N-acylethanolamines in seeds. Quantification of molecular species and their degradation upon imbibition

N-Acylethanolamines (NAEs) were quantified in seeds of several plant specie s and several cultivated varieties of a single species (cotton [Gossypium h irstutum]) by gas chromatography-mass spectroscopy. The total NAE content o f dry seeds ranged from 490 +/- 819 ng g(-1) fresh weight in pea (Pisum sat ivum cv early Alaska) to 1,608 +/- 309 ng g(-1) fresh weight in cotton (cv Stoneville 7A glandless). Molecular species of NAEs in all seeds contained predominantly 16C and 18C fatty acids, with N-linoleoylethanolamine (NAE18: 2) being the most abundant (approaching 1,000 ng g(-1) fresh weight in cott onseeds). Total NAE levels dropped drastically following 4 h of imbibition in seeds of pea, cotton, and peanut (Arachis hypogea cv Virginia), and this decline was most pronounced for NAE18:2. A novel enzyme activity was ident ified in cytosolic fractions of imbibed cottonseeds that hydrolyzed NAE18:2 in vitro. NAE degradation was optimal at 35 degrees C in 50 mM MES buffer, pH 6.5, and was inhibited by phenylmethylsulfonyl fluoride and 5,5'-dithio -bis(2-nitrobenzoic acid), which is typical of other amide hydrolases. Amid ohydrolase activity in cytosolic fractions exhibited saturation kinetics to ward the NAE18:2 substrate, with an apparent K-m of 65 mu M and a V-max of 83 nmol min(-1) mg(-1) protein. Total NAE amidohydrolase activity increased during seed imbibition, with the highest levels (about four times that in dry seeds) measured 2 h after commencing hydration. NAEs belong to the fami ly of "endocannabinoids," which have been identified as potent lipid mediat ors in other types of eukaryotic cells. This raises the possibility that th eir imbibition-induced metabolism in plants is involved in the regulation o f seed germination.