Plant Lipid Biochemistry

N-ACYLPHOSPHATIDYLETHANOLAMINEs
(NAPEs), N-ACYLETHANOLAMINES (NAEs)
AND OTHER ACYLAMIDES: METABOLISM,
OCCURRENCE AND FUNCTIONS IN
PLANTS
1. Introduction
N-Acylphosphatidylethanolamines (NAPEs), N-acylethanolamines (NAEs) and other acylamides are
nitrogen-containing lipids. Nitrogen-containing lipids are represented by a broad range of important
structural and bioactive molecules in plant tissues including NAPEs, NAEs, alkamides, sphingolipids,
and amino acid-conjugated fatty acids (e.g. N-jasmonyl-isoleucine) [1-5] (Figure 1). The synthesis and
functions of sphingolipids will be described in this web site by Edgar B. Cahoon. Here we will focus on
the metabolism, occurrence and functions of other important acylamide compounds present in plant
cells such as NAPE, NAE and alkamide.

Figure 1. Structure of different acylamides or nitrogen-containing lipids. NAPE, Nacylphosphatidylethanolamine; NAE, N-acylethanolamine; AHL, N-decanoyl-homoserine lactone.

NAPE is generally considered to be present in all major groups of organisms including plants, animals,
and some microbes. This class of glycerophospholipids is composed of phosphatidylethanolamine (PE)
with a third fatty acid linked via an amide bond to the ethanolamine head group of the PE (Figure 1). The
molecular species composition of NAPEs are complex with variations in the length and numbers of
double bonds in the three acyl chains being described recently for Arabidopsis thaliana [6]. Described in
detail below, in plants, NAPE is synthesized by N-acylation of PE by a NAPE synthase with either a free
fatty acid (FFA) [7] or acyl-CoA substrate [8] (Figure 2).

Figure 2. Metabolic pathway of NAPE/NAE formation in plants. NAPE, Nacylphosphatidylethanolamine; PLD, phospholipase D; NAE, N-acylethanolamine; LOX, lipoxygenase;
AOS, allele oxide synthase; PA, phosphatidic acid; NAAA; N-acylethanolamine-hydrolyzing acid
amidase; FAAH, fatty acid amide hydrolase; PE, phosphatidylethanolamine.

NAPEs can be used as precursors for the formation of NAEs by hydrolysis with one or more PLDs
[9,10]. NAEs are found in virtually all types of eukaryotic organisms examined. Formation of NAEs from
NAPEs has been intensively studied in mammalian systems as part of the so-called endocannabinoid
signaling pathway and several NAE types possess potent biological activities in human physiology and
behavior [11-13]. The role of NAEs in plant systems are less well characterized, but the formation and

turnover of NAEs appears to be associated with, among other stages, seed germination and seedling
establishment (discussed in more detail below).
NAEs are metabolized in animal and plant systems by either hydrolysis or oxidation (Figure 2).
Hydrolysis of NAEs by fatty acid amide hydrolase (FAAH) to free fatty acid and ethanolamine has been
characterized at the molecular level in both plants [14] and animals [15,16] whereas hydrolysis of NAEs
by additional mechanisms have been described in animals [17-20]. Similarly, in both plants and animals,
polyunsaturated NAEs can be oxidized by lipoxygenases (LOXes) to produce NAE-oxylipins [21-23]. In
mammals, additional oxidative enzymes, most notably cyclooxygenases (COX), can act on
polyunsaturated NAEs to produce ethanolamide oxylipins [24].
Contrary to NAEs (and NAPEs), the structurally related alkamides have been identified in only a few
families of plants and some fungi [25]. The biosynthetic pathway of the alkamides and their functions are
still not entirely clear. However, despite the similarity in structure to the NAEs, the formation of
alkamides is not likely to proceed through the PLD-mediated hydrolysis of NAPEs.

2. NAPE Synthesis
The acylation of the PE with an acyl donor leads to the synthesis of NAPE. Several enzyme activities
have been reported for the biosynthesis of NAPE, but only one activity has been identified at the
molecular level. In animal cells, NAPE is reported to be synthesized by N-acylation of a
phosphatidylethanolamine with the acyl group donor being a phospholipid, specifically the sn-1 position
of a glycerophospholipid molecule (from either PE or PC) [26]. Three reactions have been described for
this phospholipid-dependent acylation: 1) the Ca2+-dependent N-acyltransferase (NAT) [27], 2) the Ca 2+independent NAT [28] and 3) a phospholipase A/acyltransferase (PLA/AT) pathway [29]. For plants, two
reactions have been described (Figure 2), one that utilizes free fatty acids (FFA) as an acyl donor [7],
and the other that uses acylCoA as an acyl donor [8]. An Arabidopsis gene, At1g78690, encoding an
acyltransferase with the acylCoA-dependent NAPE synthase activity, was characterized recently.
2.1. The FFA- dependent N-acylation:
Although not identified at the molecular level, a 64KDa membrane-bound enzyme able to transfer an
acyl chain to the amine group of PE was partially purified from cottonseed by immobilized artificial
membrane chromatography [7]. This enzyme uses the FFA as acyl donor for the direct acylation of PE
by a reverse serine-hydrolase type catalytic mechanism. The enzyme exhibited complex cooperative
kinetics toward free fatty acids, but more usual Michaelis-Menten type kinetics toward PEs. This enzyme
activity was localized in the endoplasmic reticulum, Golgi and plasma membrane fractions isolated from
cotyledons of cotton seedlings.

2.2. The AcylCoA-dependent N-acylation:

In 2010, a novel plasmalemmal protein was characterized as a NAPE synthase in vitro and in
vivo in Arabidopsis thaliana. [8]. This protein belongs to the acyltransferase family and has three or four
conserved regions with the PLsC acyltransferase motif characteristic of lysophosphatic acid
acyltransferases (LPAATs) from E. coli and mammals [30]. The recombinant NAPE synthase protein
uses acyl-CoA (16:0-CoA, 18:0-CoA) for the transfer of the acyl group to the amine group of PE. No
significant activity was observed in vitrowhen the substrate used was a FFA. However, the function of
this new protein is not entirely certain, since it was described to also have lysophosphatidylglycerol
acyltransferase (LPGAT) activity, albeit only in vitro [31]. Thus, to date two different pathways have been
described for the synthesis of NAPE in plants. However, a gene encoding a protein with FFAdependent N-acylation activity remains to be identified. In terms of the acylCoA-dependent NAPE
synthase, it is still not certain if this enzyme is sensu stricto a NAPE synthase, or if this acyltransferase
protein also operates in planta for the synthesis of different glycerolipids such as phosphatidylglycerol
(PG).

3. NAE Formation.
In plants and in animals, the formation and turnover of NAE has been widely studied. In both systems,
the acyl composition of the NAEs match well with acyl compositions at the amide position of the NAPEs
[3,6,32]. These compositional similarities along with radiolabeling studies [33,34] indicate that NAPE is
the precursor for the NAE formation. In animals, a phospholipase D specific for NAPE substrate (NAPEPLD) was cloned and well characterized [35]. This enzyme cleaves the terminal phosphodiester bond of
NAPE to produce phosphatidic acid (PA) and NAE (Figure 2). In addition, other alternative pathways for
NAE formation from NAPE have been described [17-20]. These reactions involve different
phospholipases acting on NAPE, such as 1) PLC-like followed by a phosphatase (PTPN22/SHIP1) that
releases NAE, phosphate, and diacylglycerol, or 2) a deacylase (Abh4) releasing two fatty acids and
glycerophospho-N-acyl-ethanolamine, followed by the action of glycerophosphoethanolamine diesterase
(GDE1) releasing NAE and glycerol phosphate, or 3) PLA1/A2 releasing Lyso-NAPE and fatty acid,
followed by the action of PLD-like release of NAE. In plants, the formation of NAEs is known to occur
through different PLDs (below), but the precise PLD isoforms or additional mechanisms that might
operate, remain to be elucidated.
As in animals, the formation of NAEs in plants occurs via a phospholipase D-type (PLD) activity [35].
However, the PLD family in plants is composed of numerous isoforms (12 in Arabidopsis alone), and to
date none of these PLDs have been strictly associated for the hydrolysis of NAPE to NAE. Among the
12 isoforms, only three (1 PLD, 1PLD , 1PLD ) have been tested for activity toward NAPE in
vitro [10]. Further investigations are needed to assess if a functional ortholog of the animal NAPE-PLD

exists in plants. The PLD hydrolyses a variety of phospholipid substrates but is inactive toward the
NAPE under conventional assay conditions in vitro. Actually, it has been demonstrated that the NAE is a
negative regulator of the PLD activity toward other phospholipid susbstrates [36]. The PLD and can
both metabolize the NAPE to NAE in vitro and are so far considered to be two enzymes involved in the
formation of NAEs in planta. Recently it was shown in Arabdiopsis that the NAE itself can modulate the
content of NAE/NAPE via a negative feedback regulation [6]. Plants also may have additional pathways
to form NAEs; however none have been described to date.

4. NAE Degradation
NAEs are characterized as lipid mediators in many different organisms. The regulation of the levels of
NAEs, then, is a critical step in the NAE pathway in order to terminate biological effects in vivo. The
metabolism of NAEs has been extensively studied [1-4]. In animals, the hydrolysis of NAE to FFA and
ethanolamine is catalyzed by FAAH or by a N-acylethanolamine-hydrolyzing acid amidase (NAAA) [37]
(Figure 2). Alternatively, the polyunsaturated NAEs such as NAE18:2, NAE 18:3, or NAE 20:4 can be
oxygenated viaLOX or COX to produce ethanolamide oxylipins with various potential bioactivities like
vasomodulartory effects [38-39]. These compounds could have enhanced affinity with cannabinoid
receptors in comparison of their respective non-oxygenated NAEs [40]. Alternatively, NAE-oxylipins such
as prostaglandin ethanolamides may interact with novel receptors and modulate parameters such as
intraocular blood pressure [41]. Similar pathways of hydrolysis or oxidation of NAEs also are found in
plant cells.
4.1. Fatty acid amide hydrolase in plants.
The fatty acid amide hydrolase belongs to the amidase super family of proteins, and in Arabidopsis,
there are seven proteins with the amidase signature sequence. So far in Arabidopsis, only one FAAH
and one amidase (AMI-1) have been described that can hydrolyze NAE to FFA and ethanolamine
(Figure 2) [14,42]. Contrary to the animal FAAH, the reverse reaction (FFA + ethanolamine) was not
catalyzed in plants [43]. On the other hand, Arabidopsis FAAH can hydrolyze a broad range of acyl
amide and ester substratesin vitro, including the N-acylethanolamines but does not appear to hydrolyze
ceramide. A ser-ser-lys (S-S-K) catalytic triad-type mechanism is essential for the amido hydrolase
activity of this protein [3]. The implication of FAAH in NAE metabolism in plants was confirmed by
measuring the NAE content in seeds of plants with FAAH T-DNA insertions (knock outs) or in plants
over-expressing the FAAH protein [44,45]. Since then, FAAH homologues in several plants have been
characterized at the molecular and biochemical levels. Several additional FAAH candidates have been
identified and still need to be characterized to better understand the regulation of NAE functions in
plants. One other plant protein known to hydrolyze NAE in vitro is AMI-1. However, AMI-1 has only

minor activity toward NAEs in vitro in comparison to its activity toward other compounds such as indole3-acetamide and 1-naphthaleneacetamide [42].
4.2. NAE oxidation.
A secondary pathway for NAE metabolism by LOX has been described in plant cells [44]. Contrary to
the FAAH, the lipoxygenases dont cleave the NAE, but instead oxidize the acyl chain of the
polyunsaturated NAE (PU-NAE) like N-linoleoylethanolamine or (,) N-linolenoylethanolamine to form
NAE-oxylipins. The hydroperoxides of NAE compounds formed by the LOXes can be converted to
additional NAE-oxylipins by hydroperoxide lyase (HPL) and allele oxide synthase (AOS) (Figure 2)
[21]. In vitro assays with 9-LOX fromSolanum tuberosum (St), 13-LOX from Glycine max (Gm), or
combined LOX activities in Arabidopsis homogenates, all exhibited substantial NAE-oxidation toward
NAE18:2 or NAE 18:3 [23]. In vivo, the oxidation of exogenous NAE18:2 or NAE-18:3, has been also
observed in young A. thaliana seedlings [23]. It appears that this LOX pathway can compete with FAAH
in the overall metabolism of NAEs in plants [44].

5. Alkamide Metabolism
The alkamides are structurally similar to the NAEs (Figure1). They constitute a broad group of
secondary metabolites (over 200) which have been found in 10 plant families [25]. The alkamides differ
in both the nature of their acyl group, which typically are NOT similar to membrane fatty acids of the
parent plant membranes, as well as the nature of the amide head group. Despite the large number of
molecules comprising this class of lipid, no clear enzymatic pathway has been described for their
synthesis in vivo. Because the head groups of the alkamides are propyl, isopropyl, butyl or isobutyl
amides, as opposed to ethanolamides, it is unlikely that these compounds are formed from the PLDmediated hydrolysis of NAPE. Instead it is likely that they have an alternative biosynthetic origin in the
plant families in which they have been reported. Recent labeling experiments suggest that amino acids
could be the precursor for the biosynthesis of the alkamides. Thus, valine or phenylalanine may be the
precursors for the biosynthesis of some alkamides such as affinin ((2E,6Z,8E)-N-isobutyl-2,6,8decatrienamide [46]. Interestingly, FAAH may catalyze the hydrolysis of alkamides since this enzyme
has been described as a good candidate for the hydrolysis of N-acyl-homoserine lactone (Figure 1); this
remains to be tested, however.

6. Occurrence of NAPEs/NAEs and Alkamides in Plants

In general, the amounts of NAPEs and NAEs in plant tissues are relatively minor. For example, NAPE is
around 2-3% of total phospholipid content and this was relatively similar for different tissues and

different growth stages of cotton seedlings and young plants [4,32]. NAPE and NAE content is usually
highest in desiccated seeds [6], and for both lipid classes these levels declined with seedling
establishment. In Arabidopsis, the level of NAEs in seeds was 2-2.6 g/g, and this was reduced to 0.05
g/g tissue fresh weight in seedling tissues [4,47]. Further, it has been reported that the NAE 16C and
18C are predominant in dry seeds, while NAE 12C and 14C are the proportionately higher in the NAE
pool of vegetative tissues [4]. In legume seeds, NAE profiles were similar to those in non-leguminous
seeds, with concentrations ranging from 0.17 g/g to 44.6 g/g tissue fresh weight. The NAE acyl
composition generally reflects the NAPE N-acyl composition, as well as the general membrane fatty
acid compositions of plant tissues. However, the NAE and NAPE content and composition can change
significantly with development or during different stresses such as the rehydration of desiccated seed
[48], hypoxia (e.g., potato cells) [49] or pathogen elicitor perception [47].
Regarding alkamide distribution, it has been estimated that the highest concentration of affinin is up to
1% of fresh weight in the roots of H. longipes [50].

7. Amide Lipid Functions in Plants

Since the discovery of anandamide (NAE20:4) as an endogenous ligand for the cannabinoid (CB)
receptors in the mammalian nervous system [51], the studies of the functions of NAEs in animal
systems have increased dramatically. In plants, the interest of NAEs and alkamides has been sparked
by their pronounced effects on plant growth and development [52]. Work continues on the actions of
acyl ethanolamides and alkamides in plants.

7.1. NAPE functions.

Biophysical studies with NAPE indicate that it has bilayer stabilizing properties. Because it is
synthesized from fatty acids and PE, both bilayer destabilizing lipids, the synthesis of NAPE under
certain stress conditions has been proposed to neutralize the toxic effects of FFA and PE and support
the integrity and/or organization of the membrane. Therefore, the synthesis of NAPE under stress may
help to keep the membranes functional [5,49]. NAPE biosynthesis has thus described as a
cytoprotective mechanism for membranes. Still, the main function attributed to this lipid in plants and
animals is as the precursor of NAEs which carry out various lipid mediator functions in cells. On the
other hand, NAPE functions may be more complex than previously appreciated, since the
concentrations of these lipids also can be manipulated in conjunction with NAEs in Arabidopsis by
altering the expression of FAAH [6].
7.2. NAE and alkamide functions.

In animals, functions attributed to the NAE are numerous and continue to increase [1,53]. In plants the
functions of NAE are also many and diverse. NAE-12:0 is able to inhibit the PLD activity. The inhibition
of PLD by NAE-12:0 may be partly involved in the regulation of stomata aperture [36]. It was also
reported recently that NAE 12:0 can competitively inhibit LOX activities and so some physiological
effects of NAE may be indirect though the modulation of oxylipin formation [22], as has been suggested
for the NAE12:0-mediated delay in senescence of cut carnations (Dianthus caryophyllus) [54]. NAE
metabolism may also participate in transition to flowering in Arabidopsis thaliana by modulating the
transcription level of gene playing a crucial role for the flowering timing, the flowering locus T gene (FT)
[55].

Figure 3. Some proposed functions of the NAPE, NAE and alkamide in plants. NAPE, Nacylphosphatidylethanolamine; NAE, N-acylethanolamine.

Addition of NAE12:0 to Arabidopsis seedlings results in a concentration-dependent disorganization of

cell files and an inhibition of primary root growth, especially within the first few days of post-germinative
growth. At the same time, exogenous NAE12:0 also inhibits the formation of the root hairs and the
development of the lateral roots [4,52]. The arrest of early seedling growth by added NAEs and the
normal, rapid depletion of endogenous NAEs during seedling establishment have prompted speculation
that NAEs function as negative regulators of seedling growth. Indeed, an ABI3-dependent interaction of
NAE metabolism and seedling growth arrest was demonstrated in Arabidopsis indicating and

intersection between NAE metabolism and ABA signaling in seedling development [56]. Other work
suggests that NAEs like NAE-14:0 may be involved in plant defense by modulating the expression of
defense genes such as the phenylalanine ammonia lyase (PAL), or other, early-membrane signaling
events in plant pathogen interactions [47,57].
Alkamides share some apparent overlapping effects compared with NAE in plants. Exogenous Nisobutyl-decanamide (affinin) was shown to affect seedling growth, especially in root development. At
low concentrations (7M), affinin induced an increase in the length of the primary roots, the emergence
of lateral roots and the development of root hairs [25,58]. The increase of the lateral roots could be due
to a stimulation of the emergence of pre-existing lateral root primordial, or via the synthesis of new ones.
On the other hand, higher concentrations of affinin (120M) had the opposite effect on growth [25]. At
high concentrations, this alkamide inhibited elongation of the primary root, the secondary root, and root
hairs. Unlike the NAE, the decrease in root growth did not appear to be associated with a disruption of
cell/tissue organization. Nevertheless, both types of lipids can negatively affect seedling development.
Alkamides may interact with the ethylene pathway by altering the ethylene production or the signaling
action of this hormone [25]. Affinin may also interact with the cytokinin-signaling pathway to control
meristematic activity and cell differentiation events [58]. Also, like NAEs, alkamides affected plant
defense responses against pathogens [59]. Affinin reduced the necrosis area induced by pathogens and
inhibited fungal propagation. The mode of action of this alkamide for the plant defense is not fully
understood. It might interact through the jasmonic acid pathway and MAP kinase-regulated signaling.
Based on the structural similarity between alkamides and NAEs, it may be reasonable to speculate that
some of the effects of alkamides on plants might be through endogenous targets of NAEs, since NAEs
appear to be ubiquitous in the plant kingdom, and alkamides are more restricted in their distribution.

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Lionel Faure and Kent D. Chapman

Center for Plant Lipid Research, Department of Biological Sciences, University of North
TX, USA
email: chapman@unt.edu

October 12th, 2012

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