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No description available.

No description available.

N-acylethanolamines (NAEs) are fatty acid ethanolamides that occur in a wide range of eukaryotes and their composition is specific to species, developmental stage and tissue type. In plants, NAEs negatively regulate growth and mediate stress responses. The function of NAEs is terminated by a highly conserved enzyme fatty acid amide hydrolase (FAAH). In plants, FAAH homologue has been characterized only for model plant Arabidopsis. Under normal growth conditions, AtFAAH overexpressor seedlings showed enhanced growth suggesting that FAAH might act as a modulator of endogenous NAE levels and regulate growth. 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 (Solanum lycopersicum) that may lead to improved productivity. We hypothesized that there is a functional FAAH in tomato that hydrolyzes NAEs. With AtFAAH as a template and using BLASTX, we identified two putative FAAH sequences in tomato. CLUSTALW alignment showed conserved amidase signature sequence and the catalytic triad. Molecular visualization system (PyMOL) revealed that the protein structures of putative SlFAAH1and 2 were similar in domain structure to AtFAAH, with minor differences in spatial arrangement. For further biochemical characterization, 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. This research is expected to lead to characterization of NAE pathway in a crop plant. Long-term implications of this study include development of molecular and biochemical tools necessary to improve tolerance to abiotic stress and increase crop productivity.

In plants, saturated and unsaturated N-Acylethanolamines (NAEs) with acyl chains 12C to 18C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, its metabolism and functional implications in plants however, remains incomplete. Recently, anandamide (NAE 20:4), an essential fatty acid neurotransmitter in mammalian system, was identified in moss Physcomitrella patens, in addition to other types of NAEs. Bryophytes display high tolerance to abiotic stress and thus presence of anandamide in moss, but not in higher plants, suggests that NAE 20:4 might have contributed to their survival in harsh environmental conditions. Therefore, we hypothesize the anandamide metabolic pathway might play a role in mediating stress responses in P. patens. To this extent, using previously identified NAE-metabolic genes in mouse and/or Arabidopsis as templates, we identified moss orthologs for enzymes that likely participate in anandamide metabolism. We identified members of metallo-hydrolase superfamily and a/Β-hydrolase4, and five putative fatty acid amide hydrolases, which may hydrolyze N-acylphosphatidylethanolamine and NAE, respectively. Electronic fluorescent pictograph analyses of these orthologs in moss revealed differential developmental stage-specific expression patterns in gametophyte and sporophyte stages. We are currently examining expression pattern for these putative NAE-metabolic pathway genes, along with anandamide levels, in different tissues and developmental stages of moss subjected to water stress in the presence of anandamide. These transcript and metabolite levels in moss subjected to stress are expected to offer better understanding of the role of anandamide in mediating stress responses and further allow us to identify candidate genes that might participate in NAE metabolism. Our studies are aimed at functional validation of candidate genes and generating moss transgenic lines with altered NAE metabolite profile. Our long-term goal is to conduct comprehensive analyses of NAE metabolite mutants to determine their role in growth and development, and mediating stress responses in plants.

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No description available.

Identification et caractérisation de la première N-acylphosphatidyléthanolamine synthase chez A. thaliana. Les N-acylphosphatidyléthanolamines (NAPE) sont des phospholipides complexes peu abondants au sein des membranes biologiques mais largement répandues dans différents organismes. Outre ses fonctions de stabilisation des membranes ce lipide est davantage connu pour être le précurseur des N-acyléthanolamines (NAE) qui sont impliquées dans de très nombreuses voies de signalisation chez les plantes (lors de la germination, du développement racinaire, de l’induction de gène de défense, etc.) comme chez les animaux (apoptose, ligand des récepteurs endocannabinoïdes, notion de satiété, etc.). Au début de ma thèse, les gènes codant pour les enzymes impliquées dans les différentes étapes de la voie métabolique des NAE (e.g NAPE-PLD, FAAH1 et 2) ont été caractérisées exceptées le ou les gène(s) codant pour l’enzyme catalysant la synthèse de NAPE précurseurs de ces lipides. Une étude bioinformatique a permis d’identifier de nouvelles séquences codantes pour des acyltransférases putatives chez A. thaliana dont celle du gène At1g78690. La caractérisation fonctionnelle de cette enzyme a été déterminée après son expression hétérologue chez E.coli sur fractions membranaires et protéines purifiées. Puis le profil d’expression génique, la localisation cellulaire de la protéine ainsi que son activité ont été étudiés chez les plantes à partir notamment de mutants d’A. thaliana (ADN-T et « 35S »). Les résultats obtenus au cours de cette étude ont permis d’identifier et de caractériser la première NAPE synthase chez les plantes. / Discovery and characterization of an A. thaliana N-acylphosphatidylethanolamine synthase. N-acylphosphatidylethanolamine (NAPE) is a widespread, albeit minor, membrane phospholipid in various organisms. Besides its stabilizing properties to membranes bilayers, NAPE is known to be the precursor for N-acylethanolamine (NAE) synthesis. NAE have been shown to regulate a variety of physiological functions in both plants (germination, root development, gene induction, etc.) and animals (apoptosis, ligand for cannabinoid receptors, satiety properties, etc.) At the beginning of my PhD, the genes encoding the enzymes involved in the different steps of NAE metabolism were well characterized (e.g NAPE-PLD, FAAH 1 and 2), with the exception of the NAPE synthase gene(s). A bioinformatic study allowed the identification of coding sequences for putative new acyltransferases in A. thaliana, such as the At1g78690 gene. After expression in E. coli, the functional characterization of At1g78690p was carried out by analyses of the lipid content and by enzymatic assays using membrane fractions or purified proteins. The localisation of the protein and its activity were also studied in A. thaliana mutants (ADN-T and “35S”). This study shows the identification and characterization of the first NAPE-synthase in plants.

N-acylethanolamines (NAEs) are derived from a minor membrane lipid constituent N-acylphosphatidylethanolamine and are hydrolyzed by fatty acid amide hydrolases (FAAH) into free fatty acid (FFA) and ethanolamine in both plants and animals. In Arabidopsis, NAE plays an important physiological role in growth/development and response to stress. Although NAEs are reported in tomato, their metabolic pathway remains undiscovered. It is hypothesized that there is a functional FAAH in tomato that hydrolyzes NAEs. To this extent, a putative gene that likely encodes for putative SlFAAH1 protein was identified, cloned, and heterologously expressed. Amidase activity was tested using radiolabeled NAE substrates. Furthermore, expression of putative SlFAAH1 transcripts and protein activity was quantified at different developmental stages to demonstrate endogenous amidase activity in tomato seedlings. In future, molecular and biochemical characterization of tomato FAAH will further test the conserved nature of NAE metabolic pathway in plants.

N-Acylethanolamines (NAEs) are endogenous lipid metabolites that occur in a variety of dry seeds, and their levels decline rapidly during the first few hours of imbibition (Chapman et al., 1999, Plant Physiol., 120:1157-1164). Biochemical studies supported the existence of an NAE amidohydrolase activity in seeds and seedlings, and efforts were directed toward identification of DNA sequences encoding this enzyme. Mammalian tissues metabolize NAEs via an amidase enzyme designated fatty acid amide hydrolase (FAAH). Based on the characteristic amidase signature sequence in mammalian FAAH, a candidate Arabidopsis cDNA was identified and isolated by reverse transcriptase-PCR. The Arabidopsis cDNA was expressed in E. coli and the recombinant protein indeed hydrolyzed a range of NAEs to free fatty acids and ethanolamine. Kinetic parameters for the recombinant protein were consistent with those properties of the rat FAAH, supporting identification of this Arabidopsis cDNA as a FAAH homologue. Two T-DNA insertional mutant lines with disruptions in the Arabidopsis NAE amidohydrolase gene (At5g64440) were identified. The homozygous mutant seedlings were more sensitive than the wild type to exogenously applied NAE 12:0. Transgenic seedlings overexpressing the NAE amidohydrolase enzyme showed noticeably greater tolerance to NAE 12:0 than wild type seedlings. These results together provide evidence in vitro and in vivo for the molecular identification of Arabidopsis NAE amidohydrolase. Moreover, the plants with altered NAE amidohydrolase expression may provide new tools for improved understanding of the role of NAEs in germination and seedling growth.