N-Acylethanolamines in Seeds. Quantification of Molecular Species and Their Degradation upon Imbibition1

Abstract

N-Acylethanolamines (NAEs) were quantified in seeds of several plant species and several cultivated varieties of a single species (cotton [Gossypium hirstutum]) by gas chromatography-mass spectroscopy. The total NAE content of dry seeds ranged from 490 ؎ 89 ng g ؊1 fresh weight in pea (Pisum sativum 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 cottonseeds). 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 identified in cytosolic fractions of imbibed cottonseeds that hydrolyzed NAE18:2 in vitro. NAE degradation was optimal at 35°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. Amidohydrolase activity in cytosolic fractions exhibited saturation kinetics toward the NAE18:2 substrate, with an apparent K m of 65 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 family of "endocannabinoids," which have been identified as potent lipid mediators in other types of eukaryotic cells. This raises the possibility that their imbibition-induced metabolism in plants is involved in the regulation of seed germination.

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

“…This notion is supported by the identification of NAEs in a variety of plant tissues (3,4) and the observation that the levels of NAEs in plants, as in mammals, appear to change quite dramatically under certain growth and environmental conditions (5). For instance, NAEs are elevated in desiccated seeds of a variety of plant species (6), but, during imbibition and germination, NAE levels drop significantly and remain at low concentrations during subsequent seedling growth (7,8). These observations suggest that the rapid metabolism of NAEs during seed germination might be necessary for normal seedling development.…”

“…6) was added for quantification purposes. Total lipids were extracted into chloroform, filtered, and fractionated by normal-phase HPLC with a linear gradient of 2-propanol in hexane, as described (7). Endogenous NAEs were quantified against the internal deuterated standards as tetramethylsilane-ether derivatives by GC-MS (6).…”

“…NAE quantification was performed by isotope dilution mass spectrometry from extracts of desiccated seeds and 8-d-old seedlings as described (6). One hundred milligrams of desiccated Arabidopsis seeds (harvested from plants grown at the same time under identical conditions) and seedlings grown in liquid culture were crushed in a ground-glass tissue homogenizer in the presence of hot 2-propanol (70°C) to inactivate endogenous phospholipases (7). A standard mix of deuterated NAEs (50 ng each; see ref.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14] C] linoleic acid (53 mCi⅐mmol Ϫ1 in ethanol; 1Ci ϭ 37 GBq) was purchased from PerkinElmer Life Sciences (Boston, MA), and [1-14 C] lauric acid (53 mCi⅐mmol Ϫ1 in ethanol) was from Amersham Pharmacia Biosciences (Piscataway, NJ). Specific types of NAEs were synthesized from respective radiolabeled free fatty acids as described (8).…”

Molecular Identification of a Functional Homologue of the Mammalian Fatty Acid Amide Hydrolase in Arabidopsis thaliana

“…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). Individual NAEs were identified predominantly as 12C, 16C, and 18C species with N-linoleoylethanolamine (NAE 18:2) generally being the most abundant.…”