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N-Acylethanolamines in Seeds. Quantification of
Molecular Species and Their Degradation upon Imbibition1
Kent D. Chapman*,
Barney Venables,
Robert Markovic,
Raymond W. Blair Jr., and
Chris Bettinger
Department of Biological Sciences, Division of Biochemistry and
Molecular Biology, University of North Texas, Denton, Texas 76203-5220
(K.D.C., R.M., R.W.B., C.B.); and TRAC Laboratories, 113 South
Cedar, Denton, Texas 76201 (B.V.)
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 g1 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 g1 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 Km of 65 µM and a
Vmax of 83 nmol min1
mg1 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.
1
This research was supported by the U.S.
Department of Agriculture-National Research Initiative Competitive
Grants Program (grant no. 96-35304-3862) and in part by the Texas
Higher Education Coordinating Board (grant no. ARP 003594-028). C.B.
was supported by a summer research fellowship from the Texas Academy of
Math and Sciences.
*
Corresponding author; e-mail chapman{at}unt.edu; fax 940-565-4136.
Plant Physiol. (1999) 120: 1157-1164
Copyright Clearance Center: 0032-0889/99/120//08
© 1999 American Society of Plant Physiologists
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