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, 120 (4), 1157-64

N-Acylethanolamines in Seeds. Quantification Of Molecular Species and Their Degradation Upon Imbibition

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N-Acylethanolamines in Seeds. Quantification Of Molecular Species and Their Degradation Upon Imbibition

KD Chapman et al. Plant Physiol.

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 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. Amidohydrolase activity in cytosolic fractions exhibited saturation kinetics toward the NAE18:2 substrate, with an apparent K(m) of 65 &mgr;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.

Figures

Figure 4
Figure 4
Quantification of NAE in dry seeds of several diverse, cultivated varieties of upland cotton (cvs DPL5690, DPL62, M8, Cook 307–6A, Dixie Triumph, Stoneville 7A glandless, Kemp, LoneStar, and Paymaster 147). A, Total NAE content summed from individual molecular species profiles (B). Bars represent the means ± sd of three to six independent extractions and fractionations.
Figure 1
Figure 1
Representative A214 profiles (585 mV full scale) of synthetic NAE20:4 (upper trace) and crude peanut lipids (lower trace) fractionated in a 2-propanol gradient (0%–50% in hexane) by HPLC. The peak at approximately 12 min in the upper trace is NAE20:4 (confirmed by GC-MS), the standard that marks the relative retention time of NAEs in these separations. A fraction from 11 to 15 min was collected from HPLC separations of crude seed lipids (see lower trace for example); NAEs are enriched in the 11- to 15-min fraction and the majority of contaminating lipids (mostly triacylglycerols) were removed by 8 min. This represents a major “clean-up” step since, in comparison, peanut seeds contain about 45% oil by weight.
Figure 2
Figure 2
Representative electron impact mass spectra for TMS-ether derivatives of NAE18:2 isolated from pea seeds (upper) and, for comparison, our synthetic NAE18:2 quantitative standard (lower). These compounds have identical retention times on GC (21.82 min; not shown), and their electron impact mass spectra are virtually indistinguishable. Identifiable ions used for quantification purposes include the molecular ion M+ at m/z 395, fragmentation ions [M-15]+ at m/z 380, and [M-90]+ at m/z 305. For all NAEs in seed extracts that were identified and quantified by GC-MS, identical GC retention times and electron impact mass spectra were obtained authentic synthetic standards (not shown), similar to the above example.
Figure 3
Figure 3
Quantification of NAE in dry seeds of pea, soybean, peanut, castor bean, tomato, okra, cotton, and corn. A, Total NAE content summed from individual molecular species profiles (B). Bars represent the means ± sd of three to six independent extractions and fractionations.
Figure 5
Figure 5
Comparison of the relative abundance of NAE moieties of dry seed NAPE generated enzymatically (see Methods). NAPE was purified by TLC from dry seeds of peanut, pea, and the cotton cv Stoneville 7A glandless, as described previously (Chapman and Moore, 1993). Bars represent the means ± sd of three independent extractions and fractionations.
Figure 6
Figure 6
Quantification of NAE molecular species in dry (white bars) and 4-h-imbibed (hatched bars) seeds of peanut (A), pea (B), and the cotton cv Stoneville 7A glandless (C). Bars represent the means ± sd of three to six independent extractions and fractionations.
Figure 7
Figure 7
Degradation of NAE18:2 in cytosolic fractions of imbibed cottonseeds at varying pH. Assays were conducted for 10 min at 35°C, and were initiated by the addition of enzyme. Substrate (60 μm NAE18:2) was solubilized in buffer with sonication, and the final assay volume was 0.75 mL. Data points are averages of duplicate assays and are representative of replicate experiments.
Figure 8
Figure 8
Plot of NAE18:2 degradation versus the concentration of NAE18:2. Assays were for 10 min in 50 mm MES buffer, pH 6.5, and 0.05 mg of cytosolic protein in a final volume of 0.75 mL. Substrate was solubilized in buffer with sonication, and the reaction was initiated by the addition of enzyme. Data points are averages of duplicate assays and are representative of replicate experiments. The solid line represents the data fit to the Michaelis-Menten equation (MacCurveFit software, produced by Kevin Reiner).
Figure 9
Figure 9
Time course of NAE 18:2 amidohydrolase activity measured in vitro in cell fractions (10,000g, 30-min supernatant) from dry or imbibed (for 1–4 h) cottonseeds. Results were calculated as total activity in extracts from 50 seeds at each time point, and values are the average ± se from three separate experiments. Assay conditions were as described in the legend for Figure 8, except a final substrate concentration of 100 μm was used and the enzyme content was varied between 20 and 100 μg.

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