N-Acylethanolamines: Formation and Molecular Composition of a New Class of Plant Lipids1

Abstract

NAPE is a widespread, albeit minor, membrane phospholipid in animal and plant tissues (Schmid et al., 1990; Chapman and Moore, 1993). Its unusual structural features (a third fatty acid moiety linked to the amino head group of PE) impart stabilizing properties to membrane bilayers (Domingo et al., 1994; LaFrance et al., 1997). NAPE and its hydrolysis products, NAEs, are known to accumulate in vertebrate tissues under pathological conditions (for review, see Schmid et al., 1990). Recently, there has been renewed interest in NAEs because of the contention that anandamide (N-arachidonylethanolamine) is an endogenous ligand for the cannabinoid receptor in mammalian brain (Devane et al., 1992; Fontana et al., 1995;Schmid et al., 1996). The likely route for NAE formation in neural and nonneural tissues, although the matter of some debate, is via the signal-mediated hydrolysis of NAPE (DiMarzo et al., 1994;Schmid et al., 1996;Sugiura, et al., 1996).In plants little is known regarding the catabolism of NAPE. In cottonseed microsomes NAPE was metabolized to NAE or NAlysoPE by PLD-or PLA-type activities, respectively (Chapman et al., 1995b). However, the metabolic fate of NAPE in vivo and the factors that regulate NAPE hydrolysis remain largely unknown. We previously noted that the biosynthesis of NAPE was increased in elicitortreated cell suspensions of tobacco (Nicotiana tabacum L.). Here we extend our investigations with this model system to examine NAPE catabolism by plant cells in vivo. NAE was released from NAPE, and it accumulated extracellularly. We identified by GC-MS these tobacco NAEs as N-lauroylethanolamine and N-myristoylethanolamine. These NAEs were increased in elicitor-treated cell suspensions. Furthermore, we detected the enzymatic machinery capable of the release and the degradation of NAEs in tobacco cells. To our knowledge this represents the first identification of the NAE molecular species in plant cells. It is tempting to speculate that NAPE hydrolysis in elicitortreated plant cells may be involved in a signaling pathway analogous to that found in mammalian cells. MATERIALS AND METHODS Cell Cultures, Elicitor Treatment, and Lipid ExtractionsTobacco (Nicotiana tabacum L. cv KY-14) cell suspensions were subcultured every 7 d (Chapman et al., 1995a); cell suspensions in log phase were treated with elicitor (xylanase, 1 g/mL) as previously described (Chapman et al., 1995a). Control and experimental treatments were carried out on aliquots of the same population of cells. Culture supernatants were separated from cells by filtration. Cells were quick frozen in liquid N 2 , powdered in a mortar, and

Inhibition of Phospholipase Dα byN-Acylethanolamines

“…At least two isoforms (␤ and ␥) appear to be optimally activated by micromolar concentrations of Ca 2ϩ and binding of inositol-containing phospholipids (Pappan et al, 1997), and these exhibit phospholipid substrate selectivity that differ markedly from that of PLD␣ (Pappan et al, 1998). The recently described PLD␦ activity is membrane associated and activated by free oleic acid .Evidence for the physiological function of PLD␣ points to a role in the degradation/reorganization of subcellular membranes, as well as a role in signal transduction (for review, see Chapman et al, 1998). This membrane degradation is manifested at the cellular level by loss of compartmentation leading to cell death, such as in phytohormone-initiated, PLDmediated senescence (Thompson, 1988;Fan et al, 1997).…”

“…Evidence for the physiological function of PLD␣ points to a role in the degradation/reorganization of subcellular membranes, as well as a role in signal transduction (for review, see Chapman et al, 1998). This membrane degradation is manifested at the cellular level by loss of compartmentation leading to cell death, such as in phytohormone-initiated, PLDmediated senescence (Thompson, 1988;Fan et al, 1997).…”

“…Consistent with the inhibitory biochemical effects on PLD␣ in vitro, N-lauroylethanolamine, but not lauric acid, selectively inhibited abscisic acid-induced closure of stomata in epidermal peels of tobacco (Nicotiana tabacum cv Xanthi) and Commelina communis at low micromolar concentrations. Together, these results provide a new class of biochemical inhibitors to assist in the evaluation of PLD␣ physiological function(s), and they suggest a novel, lipid mediator role for endogenously produced NAEs in plant cells.Hydrolysis of membrane phospholipids by phospholipase D 2 (PLD, EC 3.1.4.4) activity in plants has been known for decades (Hanahan and Chaikoff, 1947); however, its precise physiological roles in plants are only beginning to be understood (for review, see Chapman et al, 1998;Munnik et al, 1998;Wang, 2001b). Recent evidence at the molecular level indicates that there are at least four functional isoforms of PLD in higher plants, designated ␣, ␤, ␥, and ␦, and their biochemical properties differ substantially (Wang, 2001a).…”

“…Hydrolysis of membrane phospholipids by phospholipase D 2 (PLD, EC 3.1.4.4) activity in plants has been known for decades (Hanahan and Chaikoff, 1947); however, its precise physiological roles in plants are only beginning to be understood (for review, see Chapman et al, 1998;Munnik et al, 1998;Wang, 2001b). Recent evidence at the molecular level indicates that there are at least four functional isoforms of PLD in higher plants, designated ␣, ␤, ␥, and ␦, and their biochemical properties differ substantially (Wang, 2001a).…”

“…Evidence for the physiological function of PLD␣ points to a role in the degradation/reorganization of subcellular membranes, as well as a role in signal transduction (for review, see Chapman et al, 1998).…”