In vertebrates, the endocannabinoid signaling pathway is an important lipid regulatory pathway that modulates a variety of physiological and behavioral processes. N-Acylethanolamines (NAEs) comprise a group of fatty acid derivatives that function within this pathway, and their signaling activity is terminated by an enzyme called fatty acid amide hydrolase (FAAH), which hydrolyzes NAEs to ethanolamine and their corresponding free fatty acids. Bioinformatic approaches led to the identification of plant homologues of FAAH that are capable of hydrolyzing NAEs in vitro. To better understand the role of NAEs in plants, we identified T-DNA knockouts to Arabidopsis FAAH (AtFAAH; At5g64440) and generated plants overexpressing AtFAAH. Here we show that seeds of AtFAAH knockouts had elevated levels of endogenous NAEs, and seedling growth was hypersensitive to exogenously applied NAE. On the other hand, seeds and seedlings of AtFAAH overexpressors had lower endogenous NAE content, and seedlings were less sensitive to exogenous NAE. Moreover, AtFAAH overexpressors displayed enhanced seedling growth and increased cell size. AtFAAH expression and FAAH catalytic activity increased during seed germination and seedling growth, consistent with the timing of NAE depletion during seedling establishment. Collectively, our results show that AtFAAH is one, but not the only, modulator of endogenous NAE levels in plants, and that NAE depletion likely participates in the regulation of plant growth.endocannabinoids ͉ lipids ͉ seedling growth ͉ signaling

N-Acylethanolamines (NAEs) are endogenous constituents of plant and animal tissues, and in vertebrates their hydrolysis terminates their participation as lipid mediators in the endocannabinoid signaling system. The membrane-bound enzyme responsible for NAE hydrolysis in mammals has been identified at the molecular level (designated fatty acid amide hydrolase, FAAH), and although an analogous enzyme activity was identified in microsomes of cotton seedlings, no molecular information is available for this enzyme in plants. Here we report the identification, the heterologous expression (in Escherichia coli), and the biochemical characterization of an Arabidopsis thaliana FAAH homologue. Candidate Arabidopsis DNA sequences containing a characteristic amidase signature sequence (PS00571) were identified in plant genome data bases, and a cDNA was isolated by reverse transcriptase-PCR using Arabidopsis genome sequences to develop appropriate oligonucleotide primers. The cDNA was sequenced and predicted to encode a protein of 607 amino acids with 37% identity to rat FAAH within the amidase signature domain (18% over the entire length). Residues determined to be important for FAAH catalysis were conserved between the Arabidopsis and rat protein sequences. In addition, a single transmembrane domain near the N terminus was predicted in the Arabidopsis protein sequence, similar to that of the rat FAAH protein. The putative plant FAAH cDNA was expressed as an epitope/ His-tagged fusion protein in E. coli and solubilized from cell lysates in the nonionic detergent, dodecyl maltoside. Affinity-purified recombinant protein was indeed active in hydrolyzing a variety of naturally occurring N-acylethanolamine types. Kinetic parameters and inhibition data for the recombinant Arabidopsis protein were consistent with these properties of the enzyme activity characterized previously in plant and animal systems. Collectively these data now provide support at the molecular level for a conserved mechanism between plants and animals for the metabolism of NAEs.N-Acylethanolamines (NAEs) 1 are lipid mediators that are produced from the phospholipase D-mediated hydrolysis of Nacylphosphatidylethanolamines, a minor membrane lipid constituent of cellular membranes (1). In animal systems, anandamide (NAE 20:4) acts as an endogenous ligand for cannabinoid receptors and has varied physiological roles, such as the modulation of neurotransmission in the central nervous system (2). Anandamide also activates vanilloid receptors, functions as an endogenous analgesic (3), and appears to be involved in neuroprotection (4,5). In other tissues, NAEs have been implicated in immunomodulation (6), synchronization of embryo development (7), and induction of apoptosis (8). These endogenous bioactive molecules lose their signaling activity upon hydrolysis by fatty acid amide hydrolase (FAAH; see Ref. 9).In plants NAEs are present in substantial amounts in desiccated seeds (ϳ1 g g Ϫ1 fresh weight), and their levels decline after a few hours of imbibition (10). Ind...

Saturated and unsaturated N-acylethanolamines (NAEs) occur in desiccated seeds primarily as 16C and 18C species with N-palmitoylethanolamine and N-linoleoylethanolamine (NAE 18:2) being most abundant. Here, we examined the metabolic fate of NAEs in vitro and in vivo in imbibed cotton (Gossypium hirsutum) seeds. When synthetic [1-14 C]Npalmitoylethanolamine was used as a substrate, free fatty acids (FFA) were produced by extracts of imbibed cottonseeds. When synthetic [1-14 C]NAE 18:2 was used as a substrate, FFA and an additional lipid product(s) were formed. On the basis of polarity, we presumed that the unidentified lipid was a product of the lipoxygenase (LOX) pathway and that inclusion of the characteristic LOX inhibitors nordihydroguaiaretic acid and eicosatetraynoic acid reduced its formation in vitro and in vivo. The conversion of NAE 18:2 in imbibed cottonseed extracts to 12-oxo-13-hydroxy-N-(9Z)-octadecanoylethanolamine was confirmed by gas chromatography-mass spectrometry, indicating the presence of 13-LOX and 13-allene oxide synthase, which metabolized NAE 18:2. Cell fractionation studies showed that the NAE amidohydrolase, responsible for FFA production, was associated mostly with microsomes, whereas LOX, responsible for NAE 18:2-oxylipin production, was distributed in cytosol-enriched fractions and microsomes. The highest activity toward NAE by amidohydrolase was observed 4 to 8 h after imbibition and by LOX 8 h after imbibition. Our results collectively indicate that two pathways exist for NAE metabolism during seed imbibition: one to hydrolyze NAEs in a manner similar to the inactivation of endocannabinoid mediators in animal systems and the other to form novel NAE-derived oxylipins. The rapid depletion of NAEs by these pathways continues to point to a role for NAE metabolites in seed germination.In mammalian cells, N-acylethanolamines (NAEs) have varied physiological roles. N-Arachidonylethanolamine (anandamide), a type of NAE in mammalian brain tissue, is an endogenous ligand for the cannabinoid receptor and modulates neurotransmission. Anandamide also can activate vanilloid (capsaicin) receptors and function as an endogenous analgesic (Pertwee, 2001), and appears to be involved in neuroprotection (Hansen et al., 2000;Van der Stelt et al., 2001). In other animal tissues, NAEs have been implicated in immunomodulation (Buckley et al., 2000), synchronization of embryo development (Paria and Dey, 2000), and induction of apoptosis (Sarker et al., 2000). These endogenous bioactive molecules termed "endocannabinoids" are hydrolyzed by fatty acid amidohydrolase (AHase) to terminate their signaling functions.In plants NAEs are present in substantial amounts in desiccated cotton (Gossypium hirsutum) seeds (1.6 g Ϫ1 g fresh weight), and their levels decline after a few hours of imbibition . Individual NAEs were identified predominantly as 16C and 18C species with N-palmitoylethanolamine (NAE 16:0) and N-linoleoylethanolamine (NAE 18:2) being the most abundant. NAEs in both plant and animal cells are deri...

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