Richard P. Haslam

Overexpression of Arabidopsis ECERIFERUM1 Promotes Wax Very-Long-Chain Alkane Biosynthesis and Influences Plant Response to Biotic and Abiotic Stresses

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

Reconstitution of Plant Alkane Biosynthesis in Yeast Demonstrates That Arabidopsis ECERIFERUM1 and ECERIFERUM3 Are Core Components of a Very-Long-Chain Alkane Synthesis Complex™

Very-long-chain alkanes are major components of cuticular waxes, a protective layer covering aerial surfaces of plants. This article shows that the Arabidopsis thaliana CER1 protein interacts with the wax-associated CER3 protein and with the cytochrome b5 isoforms found in the endoplasmic reticulum, and that these proteins constitute the enzymatic complex catalyzing the redox-dependent plant alkane synthesis. In land plants, very-long-chain (VLC) alkanes are major components of cuticular waxes that cover aerial organs, mainly acting as a waterproof barrier to prevent nonstomatal water loss. Although thoroughly investigated, plant alkane synthesis remains largely undiscovered. The Arabidopsis thaliana ECERIFERUM1 (CER1) protein has been recognized as an essential element of wax alkane synthesis; nevertheless, its function remains elusive. In this study, a screen for CER1 physical interaction partners was performed. The screen revealed that CER1 interacts with the wax-associated protein ECERIFERUM3 (CER3) and endoplasmic reticulum–localized cytochrome b5 isoforms (CYTB5s). The functional relevance of these interactions was assayed through an iterative approach using yeast as a heterologous expression system. In a yeast strain manipulated to produce VLC acyl-CoAs, a strict CER1 and CER3 coexpression resulted in VLC alkane synthesis. The additional presence of CYTB5s was found to enhance CER1/CER3 alkane production. Site-directed mutagenesis showed that CER1 His clusters are essential for alkane synthesis, whereas those of CER3 are not, suggesting that CYTB5s are specific CER1 cofactors. Collectively, our study reports the identification of plant alkane synthesis enzymatic components and supports a new model for alkane production in which CER1 interacts with both CER3 and CYTB5 to catalyze the redox-dependent synthesis of VLC alkanes from VLC acyl-CoAs.

Successful high‐level accumulation of fish oil omega‐3 long‐chain polyunsaturated fatty acids in a transgenic oilseed crop

Omega-3 (also called n-3) long-chain polyunsaturated fatty acids (≥C20; LC-PUFAs) are of considerable interest, based on clear evidence of dietary health benefits and the concurrent decline of global sources (fish oils). Generating alternative transgenic plant sources of omega-3 LC-PUFAs, i.e. eicosapentaenoic acid (20:5 n-3, EPA) and docosahexaenoic acid (22:6 n-3, DHA) has previously proved problematic. Here we describe a set of heterologous genes capable of efficiently directing synthesis of these fatty acids in the seed oil of the crop Camelina sativa, while simultaneously avoiding accumulation of undesirable intermediate fatty acids. We describe two iterations: RRes_EPA in which seeds contain EPA levels of up to 31% (mean 24%), and RRes_DHA, in which seeds accumulate up to 12% EPA and 14% DHA (mean 11% EPA and 8% DHA). These omega-3 LC-PUFA levels are equivalent to those in fish oils, and represent a sustainable, terrestrial source of these fatty acids. We also describe the distribution of these non-native fatty acids within C. sativa seed lipids, and consider these data in the context of our current understanding of acyl exchange during seed oil synthesis.

The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development

Very-long-chain fatty acids (VLCFAs) are synthesized as acyl-CoAs by the endoplasmic reticulum-localized elongase multiprotein complex. Two Arabidopsis genes are putative homologues of the recently identified yeast 3-hydroxy-acyl-CoA dehydratase (PHS1), the third enzyme of the elongase complex. We showed that Arabidopsis PASTICCINO2 (PAS2) was able to restore phs1 cytokinesis defects and sphingolipid long chain base overaccumulation. Conversely, the expression of PHS1 was able to complement the developmental defects and the accumulation of long chain bases of the pas2–1 mutant. The pas2–1 mutant was characterized by a general reduction of VLCFA pools in seed storage triacylglycerols, cuticular waxes, and complex sphingolipids. Most strikingly, the defective elongation cycle resulted in the accumulation of 3-hydroxy-acyl-CoA intermediates, indicating premature termination of fatty acid elongation and confirming the role of PAS2 in this process. We demonstrated by in vivo bimolecular fluorescence complementation that PAS2 was specifically associated in the endoplasmic reticulum with the enoyl-CoA reductase CER10, the fourth enzyme of the elongase complex. Finally, complete loss of PAS2 function is embryo lethal, and the ectopic expression of PHS1 led to enhanced levels of VLCFAs associated with severe developmental defects. Altogether these results demonstrate that the plant 3-hydroxy-acyl-CoA dehydratase PASTICCINO2 is an essential and limiting enzyme in VLCFA synthesis but also that PAS2-derived VLCFA homeostasis is required for specific developmental processes.

Very-Long-Chain Fatty Acids Are Involved in Polar Auxin Transport and Developmental Patterning in Arabidopsis

This work identifies the immunophilin PASTICCINO1 as a member of the complex necessary for very-long-chain fatty acid synthesis and demonstrates that fatty acids are directly involved in auxin carrier distribution during organogenesis. Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of the VLCFA elongase complex in the endoplasmic reticulum. The pas1 mutants are deficient in lateral root formation and are characterized by an abnormal patterning of the embryo apex, which leads to defective cotyledon organogenesis. Our data indicate that in both tissues, defective organogenesis is associated with the mistargeting of the auxin efflux carrier PIN FORMED1 in specific cells, resulting in local alteration of polar auxin distribution. Furthermore, we show that exogenous VLCFAs rescue lateral root organogenesis and polar auxin distribution, indicating their direct involvement in these processes. Based on these data, we propose that PAS1 acts as a molecular scaffold for the fatty acid elongase complex in the endoplasmic reticulum and that the resulting VLCFAs are required for polar auxin transport and tissue patterning during plant development.

Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids

We have engineered the diatom Phaeodactylum tricornutum to accumulate the high value omega-3 long chain polyunsaturated fatty acid docosahexaenoic acid (DHA). This was achieved by the generation of transgenic strains in which the Δ5-elongase from the picoalga Ostreococcus tauri was expressed to augment the endogenous fatty acid biosynthetic pathway. Expression of the heterologous elongase resulted in an eight-fold increase in docosahexaenoic acid content, representing a marked and valuable change in the fatty acid profile of this microalga. Importantly, DHA was shown to accumulate in triacylglycerols, with several novel triacylglycerol species being detected in the transgenic strains. In a second iteration, co-expression of an acyl-CoA-dependent Δ6-desaturase with the Δ5-elongase further increased DHA levels. Together, this demonstrates for the first time the potential of using iterative metabolic engineering to optimise omega-3 content in algae.

Functional Characterization of the Arabidopsis β-Ketoacyl-Coenzyme A Reductase Candidates of the Fatty Acid Elongase

In plants, very-long-chain fatty acids (VLCFAs; >18 carbon) are precursors of sphingolipids, triacylglycerols, cuticular waxes, and suberin. VLCFAs are synthesized by a multiprotein membrane-bound fatty acid elongation system that catalyzes four successive enzymatic reactions: condensation, reduction, dehydration, and a second reduction. A bioinformatics survey of the Arabidopsis (Arabidopsis thaliana) genome has revealed two sequences homologous to YBR159w encoding a Saccharomyces cerevisiae β-ketoacyl reductase (KCR), which catalyzes the first reduction during VLCFA elongation. Expression analyses showed that both AtKCR1 and AtKCR2 genes were transcribed in siliques, flowers, inflorescence stems, leaves, as well as developing embryos, but only AtKCR1 transcript was detected in roots. Fluorescent protein-tagged AtKCR1 and AtKCR2 were localized to the endoplasmic reticulum, the site of fatty acid elongation. Complementation of the yeast ybr159Δ mutant demonstrated that the two KCR proteins are divergent and that only AtKCR1 can restore heterologous elongase activity similar to the native yeast KCR gene. Analyses of insertional mutants in AtKCR1 and AtKCR2 revealed that loss of AtKCR1 function results in embryo lethality, which cannot be rescued by AtKCR2 expression using the AtKCR1 promoter. In contrast, a disruption of the AtKCR2 gene had no obvious phenotypic effect. Taken together, these results indicate that only AtKCR1 is a functional KCR isoform involved in microsomal fatty acid elongation. To investigate the roles of AtKCR1 in postembryonic development, transgenic lines expressing RNA interference and overexpression constructs targeted against AtKCR1 were generated. Morphological and biochemical characterization of these lines confirmed that suppressed KCR activity results in a reduction of cuticular wax load and affects VLCFA composition of sphingolipids, seed triacylglycerols, and root glycerolipids, demonstrating in planta that KCR is involved in elongation reactions supplying VLCFA for all these diverse classes of lipids.

Feedback regulation of plastidic acetyl-CoA carboxylase by 18:1-acyl carrier protein in Brassica napus

Plant seed oil represents a major renewable source of reduced carbon, but little is known about the biochemical regulation of its synthesis. The goal of this research was to identify potential feedback regulation of fatty acid biosynthesis in Brassica napus embryo-derived cell cultures and to characterize both the feedback signals and enzymatic targets of the inhibition. Fatty acids delivered via Tween esters rapidly reduced the rate of fatty acid synthesis in a dose-dependent and reversible manner, demonstrating the existence of feedback inhibition in an oil-accumulating tissue. Tween feeding did not affect fatty acid elongation in the cytosol or the incorporation of radiolabeled malonate into nascent fatty acids, which together pinpoint plastidic acetyl-CoA carboxylase (ACCase) as the enzymatic target of feedback inhibition. To identify the signal responsible for feedback, a variety of Tween esters were tested for their effects on the rate of fatty acid synthesis. Maximum inhibition was achieved upon feeding oleic acid (18:1) Tween esters that resulted in the intracellular accumulation of 18:1 free fatty acid, 18:1-CoA, and 18:1-acyl-carrier protein (ACP). Direct, saturable inhibition of ACCase enzyme activity was observed in culture extracts and in extracts of developing canola seeds supplemented with 18:1-ACP at physiological concentrations. A mechanism for feedback inhibition is proposed in which reduced demand for de novo fatty acids results in the accumulation of 18:1-ACP, which directly inhibits plastidic ACCase, leading to reduced fatty acid synthesis. Defining this mechanism presents an opportunity for mitigating feedback inhibition of fatty acid synthesis in crop plants to increase oil yield.

Functional Characterization of the Arabidopsis thaliana β-Ketoacyl-CoA Reductase Candidates of the Fatty Acid Elongase

In plants, very-long-chain fatty acids (VLCFAs; >18 carbon) are precursors of sphingolipids, triacylglycerols, cuticular waxes, and suberin. VLCFAs are synthesized by a multiprotein membrane-bound fatty acid elongation system that catalyzes four successive enzymatic reactions: condensation, reduction, dehydration, and a second reduction. A bioinformatics survey of the Arabidopsis (Arabidopsis thaliana) genome has revealed two sequences homologous to YBR159w encoding a Saccharomyces cerevisiae β-ketoacyl reductase (KCR), which catalyzes the first reduction during VLCFA elongation. Expression analyses showed that both AtKCR1 and AtKCR2 genes were transcribed in siliques, flowers, inflorescence stems, leaves, as well as developing embryos, but only AtKCR1 transcript was detected in roots. Fluorescent protein-tagged AtKCR1 and AtKCR2 were localized to the endoplasmic reticulum, the site of fatty acid elongation. Complementation of the yeast ybr159Δ mutant demonstrated that the two KCR proteins are divergent and that only AtKCR1 can restore heterologous elongase activity similar to the native yeast KCR gene. Analyses of insertional mutants in AtKCR1 and AtKCR2 revealed that loss of AtKCR1 function results in embryo lethality, which cannot be rescued by AtKCR2 expression using the AtKCR1 promoter. In contrast, a disruption of the AtKCR2 gene had no obvious phenotypic effect. Taken together, these results indicate that only AtKCR1 is a functional KCR isoform involved in microsomal fatty acid elongation. To investigate the roles of AtKCR1 in postembryonic development, transgenic lines expressing RNA interference and overexpression constructs targeted against AtKCR1 were generated. Morphological and biochemical characterization of these lines confirmed that suppressed KCR activity results in a reduction of cuticular wax load and affects VLCFA composition of sphingolipids, seed triacylglycerols, and root glycerolipids, demonstrating in planta that KCR is involved in elongation reactions supplying VLCFA for all these diverse classes of lipids.

Peroxisomal ATP Import Is Essential for Seedling Development in Arabidopsis thaliana

Several recent proteomic studies of plant peroxisomes indicate that the peroxisomal matrix harbors multiple ATP-dependent enzymes and chaperones. However, it is unknown whether plant peroxisomes are able to produce ATP by substrate-level phosphorylation or whether external ATP fuels the energy-dependent reactions within peroxisomes. The existence of transport proteins that supply plant peroxisomes with energy for fatty acid oxidation and other ATP-dependent processes has not previously been demonstrated. Here, we describe two Arabidopsis thaliana genes that encode peroxisomal adenine nucleotide carriers, PNC1 and PNC2. Both proteins, when fused to enhanced yellow fluorescent protein, are targeted to peroxisomes. Complementation of a yeast mutant deficient in peroxisomal ATP import and in vitro transport assays using recombinant transporter proteins revealed that PNC1 and PNC2 catalyze the counterexchange of ATP with ADP or AMP. Transgenic Arabidopsis lines repressing both PNC genes were generated using ethanol-inducible RNA interference. A detailed analysis of these plants showed that an impaired peroxisomal ATP import inhibits fatty acid breakdown during early seedling growth and other β-oxidation reactions, such as auxin biosynthesis. We show conclusively that PNC1 and PNC2 are essential for supplying peroxisomes with ATP, indicating that no other ATP generating systems exist inside plant peroxisomes.