescence under UV and increased permeability to tetrazolium salt compared with the WT. Seed germination and seedling establishment of kcs17 were more delayed by salt and osmotic stress treatments than the WT. KCS17 formed homo- and hetero-interactions with KCR1, PAS2, and ECR, but not with PAS1. Therefore, KCS17-mediated VLCFA synthesis is required for suberin layer formation in Arabidopsis seed coats.

Genome-wide identification of KCS gene family in Carya illinoinensis and their roles under abiotic stress conditions

Pecan (Carya illinoinensis) is an important economic tree species of genus Carya in the Juglandaceae. Its nuts are rich in nutrients, becoming one of the consumer's favorite ones. The pecan production is facing serious threats from drought, soil salinization and acidification. β-Keto-acyl-CoA synthase (KCS) is an important enzyme responsible for the formation of very long chain fatty acid (VLCFA) and its derivatives. However, the characteristic of CiKCS genes family in pecan and their function under abiotic stress remain greatly unknown. Here, 30 CiKCSs were identified and distributed in the 14 chromosomes, subjected to gene duplication and strong purification selection. The cis-regulatory elements in the 2 kb promoters of these CiKCSs mainly correlated with plant growth, development and stress response. Notably, CiKCS3/4/5/6/24/28 genes expressions were significantly up-regulated under drought, salt and acid stresses. Interestingly, the expression of CiKCS12 under acid stress were down-regulated. CiKCS3/5/6/12 were further shown to be mainly located in the cytoplasm, had no self-activation activity and could not interact with each other. Heterologous expression of these four genes improved VLCFA levels in tobacco and mediated the tolerance of yeast to osmotic, salt, and acid treatments. These results preliminarily suggested that CiKCS3/5/6/12 might function in the cytoplasm through monomeric forms and improve the tolerance of pecan to abiotic stress mainly by mediating VLCFA synthesis. These findings will deepen the understanding of the characteristic and function of CiKCS genes, and provide new direction and reference for the stress-resistant breeding of pecan.

A very long chain fatty acid responsive transcription factor, MYB93, regulates lateral root development in Arabidopsis.

Lateral roots (LRs) are critical to root system architecture development in plants. Although the molecular mechanisms by which auxin regulates LR development have been extensively studied, several additional regulatory systems are hypothesized to be involved. Recently, the regulatory role of very long chain fatty acids (VLCFAs) has been shown in LR development. Our analysis showed that LTPG1 and LTPG2, transporters of VLCFAs, are specifically expressed in the developing LR primordium (LRP), while the number of LRs is reduced in the ltpg1/ltpg2 double mutant. Moreover, late LRP development was hindered when the VLCFA levels were reduced by the VLCFA synthesis enzyme mutant, kcs1-5. However, the details of the regulatory mechanisms of LR development controlled by VLCFAs remain unknown. In this study, we propose a novel method to analyze the LRP development stages with high temporal resolution using a deep neural network and identify a VLCFA-responsive transcription factor, MYB93, via transcriptome analysis of kcs1-5. MYB93 showed a carbon chain length-specific expression response following treatment of VLCFAs. Furthermore, myb93 transcriptome analysis suggested that MYB93 regulated the expression of cell wall organization genes. In addition, we also found that LTPG1 and LTPG2 are involved in LR development through the formation of root cap cuticle, which is different from transcriptional regulation by VLCFAs. Our results suggest that VLCFA is a regulator of LRP development through transcription factor-mediated regulation of gene expression and the transportation of VLCFAs is also involved in LR development through root cap cuticle formation.

Genome-Scale Analysis of the Grapevine KCS Genes Reveals Its Potential Role in Male Sterility.

Very long-chain fatty acid (VLCFA) synthesis in plants, is primarily rate-limited by the enzyme 3-ketoacyl CoA synthase (KCS), which also controls the rate and carbon chain length of VLCFA synthesis. Disruption of VLCFA during pollen development, may affect the pollen wall formation and ultimately lead to male sterility. Our study identified 24 grapevine KCS (VvKCS) genes and provided new names based on their relative chromosome distribution. Based on sequence alignment and phylogenetic investigation, these genes were grouped into seven subgroups, members of the same subgroup having similar motif structures. Synteny analysis of VvKCS genes, showed that the segmental duplication events played an important role in expanding this gene family. Expression profiles obtained from the transcriptome data showed different expression patterns of VvKCS genes in different tissues. Comparison of transcriptome and RT-qPCR data of the male sterile grape 'Y-14' and its fertile parent 'Shine Muscat', revealed that 10 VvKCS genes were significantly differentially expressed at the meiosis stage, which is a critical period of pollen wall formation. Further, joint analysis by weighted gene co-expression network analysis (WGCNA) and Kyoto Encyclopedia of Genes and Genomes (KEGG), revealed that five of these VvKCS (VvKCS6/15/19/20/24) genes were involved in the fatty acid elongation pathway, which may ultimately affect the structural integrity of the pollen wall in 'Y-14'. This systematic analysis provided a foundation for further functional characterization of VvKCS genes, with the aim of grapevine precision breeding improvement.

Computational insight into structural basis of human ELOVL1 inhibition

Very long-chain fatty acids (VLCFAs) play a direct role in the development of a neurological disorder, X-linked adrenoleukodystrophy (X-ALD). Since ELOVL1 catalyzes the rate-limiting step of the synthesis of VLCFAs, it has emerged as an attractive target for the treatment of X-ALD. Recently two potent inhibitors, compound 22 (C22) and compound 27 (C27) have been reported to specifically inhibit human ELOVL1 but their structural basis of inhibition has not been explored. In the present study, we have used a homology model of human ELOVL1 to deduce the binding site and binding modes of C22 and C27. We have employed computational approaches to characterize the binding of C22 and C27. Initially, binding of hexacosanoyl-CoA (C26:0-CoA) to ELOVL1 was modelled and further validated by molecular dynamics (MD) simulation. We observed that the fatty acid tail of C26: CoA protrudes from a unique opening located at the occluded end of ELOVL1. Structural comparison of ELOVL1 with the crystal structure of ELOVL7 revealed that the unique opening was not present in human ELOVL7. Combined blind and focused molecular docking approaches revealed that C22 and C27 exhibit favourable binding in the same unique opening. Further, MD simulations and free binding energy calculations confirmed that C22 and C27 maintain the favourable binding in the unique opening of ELOVL1. Overall, our findings suggest that selective human ELOVL1 inhibitors block the binding of long tails of VLCFAs near the occluded end of ELOVL1. Present study will be helpful in the discovery and design of novel, selective and potent inhibitors of human ELOVL1.

Hello darkness, my old friend: 3-KETOACYL-COENZYME A SYNTHASE4 is a branch point in the regulation of triacylglycerol synthesis in Arabidopsis thaliana.

Plant lipids are important as alternative sources of carbon and energy when sugars or starch are limited. Here, we applied combined heat and darkness or extended darkness to a panel of ∼300 Arabidopsis (Arabidopsis thaliana) accessions to study lipid remodeling under carbon starvation. Natural allelic variation at 3-KETOACYL-COENZYME A SYNTHASE4 (KCS4), a gene encoding an enzyme involved in very long chain fatty acid (VLCFA) synthesis, underlies the differential accumulation of polyunsaturated triacylglycerols (puTAGs) under stress. Ectopic expression of KCS4 in yeast and plants proved that KCS4 is a functional enzyme localized in the endoplasmic reticulum with specificity for C22 and C24 saturated acyl-CoA. Allelic mutants and transient overexpression in planta revealed the differential role of KCS4 alleles in VLCFA synthesis and leaf wax coverage, puTAG accumulation, and biomass. Moreover, the region harboring KCS4 is under high selective pressure and allelic variation at KCS4 correlates with environmental parameters from the locales of Arabidopsis accessions. Our results provide evidence that KCS4 plays a decisive role in the subsequent fate of fatty acids released from chloroplast membrane lipids under carbon starvation. This work sheds light on both plant response mechanisms and the evolutionary events shaping the lipidome under carbon starvation.

Increased expression of ELOVL7 contributes to production of inflammatory cytokines in THP-1 cell-derived M1-like macrophages.

The elevation of intracellular very long-chain fatty acids (VLCFAs) augments pro-inflammatory activity of macrophages. VLCFAs are considered to function as regulators in macrophage inflammatory responses; however, the precise mechanism of regulating the production of VLCFAs is unclear. In this study, we focused on elongation of the very‑long‑chain fatty acid protein (ELOVL) family, rate-determining enzymes for VLCFA synthesis, in macrophages. ELOVL7 mRNA was upregulated in human monocytic THP-1 cell-derived M1-like macrophages. Metascape analysis using the RNA-seq data set showed the involvement of NF-κB and STAT1 in transcriptional regulation of ELOVL7 highly correlated genes. Gene ontology (GO) enrichment analysis suggested that ELOVL7 highly correlated genes were closely associated with multiple pro-inflammatory responses, including response to virus and positive regulation of NF-κB signaling. Consistent with RNA-seq analysis, the NF-κB inhibitor BAY11-7082, but not the STAT1 inhibitor fludarabine, canceled ELOVL7 upregulation in M1-like macrophages. ELOVL7 knockdown decreased interleukin (IL)-6 and IL-12/IL-23 p40 production. Moreover, RNA-seq analysis of plasmacytoid dendritic cells (pDCs) revealed that ELOVL7 was upregulated in pDCs treated with TLR7 and TLR9 agonists. In conclusion, we propose that ELOVL7 is a novel pro-inflammatory gene that is upregulated by inflammatory stimuli, and regulates M1-like macrophage and pDC functions.

Stearate-derived very long-chain fatty acids are indispensable to tumor growth.

Reprogramming of lipid metabolism is emerging as a hallmark of cancer, yet involvement of specific fatty acids (FA) species and related enzymes in tumorigenesis remains unclear. While previous studies have focused on involvement of long-chain fatty acids (LCFAs) including palmitate in cancer, little attention has been paid to the role of very long-chain fatty acids (VLCFAs). Here, we show that depletion of acetyl-CoA carboxylase (ACC1), a critical enzyme involved in the biosynthesis of fatty acids, inhibits both de novo synthesis and elongation of VLCFAs in human cancer cells. ACC1 depletion markedly reduces cellular VLCFA but only marginally influences LCFA levels, including palmitate that can be nutritionally available. Therefore, tumor growth is specifically susceptible to regulation of VLCFAs. We further demonstrate that VLCFA deficiency results in a significant decrease in ceramides as well as downstream glucosylceramides and sphingomyelins, which impairs mitochondrial morphology and renders cancer cells sensitive to oxidative stress and cell death. Taken together, our study highlights that VLCFAs are selectively required for cancer cell survival and reveals a potential strategy to suppress tumor growth.

A Novel Cell Death Mechanism Involving the Sphingosine‐to‐Glycerophospholipid Pathway

Apoptosis is the most well‐studied form of cell death, but there exist other cell death pathways that also have roles in normal physiology and disease. It is unclear what other forms of cell death remain to be discovered and how these pathways could inform our understanding of cell and molecular biology. Caspase‐independent lethal 56 (CIL56) is a compound that kills cells via a novel cell death mechanism. We conducted a genome‐wide CRISPR screen to identify novel regulators of this death pathway. From the screen, we identified trans‐2‐enoyl‐CoA reductase (TECR) as a key regulator of CIL56‐induced death. TECR is localized to the endoplasmic reticulum and is required for very long‐chain fatty acid (VLCFA) synthesis. When the gene encoding TECR is disrupted, cells become resistant to CIL56‐induced death. We find that VLCFA synthesis is dispensable for CIL56‐induced death. On the contrary, TECR has a non‐canonical role in the conversion of sphingosine to palmitate in the sphingosine‐to‐glycerophospholipid pathway. Through chemical complementation analyses, we find that this pathway is important for death induction by CIL56. It remains unclear how this pathway could be acting to execute cell death. It is known that palmitate produced in the endoplasmic reticulum can be incorporated into ceramide species, which have long been associated with cell death. Preliminary lipidomic analyses reveal a dramatic increase in palmitoyl‐ceramide upon CIL56 treatment. We are investigating the role of this species in CIL56‐induced cell death to further characterize the mechanism of death and its importance.

Synthesis of C20-38 Fatty Acids in Plant Tissues.

Very-long-chain fatty acids (VLCFA) are involved in a number of important plant physiological functions. Disorders in the expression of genes involved in the synthesis of VLCFA lead to a number of phenotypic consequences, ranging from growth retardation to the death of embryos. The elongation of VLCFA in the endoplasmic reticulum (ER) is carried out by multiple elongase complexes with different substrate specificities and adapted to the synthesis of a number of products required for a number of metabolic pathways. The information about the enzymes involved in the synthesis of VLCFA with more than 26 atoms of Carbon is rather poor. Recently, genes encoding enzymes involved in the synthesis of both regular-length fatty acids and VLCFA have been discovered and investigated. Polyunsaturated VLCFA in plants are formed mainly by 20:1 elongation into new monounsaturated acids, which are then imported into chloroplasts, where they are further desaturated. The formation of saturated VLCFA and their further transformation into a number of aliphatic compounds included in cuticular waxes and suberin require the coordinated activity of a large number of different enzymes.

Genome-Wide Identification and Expression Profiling of KCS Gene Family in Passion Fruit (Passiflora edulis) Under Fusarium kyushuense and Drought Stress Conditions.

Plant and fruit surfaces are covered with cuticle wax and provide a protective barrier against biotic and abiotic stresses. Cuticle wax consists of very-long-chain fatty acids (VLCFAs) and their derivatives. β-Ketoacyl-CoA synthase (KCS) is a key enzyme in the synthesis of VLCFAs and provides a precursor for the synthesis of cuticle wax, but the KCS gene family was yet to be reported in the passion fruit (Passiflora edulis). In this study, thirty-two KCS genes were identified in the passion fruit genome and phylogenetically grouped as KCS1-like, FAE1-like, FDH-like, and CER6-like. Furthermore, thirty-one PeKCS genes were positioned on seven chromosomes, while one PeKCS was localized to the unassembled genomic scaffold. The cis-element analysis provides insight into the possible role of PeKCS genes in phytohormones and stress responses. Syntenic analysis revealed that gene duplication played a crucial role in the expansion of the PeKCS gene family and underwent a strong purifying selection. All PeKCS proteins shared similar 3D structures, and a protein–protein interaction network was predicted with known Arabidopsis proteins. There were twenty putative ped-miRNAs which were also predicted that belong to nine families targeting thirteen PeKCS genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation results were highly associated with fatty acid synthase and elongase activity, lipid metabolism, stress responses, and plant-pathogen interaction. The highly enriched transcription factors (TFs) including ERF, MYB, Dof, C2H2, TCP, LBD, NAC, and bHLH were predicted in PeKCS genes. qRT-PCR expression analysis revealed that most PeKCS genes were highly upregulated in leaves including PeKCS2, PeKCS4, PeKCS8, PeKCS13, and PeKCS9 but not in stem and roots tissues under drought stress conditions compared with controls. Notably, most PeKCS genes were upregulated at 9th dpi under Fusarium kyushuense biotic stress condition compared to controls. This study provides a basis for further understanding the functions of KCS genes, improving wax and VLCFA biosynthesis, and improvement of passion fruit resistance.

Protein-protein interactions in fatty acid elongase complexes are important for very-long-chain fatty acid synthesis.

Fatty acid elongase (FAE), which catalyzes the synthesis of very-long-chain fatty acids (VLCFAs), is a multiprotein complex; however, little is known about its quaternary structure. In this study, bimolecular fluorescence complementation and/or yeast two-hybrid assays showed that homo-interactions were observed in β-ketoacyl-CoA synthases (KCS2, KCS9, and KCS6), Eceriferum2-like proteins [CER2 and CER2-Like2 (C2L2)], and FAE complex proteins (KCR1, PAS2, ECR, and PAS1), except for CER2-Like1 (C2L1). Hetero-interactions were observed between KCSs (KCS2, KCS9, and KCS6), between CER2-LIKEs (CER2, C2L2, and C2L1), and between FAE complex proteins (KCR1, PAS2, ECR, and PAS1). PAS1 interacts with FAE complex proteins (KCR1, PAS2, and ECR), but not with KCSs (KCS2, KCS9, and KCS6) and CER2-LIKEs (CER2, C2L2, and C2L1). Asp308 and Arg309-Arg311 of KCS9 were essential for the homo-interactions of KCS9 and hetero-interactions between KCS9 and PAS2 or ECR. Asp339 of KCS9 is involved in its homo- and hetero-interactions with ECR. Complementation analysis of the Arabidopsis kcs9 mutant by the expression of amino acid-substituted KCS9 mutant genes showed that Asp308 and Asp339 of KCS9 are involved in the synthesis of C24 VLCFAs from C22. This study suggests that protein-protein interaction in FAE complexes is important for VLCFA synthesis and provides insight into the quaternary structure of FAE complexes for efficient synthesis of VLCFAs.

Discovery and Optimization of Pyrazole Amides as Inhibitors of ELOVL1.

Accumulation of very long chain fatty acids (VLCFAs) due to defects in ATP binding cassette protein D1 (ABCD1) is thought to underlie the pathologies observed in adrenoleukodystrophy (ALD). Pursuing a substrate reduction approach based on the inhibition of elongation of very long chain fatty acid 1 enzyme (ELOVL1), we explored a series of thiazole amides that evolved into compound 27─a highly potent, central nervous system (CNS)-penetrant compound with favorable in vivo pharmacokinetics. Compound 27 selectively inhibits ELOVL1, reducing C26:0 VLCFA synthesis in ALD patient fibroblasts, lymphocytes, and microglia. In mouse models of ALD, compound 27 treatment reduced C26:0 VLCFA concentrations to near-wild-type levels in blood and up to 65% in the brain, a disease-relevant tissue. Preclinical safety findings in the skin, eye, and CNS precluded progression; the origin and relevance of these findings require further study. ELOVL1 inhibition is an effective approach for normalizing VLCFAs in models of ALD.