Emerging Physiological Roles for N-Acylphosphatidylethanolamine Metabolism in Plants: Signal Transduction and Membrane Protection

Chemistry and Physics of Lipids 108 (2000) 221–230 www.elsevier.com/locate/chemphyslip

Review Emerging physiological roles for N-acylphosphatidylethanolamine metabolism in plants: signal transduction and membrane protection

Kent D. Chapman *

Department of Biological Sciences, Di6ision of Biochemistry and Molecular Biology, Uni6ersity of North Texas, Denton, TX 76203-5220 USA Received 27 March 2000; received in revised form 24 May 2000; accepted 24 May 2000

Abstract

The activation of N-acylphosphatidylethanolamine (NAPE) metabolism in plants appears to be associated mostly with cellular stresses. In response to pathogen elicitors, NAPE is hydrolzyed by phospholipase-D (PLD), and corresponding medium-chain, saturated N-acylethanolamines (NAEs) are released by plant cells where they act as lipid mediators to modulate ion flux and activate defense gene expression. In desiccated seeds of higher plants, long-chain, saturated and unsaturated NAEs are prevalent, but are rapidly metabolized during the first few hours of imbibition, a period of substantial osmotic stress. NAPE synthesis is increased in seeds during this same period of rapid rehydration. A membrane-bound enzyme designated NAPE synthase has been purified from imbibed cottonseeds and its unusual biochemical properties suggest that it may scavenge free fatty acids in vivo. This feature of NAPE metabolism may be unique to higher plants a may be a mechanism for the rapid recycling of fatty acids back into membrane-associated NAPE. Altogether, increasing evidence indicates that NAPE metabolism in plants shares functional similarities with NAPE metabolism in animal systems, including signal transduction and cellular protection. In particular, the emerging role of released NAEs as lipid mediators in plant defense signaling represents an intriguing parallel to ‘endocannabinoid signaling’ in several mammalian cell types. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: N-acylethanolamine; N-acylphosphatidylethanolamine; Fatty acid metabolism; Plant-defense signaling; Membrane protection

Abbre6iations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; NAE, N-acylethanolamine; NAPE, N-acylphosphatidylethanolamine; PLD, phospholipase D. * Tel.: +1-940-5652969; fax: +1-940-5654136. E-mail address: [email protected] (K.D. Chapman).

1. Introduction and perspective min exposure to the fungal elicitor protein-xylanase (Chapman et al., 1998). Xylanase has been shown N-Acylphosphatidylethanolamine (NAPE) was to elicit a variety of cellular responses similar to first reported in the mid 1960s as a minor con- defense responses of intact plants to pathogens stituent in wheat flour (Bomstein, 1965) and its (Anderson et al., 1990; Bailey et al., 1993; Hanania occurrence soon became associated with seeds of and Avni, 1997; Furman-Matarasso et al., 1999), higher plants (Dawson et al., 1969). However in the and its activation of NAPE catabolism suggested early 1970s, an unfortunate misidentification of a possible role in signaling of plant pathogen NAPE (actually phosphatidylmethanol produced perception. The release of NAE into the cell culture as an artifact during lipid extractions; Roughan et medium was confirmed by GC–MS and discovered al., 1978) in a series of metabolic labeling studies to be mostly N-myristoyl- and N-lauroyl- with developing seeds (Wilson and Rinne, 1974), ethanolamine (Chapman et al., 1998). Almost no left many plant lipid biochemists doubting the NAE was recovered with cells. Although poor existence of this unusual phospholipid as an en- recovery from TLC plates and poor GC–MS dogenous component of plant tissues. Not until the properties of the O-acetylated NAEs, left these 1990s, after much work with NAPE metabolism results qualitative, it nonetheless marked the first had been done in mammalian tissues, was the report of a signal-mediated formation of NAE by existence and metabolism of NAPE in plants re-ex- plant cells, and indicated that released NAEs accu- amined. Experimental evidence from a combina- mulated extracellularly. These preliminary results tion of biochemical and biophysical approaches now have been supported by more quantitative indeed supported the existence of NAPE as a experiments with leaves of intact tobacco plants minor, endogenous constituent of plant seeds treated either with xylanase or cryptogein elicitors (Chapman and Moore, 1993a; Sandoval et al., (Tripathy et al., 1999). For example, NAE14:0 1995). In fact, radiolabeling experiments with [1,2- content in tobacco leaves increased from 694to 14C]-ethanolamine in vivo indicated that a variety 64929 ng g−1 fresh weight following infiltration of plant cells and tissues (not just seeds) had the of xylanase and incubation for 10 min. capacity to synthesize NAPE de novo via a PE A microsomal PLD-type activity was identified intermediate (Chapman and Moore, 1993a). In in tobacco cell suspensions that catalyzed the addition, recent evidence has indicated that NAPE formation of [14C]NAE from [14C]NAPE, and its serves as a precursor for the signal-activated forma- activity was stimulated about 20-fold by masto- tion of NAEs (Chapman et al., 1998) which appear paran, suggesting that G-proteins might be in- to function as lipid mediators in plant cell signaling volved in the activation of this pathway (Chapman (Tripathy et al., 1999). et al., 1998). Several PLD isoforms have been cloned from plants, and a survey of expressed Arabidopsis recombinant PLD activities in vitro 2. NAPE catabolism in elicited cell cultures and revealed some interesting differences among the plants individual isoforms in their capacity for NAPE hydrolysis (Pappan et al., 1998). The PLDa iso- N-Acylation of PE in plant cells was stimulated form, mostly associated with membrane degrada- for several h following treatment with pathogen tive processes, was inactive toward NAPE, while elicitors (Chapman et al., 1995), which raised the both the PLDb and g isoforms formed NAE from possibility that NAPE metabolism was involved in NAPE. In fact, the PLDg isoform actually ap- signaling pathogen perception. In radiolabeling peared to prefer NAPE over PC as a substrate experiments in vivo with tobacco cell suspensions, under equivalent reaction conditions. Thus it ap- [14C]NAE (labeled on the ethanolamine carbons) pears that PLDb and/or g is/are likely to be increased approximately six-fold in the culture involved in the formation of NAEs in plants. medium whereas [14C]NAPE associated with cells However, these in vitro studies should be inter- decreased by about five-fold following a brief 10 preted with some caution since the PLDa K.D. Chapman / Chemistry and Physics of Lipids 108 (2000) 221–229 223 isoform, previously thought to require millimolar several abiotic environmental stresses), and allows amounts of Ca2+ for optimum activity, was re- for the metabolic channeling of carbon into antimi- cently reported to be active in vitro at near-physi- crobial phenylpropanoids (Rasmussen and Dixon, ological Ca2+ concentrations in acidic pH (Pappan 1999). The activation of PAL expression was re- and Wang, 1999). cently shown to involve a NO-mediated pathway that when activated in conjunction with the production of reactive oxygen intermediates (ROI) 3. NAEs as lipid mediators in plant defense leads to hypersensitive cell death and confinement signaling of the pathogen (Alvarez et al., 1998; Delledonne et al., 1998; Durner et al., 1998). Exogenously A number of predictable short (min)- and long- applied NAE14:0 activated PAL expression inde- term (h–d) cellular responses are known to occur pendent of the addition of elicitors both in cell when resistant host plants encounter pathogens, suspensions and in leaves of tobacco plants (Tripa- and these events collectively help to establish sys- thy et al., 1999). Activation of PAL expression was temic immunity in the plant to subsequent patho- evident at submicromolar concentrations of gen invasion (Dixon and Lamb, 1990; Dixon et al., NAE14:0 but was not activated by even high 1994; Alvarez et al., 1998). Many of these defense micromolar concentrations of myristic acid (Tripa- responses (although not all) can be invoked in cell thy et al., 1999). Even more interesting, like the suspension cultures by the addition of pathogen alkalinization response, the effect of NAE14:0 was elicitors such as the fungal protein xylanase (Fur- reduced in the presence of equimolar concentra- man-Matarasso et al., 1999). tions of AM281 (Tripathy and Chapman; unpub- Among the earliest detectable changes of plant lished results). In summary, we hypothesize that cells in response to elicitor treatment is rapid ion PLD-mediated NAE release which is triggered in flux (influx of H+ and Ca2+ and efflux of K+)at vivo by perception of pathogen elicitors, functions the plasma membrane (Ebel and Scheel, 1992). in both the attentuation of the primary signal, and Addition of exogenous NAE14:0 to tobacco cells activation of defense gene expression likely through reduced the characteristic medium alkalinization the action of a CB-like receptor, which may involve triggered by a number of elicitors (Tripathy et al., the NO second messenger system (see Fig. 1). 1999). A number of synthetic NAE species were effective at inhibiting this alkalinization response at micromolar levels, but inhibition was time- and 4. NAE quantification and catabolism in seeds concentration-dependent for NAE14:0 into the high nanomolar range. Inhibition of medium alka- Medium-chain, saturated NAEs appear to pre- linization by exogenous NAE14:0 was blocked by dominate in cell suspensions and leaves, and this adding the CB receptor antagonist, AM281 (Tripa- prompted us to identify and quantify the types of thy and Chapman; unpublished results), suggesting NAEs in another plant system for which we had that NAE action is from outside the cell via a cell considerable data on NAPE metabolism — seeds surface receptor. We postulate that in vivo levels of of higher plants. Individual NAEs were recently NAE14:0 that accumulate in the minutes following identified and quantified in seeds of a variety of elicitor treatment are part of the mechanism to plant species and several cultivars of a single species attenuate the cellular perception of pathogen elici- (cotton) by GC–MS (see Table 1; data from tors. Chapman et al., 1999). The total NAE content of dry seeds ranged from 490989 ng g−1 fresh weight 3.1. Acti6ation of defense gene expression in pea to 16089309 ng g−1 fresh weight in cotton. NAEs in all seeds were comprised of predominantly Activation of phenylalanine–ammonia lyase 16C and 18C fatty acids, with N-linoeloyl- (PAL) gene expression is among the long-term ethanolamine generally being the most abundun- responses of plant cells to pathogen elicitors (and dant. The acyl composition of NAE in seeds was 224 K.D. Chapman / Chemistry and Physics of Lipids 108 (2000) 221–229 qualitatively similar to the N-acylcomposition of contained an enzyme activity that metabolized seed NAPE species, implying a metabolic relation- NAE 18:2 in vitro. The cottonseed enzyme exhib- ship between these lipid classes (Chapman et al., ited biochemical properties similar with other m 1999). Total NAE levels dropped dramatically amide hydrolases, and had apparent Km of 65 M −1 −1 following 4 h imbibition in seeds of several species for NAE18:2 and Vmax of 83 nmol min mg plant species. Cytosolic extracts of imbibed seeds protein. Since NAEs are potent lipid mediators in other types of eukaryotic cells, it is possible that their imbibition-induced catabolism may play a role in the regulation of seed germination. At the very least, the differences in NAE content and composition in different plant cell types are sug- gestive of different physiological functions for NAEs in seeds and leaves.

5. NAPE biosynthesis

5.1. Acti6ation following abiotic and biotic physiological stresses

Physiological conditions which induce the synthesis of NAPE may provide clues to new roles Fig. 1. Hypothetical model illustrating the metabolism of for NAPE metabolism in plant cells. Increased NAPE/NAE in signal transduction of pathogen elicitor per- NAPE synthesis appears to be associated with ception in tobacco. Elicitors, such as xylanase (and cryp- certain abiotic and biotic physiological stresses in togein), are perceived by receptors at the cell surface (Hanania and Avni, 1997; Furman-Matarasso et al., 1999). Elicitor plants. Increased NAPE content and/or NAPE perception activates, among other things, the PLD-mediated synthase activity was associated with dehydration/ hydrolysis of cellular NAPE and the corresponding, rapid rehydration stress in imbibing seeds (Sandoval et extracellular accumulation of NAE (Chapman et al., 1998). al., 1995), and also was associated with chilling Microsomal NAE formation in vitro was stimulated 20-fold by stress in seedlings during post-germinative growth the addition of mastoparan implicating the involvement of G-proteins in elicitor-regulated NAPE-PLD activation (Chap- (Chapman and Sprinkle, 1996). In cell suspen- man et al., 1998). NAPE was hydrolyzed by either the PLDb sions of tobacco treated with fungal elicitors, or g isoforms in vitro, but not by the PLDh isoform (Pappan NAPE biosynthesis was increased about 2-fold et al., 1998), suggesting the two recently discovered, polyphos- after2hoftreatment Chapman et al., 1995). phoinositide-regulated PLDs may be involved in elicitor sig- While it is possible that NAPE might be synthe- naling. NAE14:0 levels rose 10–50 fold in elicitor treated tobacco leaves, and these in vivo concentrations were sufﬁcient sized as part of a protective mechanism in cell to activate the expression of phenylalanine–ammonia lyase membranes, it is also possible that the increase in (PAL) gene expression independent of elicitor treatment (Tri- NAPE biosynthesis under these conditions was to pathy et al., 1999). Based on pharmacological experiments replenish NAPE consumed during signal-medi- with AM281 (Tripathy and Chapman, unpublished results), ated depletion (and formation of NAE; see we postulate that NAE activation (+) of PAL expression (and attenuation (−) of the alkalinization response) is medi- above). ated by a CB-like receptor at the cell surface. Intracellular NO production may be a downstream target of NAE signaling, 5.2. Reconstitution in 6itro since NO was recently shown to activate PAL gene expression, but not other classes of ‘defense genes’ (Delledonne et al., As a ﬁrst step toward better understanding the 1998; Durner et al., 1998). It is not yet clear whether NAE release is necessary for PAL activation, and there may be regulation of NAPE metabolism in plants, one additional pathways independent of NAPE metabolism to goal has been to isolate and characterize the activate PAL expression in response to elicitor perception. regulatory properties of the NAPE biosynthetic K.D. Chapman / Chemistry and Physics of Lipids 108 (2000) 221–229 225

Table 1 Summary of N-acylethanolamines identiﬁed and quantiﬁed by GC–MS (TMS–ehter derivatives following normal-phase HPLC fractionation) in seeds (dry and imbibed) of various plantsa

a (Data are summarized from Chapman et al., 1999). machinery in plant cells. NAPE synthesis was A surprising feature of NAPE synthesis in reconstituted in vitro in extracts from imbibed plants is that it proceeds by a direct N-acylation cottonseeds, and confirmed to proceed via a PE of PE with unesterified free fatty acids. This was intermediate consistent with results from experi- first suspected in reconstitution experiments in ments in vivo (Chapman and Moore, 1993a). which [1- 14C]palmitic acid was preferred as a NAPE formation was associated with microsomes donor for the specific N-acylation of PE — and proceeded in vitro in a time- and protein-depen- preferred by 2–3 orders of magnitude over either dent manner from PE that was radiolabeled in vivo [14C]palmitoylCoA or [14C]diacylPC (Chapman (from exogenously applied [1,2-14C]-ethanolamine) and Moore, 1993b). The enzyme that catalyzed seedling tissues prior to cell fractionation. Further, this reaction, designated NAPE synthase, was NAPE was synthesized in cottonseed microsomes membrane-bound and confined to the ER, Golgi, from PE that was synthesized in vitro via the and plasma membranes of several plant systems nucleotide pathway (microsomes preincubated with (Chapman and Sriparameswaran, 1997). Prelimin- [14C]CDPethanolamine) or from PE synthesized via ary characterization of the cottonseed microsomal the exchange pathway (microsomes preincubated NAPE synthase indicated biphasic, saturable with [2-14C]-ethanolamine). kinetics with respect to palmitic acid, suggesting 226 K.D. Chapman / Chemistry and Physics of Lipids 108 (2000) 221–229 the presence of both high- and low-affinity sites taken a more traditional biochemical approach to for the free fatty acid substrate. The cottonseed the study of this enzyme’s function. In a collabo- NAPE synthase was solubilized from microsomal rative effort with Dr Charles Pidgeon’s labora- membranes in dodecylmaltoside in an active form, tory, we affinity-purified the dodecylmaltoside- and was comprised of three charge isoforms solubilized cottonseed NAPE synthase to appar- (Chapman and Moore, 1994). However, the active ent homogeneity by immobilized artificial mem- enzyme was later purified to apparent homogene- brane (IAM) chromatography employing an ether ity by immobilized artificial membrane (IAM) analogue of PE as the immobilized ligand (Cai et chromatography and was composed of a 64 kDa al., 1995). Solubilized NAPE synthase bound to subunit only (below), and so the nature of the the PE surface and was eluted by a ‘pulse’-gradi- charge isoforms separated by preparative ioselec- ent of dimyristoylPE in detergent. The eluted tric focusing remains unclear. active enzyme fraction was comprised of a single The physiological significance of the utilization of protein band migrating at 64 000 Mr in denatur- free fatty acids to synthesize NAPE is not known. ing PAGE. This protein band was specifically However, we have speculated that this may be a photoaffinity-labeled by the free fatty acid ana- protective mechanism in cell membranes for the logue, 125I-ASD (12-[(4-azidosalicyl)amino] dode- scavenging of potentially cytotoxic free fatty acids. canoic acid) — a compound which was shown to Another related possiblitiy is that this may be a interact with the free fatty acid substrate binding mechanism for rapidly recycling of fatty acids back site of the cottonseed NAPE synthase (McAndrew into NAPE following activation of the phosphodi- et al., 1995). esterase/amidohydrolase activities; i.e. a mechanism For kinetics studies an alternative IAM surface for confining NAPE-NAE signaling to appropriate (PE analogue lacking the glycerol backbone) was membrane domains which prevents the unregulated release of free fatty acids throughout plant cells (see Fig. 2). The intracellular location of NAPE biosynthesis was confined to the endomembrane systems and plasma membranes in cells of several plant tissue sources (Chapman and Sriparameswaran, 1997). Localized activation of parts, or all, of the so-called N-acylation-phosphodiesterase pathway (Schmid et al., 1996) may allow for the participation of NAPE metabolism in regulating cell signaling functions and/or fatty acid scavenging in membranes depending upon cellular needs, enzyme localization, and/or protein-protein associations. Since animal systems seem to synthesize NAPE by a transacylase activity, the scavenging of free fatty acids for protection or cell signaling may be a unique utilization of NAPE metabolism by plant cells. Certainly, some interesting physiological questions will be answered as more molecular information of NAPE metabolism becomes available. Fig. 2. Speculative scheme for the recycling of free fatty acids 5.3. Purification and characterization of the via NAPE metabolism. NAPE is hydrolyzed by PLD to form NAE which is hydrolyzed to free fatty acid by fatty acid amide membrane-bound NAPE synthase hydrolase (FAAH). Free fatty acids are incorporated back into the N-acyl position of NAPE by the NAPE synthase in There are no known homologues of the plant plant cells. This cycle could be used for signal transduction or NAPE synthase in DNA databases, and we have to scavenge free fatty acids depending upon cellular demands. K.D. Chapman / Chemistry and Physics of Lipids 108 (2000) 221–229 227 employed for the isolation of NAPE synthase so acterization of the plant NAPE synthase and its that the enzyme could be prepared in the absence relationship (if any) to the mammalian fatty acid of both substrates (McAndrew and Chapman, amide hydrolase (Cravatt et al., 1996) or the 1998). Initial velocity measurements made with transacylase (Schmid et al., 1990, 1996) will await various free fatty acid substrates revealed non- cloning of the corresponding cDNA sequence. Michaelis–Menten, biphasic kinetics with appar- Regardless, most certainly the regulation of NAPE ent high and low affinity sites (each exhibiting synthesis has important implications for the pro- positive cooperativity), similar to that observed in duction of NAE lipid mediators in eukaryotic cells microsomal membranes (Chapman and Moore, and warrants further attention. 1993b). This suggested that the unusual kinetics were likely a property of the enzyme itself and not a result of the membrane environment. The NAPE 6. Summary and future directions synthase was most active toward linoleic and palmitic acids and least active toward stearic and While our understanding of NAPE/NAE func- myristic acids which was consistent with the relation in plants lags far behind that in animal tive N-acyl fatty acid profile in cottonseed NAPE systems, substantial evidence indicates that NAPE molecular species (Chapman et al., 1999). By con- metabolism in cells of higher plants functions in trast the NAPE synthase enzyme did not exhibit the formation of NAE lipid mediators in a manner biphasic kinetics with respect to its PE substrate similar to the generation of NAEs in various and showed no appreciable differences in activity animal cell types. First, NAE appears to be re- toward ‘natural’ (16:0/18:2 PE, most abundant PE leased extracellularly in plant systems and its accu- in cottonseed membranes) or ‘artificial’ (18:1/18:1 mulation is at the expense of a reduction in cellular PE, not present in appreciable quantities in cotton- NAPE (Chapman et al., 1998). Second, GC–MS seed membranes) PE species (McAndrew and quantification procedures have identified tissue Chapman, 1998). The synthesis of NAPE by the specific differences in types and quantities of purified enzyme preparation did not occur if [14C] NAEs in plant systems (Chapman et al., 1998, palmitoylCoA or [14C] dipalmitoylPC were sup- 1999) and those that appear to function in defense- plied as the acyl donor, and niether ethanolamine related signaling in leaves are medium-chain, satu- nor sphingosine functioned as an acyl acceptor. rated NAEs (e.g. NAE14:0; Tripathy et al., 1999). Chemical modification experiments suggested In addition, PLD-mediated formation of NAE that the NAPE synthase probably forms an acyl from NAPE was reconstituted in tobacco cell enzyme intermediate via a single serine residue as membranes (Chapman et al., 1998), and a survey it catalyzes the formation of an amide bond be- of recombinant plant PLD isoforms suggests that tween the ethanolamine headgroup of PE and the the PLDb and/or g isoforms may be responsible carboxyl of the free fatty acid (McAndrew and for the formation of NAE in vivo (Pappan et al., Chapman, 1998). A serine protease transition state 1998). Even the turnover of NAE appears to be by analogue, diisopropylflourophoshate (DFP), inac- a similar mechanism in plant and animal cells. tivated the purified NAPE synthase in a time- and NAE amidohydrolase activities have been re- concentration-dependent manner. Free palmitic ported to occur in several plant systems, and these acid offered protection from DFP inactivation and may catalyze the ‘inactivation’ of NAEs in vivo. [14C]-DFP incorporation into TCA-precipitable Although much work is still required, the emerging protein. These studies strongly suggested that the similarities suggest that NAE signaling may be an NAPE synthase acts much like a reverse serine evolutionarily conserved transduction pathway protease, and is probably an entropy-driven reac- important for cellular communication in all multi- tion promoted by hydrophobic interactions as cellular eukaryotes. suggested by Kruszka and Gross (1994) for the Certainly, a clearer understanding of the regula- ATP- and CoA-independent synthesis of N- tion of NAE signaling in plants will require a arachidonylethanolaminde. Further detailed char- detailed examination of its turnover. Efforts are 228 K.D. Chapman / Chemistry and Physics of Lipids 108 (2000) 221–229 underway to characterize this turnover in plants, Acknowledgements and preliminary indications are that the biochemical properties of the plant NAE amidohydrolase Work on NAPE metabolism in the author’s is similar (Chapman et al., 1999) to the well-char- laboratory has been supported by the USDA- acterized mammalian enzyme (Cravatt et al., NRICGP, and in part by the Texas Higher Edu- 1996). One potentially important difference is that cation Coordinating Board. the plant enzyme appears to predominantly local- ize to cytosol-enriched fractions, but this may be an artifact of tissue homogenization and cell frac- References tionation. It should be pointed out that DNA sequences with homology to mammalian FAAH Alvarez, M.E., Pennell, R.I., Meijer, P.J., Ishikawa, A., Dixon, are appearing in the databases (e.g. Arabidopsis R.A., Lamb, C., 1998. Reactive oxygen intermediates me- putative amidase, Genbank Accession no. diate a systemic signal network in the establishment of Y10342), and molecular approaches should help plant immunity. Cell 92, 773–784. Anderson, J.D., Bailey, B.A., Dean, J.F.D., Taylor, R., 1990. to clarify the role of the amidohydrolase in NAE A fungal endoxylanase elicits ethylene biosynthesis in to- turnover in plants. One interesting aspect of NAE bacco (Nicotiana tabacum L. cv. Xanthi) leaves. In: Flores, turnover for which there is no data in plants, is H.E., Arteca, R.N., Shannon, J.C. (Eds.), Polyamines and the mechanism(s) for NAE movement across Ethylene: Biochemistry, Physiology and Interactions. membranes. The data do seem to indicate that American Society of Plant Physiologists, Rockville, MD, NAEs accumulate extracellularly in response to pp. 146–156. Bailey, B.A., Korcak, R.F., Anderson, J.D., 1993. Sensitivity perception of pathogen elicitors, and that there to an ethylene biosynthesis-inducing endoxylanase in Nico- are active amidohydrolase activities intracellu- tiana tabacum L. cv. Xanthi is controlled by a single larly. But no information exists for an NAE dominant gene. Plant Physiol. 101, 1081–1088. transport mechanism, and this likely will reveal Bomstein, R.A., 1965. A new class of phosphatides isolated some important factors of NAE regulation in from soft wheat flour. Biochem. Biophys. Res. Commun. plants. 21, 49–54. Cai, S., McAndrew, R.S., Leonard, B.P., Chapman, K.D., Efforts to identify the molecular target(s) of Pidgeon, C., 1995. Rapid purification of cottonseed mem- NAE action in plant cells have only just begun, brane-bound N-acylphosphatidylethanolamine synthase by but there now is support for biological activity of immobilized artificial membrane chromatography. J. Chro- NAEs in plants at physiological concentrations, matog. A 696, 49–62. specifically in signal transduction of pathogen Chapman, K.D., Conyers-Jackson, A., Moreau, R.A., Tripa- elicitor recognition. NAE14:0 at submicromolar thy, S., 1995. Increased N-acylphosphatidylethanolamine biosynthesis in elicitor-treated tobacco cells. Physiol. Plant concentrations is capable of activating plant de- 95, 120–126. fense-gene expression in a manner similar to Chapman, K.D., Moore, T.S., Jr, 1993a. N-acylphos- pathogen elicitors which promote the release of phatidylethanolamine synthesis in plants: occuurrence, NAE (Tripathy et al., 1999). Future research molecular composition, and phospholipid origin. Arch. should focus on identification of NAE receptors Biochem. Biophys. 301, 21–33. in plant membranes, as well as the molecular Chapman, K.D., Moore, T.S., Jr, 1993b. Catalytic properties manipulation of this signaling pathway, so the of a newly-discovered acyltransferase the synthesizes N- acylphosphatidylethanolamine in cottonseed microsomes. precise role of NAE in plant defense signaling can Plant Physiol. 102, 761–769. be determined. It is likely that this NAE signaling Chapman, K.D., Moore, T.S., Jr, 1994. Isozymes of cotton- pathway will be found to be involved in other seed microsomal N-acylphosphatidylethanolamine syn- signal transduction pathways in plant cells. In thase: detergent solubilization and electrophoretic particular, as the roles of PLD become clarified in separation of active enzymes with different properties. plant cell signaling it is likely that other stimulus- Biochim. Biophys. Acta 1211, 29–36. Chapman, K.D., Sprinkle, W.B., 1996. Developmental, tissue- response pathways will be identified which are specific and environmental factors regulate the biosynthesis mediated in part by the metabolism of NAPE and of N-acylphosphatidylethanolamine in cotton (Gossypium NAE. hirsutum L.). J. Plant Physiol. 149, 277–284. K.D. Chapman / Chemistry and Physics of Lipids 108 (2000) 221–229 229

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