Manipulation of Arabidopsis fatty acid amide hydrolase expression modifies plant growth and sensitivity to N -acylethanolamines

Abstract

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

Synthesis of Phenoxyacyl-Ethanolamides and Their Effects on Fatty Acid Amide Hydrolase Activity

“…This inhibitory effect of NAEs on seedling growth occurs in part through a complex interaction with ABA (abscisic acid) signaling machinery during the embryo-to-seedling transition that remains incompletely understood (14 -16). On the other hand, reductions of endogenous seed NAE levels through the overexpression of FAAH in Arabidopsis results in enhanced seedling growth and increased size of roots, cotyledons, and other plant organs (13). Other physiological processes have been attributed to FAAH-mediated alteration of NAE levels in plants, such as flowering time, which is induced by the expression and translocation of the FT (flowering locus T) protein from leaves to the vegetative meristem.…”

“…In plants, NAEs are involved in seedling establishment and growth (6). Exogenously applied N-lauroylethanolamine (NAE 12:0) arrests seedling growth, and this is evident through marked changes in root architecture and elongation (12,13). This inhibitory effect of NAEs on seedling growth occurs in part through a complex interaction with ABA (abscisic acid) signaling machinery during the embryo-to-seedling transition that remains incompletely understood (14 -16).…”

“…Genetic approaches have been developed to reduce FAAH expression (FAAH knockouts) in mice and Arabidopsis (13,29), although this approach has shown limited success, especially in plants where it appears that there are redundant pathways for NAE catabolism and where it has been difficult to raise endogenous NAE levels dramatically in vivo. On the other hand, it has been possible to overexpress FAAH in plants and reduce NAE levels to some extent to influence several physiological processes, including growth, defense, and flowering (13,16,18). However, there is limited information on chemical compounds that reduce the NAE content in plant and animal systems via enhanced FAAH activity.…”

“…Utilization of general serine protease inhibitors and/or specific inhibitors of FAAH activity, such as phenylmethylsulfonyl fluoride (PMSF), 5Z,8Z,11Z,14Z-eicosatetraenyl-methyl ester phosphonofluoridic acid (MAFP), and 3Ј-(aminocarbonyl)-[1,1Ј-biphenyl]-3-yl)-cyclohexylcarbamate (URB597), have been reported to elevate endogenous levels of NAE and to extend or amplify processes regulated by NAE signaling (28). Genetic approaches have been developed to reduce FAAH expression (FAAH knockouts) in mice and Arabidopsis (13,29), although this approach has shown limited success, especially in plants where it appears that there are redundant pathways for NAE catabolism and where it has been difficult to raise endogenous NAE levels dramatically in vivo. On the other hand, it has been possible to overexpress FAAH in plants and reduce NAE levels to some extent to influence several physiological processes, including growth, defense, and flowering (13,16,18).…”

Cytokinin Receptors Are Involved in Alkamide Regulation of Root and Shoot Development in Arabidopsis

“…Anandamide, the main ligand of the cannabinoid receptor in mammals regulates many cellular and developmental responses including the modulation of neurotransmission in the central nervous system, synchronization of embryo development, and vasodilation (for review, see Pertwee, 2006). It appears that NAE signaling has important biological functions in plants, such as germination (Wang et al, 2006;Teaster et al, 2007), defense responses (Chapman et al, 1998), and root development (Blancaflor et al, 2003), but research is still at a relatively early stage.…”