N-acylethanolamines (NAEs) are a diverse family of signaling lipids that occur in eukaryotes and their presence is specific to developmental stage and tissue type. In plants, NAEs with an acyl chain ranging from C12 to C18 are common with NAE 18:2 generally being the most abundant type, particularly in desiccated seeds. In Arabidopsis, NAEs negatively regulate growth and mediate stress responses via abscisic acid-dependent and -independent signaling pathway. The function of NAEs is terminated by a highly conserved fatty acid amide hydrolase (FAAH). Because of the significant role NAEs were shown to play in model plant Arabidopsis it is pertinent to elucidate this conserved metabolic pathway in crop species such as tomato. It is hypothesized that NAE pathway occurs in tomato and that there is a functional FAAH that hydrolyzes NAEs.To test this hypothesis, NAE content and composition will be determined in various tissues and developmental stages of tomato by selective lipidomic analysis. Furthermore, a functional homolog of AtFAAH has been identified in tomato and will be biochemically characterized.Thus far, full-length coding sequence of SlFAAH1 and SlFAAH2 were isolated and cloned into a heterologous expression system. The expressed protein will be characterized for its hydrolytic activity against radiolabelled NAE substrates. Temporal expression of SlFAAH1 and SlFAAH2 in different tissues will also be analyzed by quantitative PCR to correlate with the NAE levels. The molecular and biochemical characterization of FAAH in addition to determining the composition of NAEs in tomato will further validate the conserved nature of NAE metabolic pathway in plants.

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N-acylethanolamines (NAEs) including anandamide are lipid derivative molecules, which play vital roles in physiological and developmental processes in plants and animals and mediate stress responses. In mammals, NAEs are synthesized from hydrolysis of their precursor molecule N-acylphosphatidylethanolamine (NAPE) by NAPE-specific phospholipaseD (NAPE-PLD). All NAEs including anandamide (NAE20:4) are hydrolyzed by fatty acid amide hydrolase (FAAH) into free fatty acid and ethanolamine. To date, different NAEs including anandamide have been identified in Physcomitrella patens but its metabolic pathway remains undiscovered. It is hypothesized that NAE metabolic pathway in P. patens is conserved and is similar to that of other eukaryotic systems. To this extent, putative PpNAPE-PLD and PpFAAH were identified and cloned for heterologous expression and characterization. Expression of PpFAAH was further verified by Western blot analysis. Future studies will involve biochemical characterization of putative PpNAPE-PLD and PpFAAH, to establish the evolutionarily conserved nature of NAE functions in early land plants.

N-acylethanolamines (NAEs) with C12-C18 acyl chain are ubiquitous in seed plants and play a role in mediating abscisic acid (ABA)-dependent or -independent responses to stress. In moss Physcomitrella patens, using selective lipidomics approach, we recently identified the occurrence of anandamide or N-arachidonylethanolamide (NAE 20:4) and its precursors that were previously not reported in plants. Occurrence of anandamide in moss provides us with a unique opportunity to address if early land plants retained NAE-mediated signaling mechanism that is akin to animals but not to vascular plants. It is hypothesized that a distinctive NAE profile and metabolic pathway occurs in P. patens. To this extent, putative genes that might be responsible for anandamide metabolic pathway were identified and their expression levels were determined for three developmental stages of moss. The NAE metabolite levels and transcript levels for putative genes were higher in protonema stage and anandamide showed higher growth inhibitory effects, chlorophyll reduction, and putative gene induction than NAE 12:0, compared to ABA, when applied exogenously.

Il est aujourd'hui bien établi que les amibes libres jouent le rôle de vecteurs à la bactérie pathogène Legionella pneumophila favorisant ainsi son développement et sa propagation dans l'environnement. Ainsi, et jusqu'à maintenant, toutes les espèces d'amibes libres étudiées ont démontré une capacité à soutenir et favoriser la croissance de la bactérie responsable des légionelloses. Toutefois, l'ensemble des études a porté sur un nombre restreint d'espèces amibiennes, et leurs capacités relatives à soutenir la croissance bactérienne n'ont que très peu été abordées. Nous avons comparé la capacité de différentes espèces amibiennes à soutenir la prolifération de différentes souches de L. pneumophila du sérogroupe 1. Ces études ont mis en évidence les propriétés particulières d'une souche d'amibe appartenant à l'espèce Willaertia magna. Cette souche présente, au contraire de toutes les autres espèces, la capacité à inhiber et diminuer la prolifération de certaines souches de L. pneumophila. Nous avons par ailleurs pu démontrer l'existence d'une phagocytose interamibienne entre différentes espèces d'amibes, un phénomène jamais mis en évidence à notre connaissance. Les conséquences de cette phagocytose interamibienne sur la croissance et la prolifération de L. pneumophila sont aussi rapportées. La microscopie électronique suggère fortement que la bactérie L. pneumophila ne peut inhiber la fusion phagolysosomale chez W. magna à l'inverse du phénomène observé chez les espèces amibiennes permissives. Ces observations démontrent l'importance de phénomènes membranaires dans la capacité des bactéries à parasiter leur hôte amibien. L'analyse comparée de la composition lipidique des membranes de différentes espèces amibiennes montre chez W. magna une expression élevée de phosphatidylcholine. L'inhibition de la voie de biosynthèse de ce phospholipide par méthylation de phosphatidyléthanolamine résulte en une forte diminution de la croissance amibienne, suggérant que cette voie métabolique joue un rôle important dans les capacités de résistance de W. magna.

An understanding of the potential roles as lipid mediators of a family of bioactive metabolites called N-acylethanolamines (NAEs) depends on their accurate identification and quantification. The levels of 18C unsaturated NAEs (e.g. NAE18:2, NAE 18:3, etc.) in wild-type seeds (about 2000 ng/g fw) generally decreased by about 80% during germination and post-germinative growth. In addition, results suggest NAE-degradative fatty acid amide hydrolase (FAAH) expression does not play a major role in normal NAE metabolism as previously thought. Seedlings germinated and grown in the presence of abscisic acid (ABA), an endogenous plant hormone, exhibited growth arrest and secondary dormancy, similar to the treatment of seedlings with exogenous N­lauroylethanolamine (NAE12:0). ABA-mediated growth arrest was associated with higher levels of unsaturated NAEs. Overall, these results are consistent with the concept that NAE metabolism is activated during seed germination and suggest that the reduction in unsaturated NAE levels is under strict temporal control and may be a requirement for normal seed germination and post-germinative growth.

N-Acylethanolamines (NAEs) are bioactive acylamides that are present in a wide range of organisms. Because NAE levels in seeds decline during imbibition similar to ABA, a physiological role was predicted for these metabolites in Arabidopsis thaliana seed germination and seedling development. There is also a corresponding increase of AtFAAH (fatty acid amide hydrolase), transcript levels and activity, which metabolizes NAE to ethanolamine and free fatty acids. Based on whole genome microarray studies it was determined that a number of up-regulated genes that were responsive to NAE were also ABA responsive. NAE induced gene expression in these ABA responsive genes without elevating endogenous levels of ABA. It was also determined that many of these NAE/ABA responsive genes were associated with an ABA induced secondary growth arrest, including ABI3. ABI3 is a transcription factor that regulates the transition from embryo to seedling growth, the analysis of transcript levels in NAE treated seedlings revealed a dose dependent, inverse relationship between ABI3 transcript levels and growth, high ABI3 transcript levels were associated with growth inhibition. Similar to ABA, NAE negatively regulated seedling growth within a narrow window of early seedling establishment. When seedlings are exposed to NAE or ABA within the window of sensitivity, the induction of genes normally associated with the ungerminated desiccation tolerant state resumed. The NAE tolerant FAAH overexpressor and the NAE sensitive FAAH knockout both had a NAE/ABA sensitive window similar to the wild type A. thaliana. The abi3-1 ABA insensitive mutant does not undergo growth arrest upon exposure to ABA, but NAE did induce growth arrest when treated within the sensitivity window. This evidence showed that although NAE functions within an ABA dependent pathway, it also functions in an ABA independent signaling pathway. The FAAH overexpressor is tolerant to NAE through its ability to quickly metabolize NAE from the growth media, yet it is hypersensitive to ABA. The FAAH overexpressor also displayed hypersensitivity to GA, which improved its delayed germination in non-stratified seed, while the FAAH knock out showed GA insensitivity. Overall, these results showed that NAE functions as a negative regulator of germinating seed and seedling growth in ABA dependent and independent signaling pathways, and that altered NAE metabolism may interfere with ABA/GA perception in germinating seed.

Il est aujourd’hui bien établi que les amibes libres jouent le rôle de vecteurs à la bactérie pathogène Legionella pneumophila favorisant ainsi son développement et sa propagation dans l’environnement. Ainsi, et jusqu’à maintenant, toutes les espèces d’amibes libres étudiées ont démontré une capacité à soutenir et favoriser la croissance de la bactérie responsable des légionelloses. Toutefois, l’ensemble des études a porté sur un nombre restreint d’espèces amibiennes, et leurs capacités relatives à soutenir la croissance bactérienne n’ont que très peu été abordées. Nous avons comparé la capacité de différentes espèces amibiennes à soutenir la prolifération de différentes souches de L. pneumophila du sérogroupe 1. Ces études ont mis en évidence les propriétés particulières d’une souche d’amibe appartenant à l’espèce Willaertia magna. Cette souche présente, au contraire de toutes les autres espèces, la capacité à inhiber et diminuer la prolifération de certaines souches de L. pneumophila. Nous avons par ailleurs pu démontrer l’existence d’une phagocytose interamibienne entre différentes espèces d’amibes, un phénomène jamais mis en évidence à notre connaissance. Les conséquences de cette phagocytose interamibienne sur la croissance et la prolifération de L. pneumophila sont aussi rapportées. La microscopie électronique suggère fortement que la bactérie L. pneumophila ne peut inhiber la fusion phagolysosomale chez W. magna à l’inverse du phénomène observé chez les espèces amibiennes permissives. Ces observations démontrent l’importance de phénomènes membranaires dans la capacité des bactéries à parasiter leur hôte amibien. L’analyse comparée de la composition lipidique des membranes de différentes espèces amibiennes montre chez W. magna une expression élevée de phosphatidylcholine. L’inhibition de la voie de biosynthèse de ce phospholipide par méthylation de phosphatidyléthanolamine résulte en une forte diminution de la croissance amibienne, suggérant que cette voie métabolique joue un rôle important dans les capacités de résistance de W. magna / Free living amoeba is a known vector of L. pneumophila in the environment and it has been shown to favour bacterial growth. Until now, all studied amoeba species showed a capacity to support the growth of the bacterium responsible for the Legionnaire’s disorder. However, these studies were related to a restricted number of amoebic species, and their relative capacity to support the bacterial growth. We compared the capacity of various amoebic species to support the proliferation of various strains of L. pneumophila. These studies highlighted the particular properties of a strain belonging to the Willaertia magna species. This amoeba has, contrary to all the other species, the capacity to inhibit and decrease L. pneumophila proliferation. We also demonstrate the existence of an inter-amoebic phagocytosis between various species of amoebas, a phenomenon never studied to our knowledge. The consequences of this inter-amoebic phagocytosis on the growth and proliferation of L. pneumophila are also reported. Electron microscopy strongly suggests that the bacterium cannot inhibit the phagolysosomal fusion in W. magna contrary to permissive amoebic species. These observations suggest the importance of membrane phenomena in the capacity of the bacteria to infest their amoebic host. Compared analysis of the lipidic composition of various amoebic species shows in W. magna a high expression of phosphatidylcholine the major phospholipid. The inhibition of the PE N-methyltransferase biosynthesis pathway of this phospholipid results in a strong reduction of the amoebic growth, suggesting that this metabolic pathway plays an important role in the resistance capacity of W. magna to L.pneumophila.

Anandamide (NAE 20:4) or arachidonlyethanolamine (AEA) is the most widely studied N-acylethanolamine (NAE) because it mediates several physiological functions in mammals. In vascular plants, 12-18C NAEs inhibit growth in an abscisic acid (ABA)-dependent and -independent manner. Anandamide, which is unique to bryophyte Physcomitrella patens, inhibited gametophyte growth and reduced chlorophyll content when applied exogenously. It is hypothesized that anandamide mediates its responses through morphological and cellular changes. Following growth inhibition by short-term anandamide-treatment, microscopic analyses revealed relocated chloroplasts and depolymerized F-actin in protonemal tips. Long-term treatment showed partially bleached gametophyte cells with degraded and browning chloroplasts. These anandamide-mediated responses have physiological implications as AEA may function as a signal for gametophytes to activate secondary dormancy as seen with ABA. Future studies will investigate the role of AEA in mediating stress responses and possible interaction with ABA.

Classically, endogenous fatty acid ethanolamides and their derivatives that bind to the cannabinoid receptors and trigger a signalling pathway are referred to as endocannabinoids. Although derivatives of arachidonic acid, including arachidonylethanolamine or anandamide, are the known endogenous ligands for cannabinoid receptors, other fatty acid ethanolamides or N-acylethanolamines (NAE) that vary in carbon chain length and saturation occur ubiquitously in eukaryotic organisms and play an important role in their physiology and development. The metabolic pathway for NAEs is highly conserved among eukaryotes and well characterised in mammalian systems. Although NAE pathway is only partly elucidated in plants, significant progress has been made in the past 20 years in understanding the implications of the metabolism of saturated and unsaturated endocannabinoid-like molecules in plant development and growth. The latest advancements in the field of plant endocannabinoid research are reviewed. Key Concepts Endocannabinoids are endogenous ligands of cannabinoid receptors in mammalian systems. Endocannabinoids belong to a class of small bioactive lipid molecules that are derivatives of fatty acids including their ethanolamides, referred to as N-acylethanolamines. N-Acylethanolamines are ubiquitous and their metabolic pathway is highly conserved among eukaryotes. In higher plants, only 12–18C N-acylethanolamines have been identified and their metabolic pathway is partly elucidated. The endocannabinoid-like lipids play an important role in seed germination, seedling development, flowering and cellular organisation. In plants, N-acylethanolamines also participate in mediating responses to biotic and abiotic stress.