FEBS Letters xxx (2010) xxx–xxx

journal homepage: www.FEBSLetters .org

Lauroylethanolamide is a potent competitive inhibitor of lipoxygenase activity

Jantana Keereetaweep a,1, Aruna Kilaru a,1,2, Ivo Feussner b, Barney J. Venables a, Kent D. Chapman a,*

a University of North Texas, Center for Plant Lipid Research, Department of Biological Sciences, Denton, TX 76203, USAb Georg-August-University, Albrecht-von-Haller-Institute for Plant Sciences, Department of Plant Biochemistry, D-37077 Göttingen, Germany

a r t i c l e i n f o

Article history:Received 30 May 2010Revised 3 June 2010Accepted 7 June 2010Available online xxxx

Edited by Ulf-Ingo Flügge

Keywords:Jasmonic acid (JA)LauroylethanolamideLipoxygenaseLOX inhibitorN-Acylethanolamine

0014-5793/$36.00 � 2010 Federation of European Biodoi:10.1016/j.febslet.2010.06.008

Abbreviations: FAAH, fatty acid amide hydrolase;lauric acid; FFA 18:2, linoleic acid; FFA 18:3, a-linolenknockout; LOX, lipoxygenase; NAE, N-acylethanolamthanolamide; NAE 18:2, N-linoleoylethanolamide; Nthanolamide; NDGA, nordihydro guaiaretic acidpolyunsaturated; PUFA, polyunsaturated fatty acid; W

* Corresponding author. Address: University of NortResearch, Department of Biological Sciences, 1155 UnTX 76203, USA. Fax: +1 940 369 8656.

E-mail address: chapman@unt.edu (K.D. Chapman1 These authors contributed to this work equally.2 Present address: Michigan State University, Depar

Lansing, MI 48824, USA.

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a b s t r a c t

The lipoxygenase (LOX) pathway was proposed to compete with hydrolysis and be partly responsiblefor the metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). Treatment of Arabidopsisseedlings with lauroylethanolamide (NAE 12:0) resulted in elevated levels of PU-NAE species, andthis was most pronounced in plants with reduced NAE hydrolase activity. Enzyme activity assaysrevealed that NAE 12:0 inhibited LOX-mediated oxidation of PU lipid substrates in a dose-dependentand competitive manner. NAE 12:0 was 10–20 times more potent an inhibitor of LOX activities thanlauric acid (FFA 12:0). Furthermore, treatment of intact Arabidopsis seedlings with NAE 12:0 (but notFFA 12:0) substantially blocked the wound-induced formation of jasmonic acid (JA), suggesting thatNAE 12:0 may be used in planta to manipulate oxylipin metabolism.� 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

N-Acylethanolamines (NAEs) are ethanolamide-conjugatedfatty acid derivatives and their metabolism is a central part ofthe endocannabinoid signaling pathway in mammals [1]. As a con-sequence, NAEs regulate a broad range of physiological processesin vertebrates including neurotransmission, satiety, immune func-tion, embryo implantation, and apoptosis [2–5]. The biologicalactivities of NAEs are controlled through a balance between theirformation and degradation, although the precise machinery andmolecular regulation of these processes is only partly understood.

In plants, the functional role(s) of NAE metabolism is (are) onlyrecently being explored [6]. NAEs occur at their highest levels(ppm levels) in desiccated seeds and these levels decline withthe progression of germination and seedling growth suggesting a

chemical Societies. Published by E

FFA, free fatty acid; FFA 12:0,ic acid; JA, jasmonic acid; KO,

ine; NAE 12:0, N-lauroyle-AE 18:3, a/c-N-linolenoyle-; OE, overexpressor; PU,T, wild type

h Texas, Center for Plant Lipidion Circle #305220, Denton,

).

tment of Plant Biology, East

, J., et al. Lauroylethanolamide

possible role in seedling establishment [7]. NAE species in plantscontain 12–18C acyl groups with the unsaturated 18C NAE types(NAE 18:1, NAE 18:2, NAE 18:3) being the most abundant [8,9].Polyunsaturated NAE (PU-NAE; e.g., 18:2, 18:3) species comprisedmore than 75% of the total NAE pool in Arabidopsis seeds, and theirproportionate levels were depleted substantially during seedlingestablishment, in part by a fatty acid amide hydrolase (FAAH;[10–12]). However, more recent studies with faah T-DNA knockoutplants (KO) that lacked FAAH activity showed that the PU-NAEcontent continued to decline with germination and post-germina-tion growth, suggesting the likelihood of an alternative NAE meta-bolic pathway [12]. Previously, activity of lipoxygenase (LOX)toward NAE 18:2 and NAE 18:3 substrates was demonstratedin vitro [13]. Further, Shrestha et al. [11] showed that both hydro-lase and LOX pathways were able to metabolize NAEs in cottonseeds, proposing competing pathways that cooperated in thedepletion of NAEs during seedling growth.

Recent studies showed that N-lauroylethanolamide (NAE 12:0)has potent growth inhibition properties when applied exogenouslyat low micromolar concentrations to Arabidopsis thaliana seedlings[12,14] (see also Fig. 1). Several possible mechanisms for this ac-tion have been suggested including inhibition of phospholipase D(PLDalpha) [15], interaction with phytohormone signaling path-ways [16], and modulation of ABI3 transcript levels [17]. However,NAEs have multiple targets in animal systems and perturbation ofNAE levels is known to affect a broad range of processes in animals[18]. It is likely that NAE metabolism interacts with several differ-ent targets in plant systems, including inhibition of LOX enzymes.

lsevier B.V. All rights reserved.

is a potent competitive inhibitor of lipoxygenase activity. FEBS Lett. (2010),

Fig. 1. Arabidopsis seedlings grown on solid media oriented horizontally containing either NAE 12:0 (35 lM) or NDGA (25 lM) showed significant retardation of seedlingdevelopment when compared with DMSO solvent control (0.1%) (A). faah knockouts (KO) were hypersensitive to NAE 12:0, whereas FAAH overexpressing lines, OE-7 and OE-11, were tolerant to NAE 12:0 compared with wild type (WT) or vector-only controls (VC). Quantification of primary root length of 8-d old seedlings in plates orientedvertically emphasized the marked inhibition of root elongation by NAE and NDGA (B). NAE quantification (C and D) indicated that NAE 12:0 and NDGA increased thepolyunsaturated NAE content. In faah knockouts (KO), which were hypersensitive to NAE 12:0 (compared to WT or FAAH OE), levels of NAE 18:2 and NAE 18:3 weresubstantially higher than those of untreated or treated FAAH OE and WT seedlings. Seedlings with altered NAE metabolism showed similar growth responses on NDGA (A andB) compared with WT and their endogenous PU-NAE levels were similar to each other but higher than the untreated seedlings (C and D). Root lengths are the average andstandard deviation of 30–32 seedlings, and results are representative of multiple experimental trials. PU-NAE quantitative data are the means ± S.D. of three biologicalreplicates.

2 J. Keereetaweep et al. / FEBS Letters xxx (2010) xxx–xxx

LOX is a dioxygenase enzyme commonly found in plant and ani-mal kingdoms and catalyzes the oxidation of polyunsaturated freefatty acids (PUFAs) to produce hydroperoxides [19]. In plants, LOXisoforms mostly exhibit substrate preferences for peroxidation ateither the C9 or C13 position of 18C acyl chain and along with sub-sequent enzymes in the LOX pathway, generate a variety of oxyli-pin metabolites. Perhaps most widely studied is the 13-LOXpathway, which is ubiquitous in plants. Peroxidation, reductionand cyclization of 13-hydroperoxide of a-linolenic acid (FFA

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18:3) followed by b-oxidation leads to the formation of jasmonicacid (JA). This pathway has been shown to be important in woundresponses, defenses against herbivory and some pathogens, and inanther development [20,21]. Though 9-LOXs are less well charac-terized, their role in root development has recently been reported[22]. Further there is mounting evidence for oxidation of esterifiedor amide linked fatty acids in addition to the conventional FFA pre-cursors [13,23,24]. Collectively, the wide range of substrate prefer-ences of LOX isoforms and pathway enzymes create the need to

is a potent competitive inhibitor of lipoxygenase activity. FEBS Lett. (2010),

J. Keereetaweep et al. / FEBS Letters xxx (2010) xxx–xxx 3

consider a broader contextual framework for oxylipin formation,metabolism and function.

Considering the extent of PU-NAE depletion in Arabidopsis seed-lings, even in the absence of the majority of the hydrolase activityin faah knockouts, it is possible that LOX may oxidize NAE 18:2 andNAE 18:3 during seedling growth. Here we expand previous obser-vations that LOX enzymes metabolize NAE 18:2 and NAE 18:3, tocharacterize and compare 9-LOX activity toward these ethanola-mides with 13-LOX activity, and further demonstrate a novel, po-tent inhibition of LOX enzymes in general by a saturatedmedium-chain NAE (NAE 12:0). This phenomenon provides anexplanation for the elevated levels of PU-NAEs in seedlings treatedwith this compound, especially exacerbated in faah knockouts, andalso points to novel uses for this chemical inhibitor at micromolarconcentrations to influence LOX metabolite levels, like JA, inplanta.

Table 1Kinetic parameters of 13- and 9-LOX with different substrates.

Enzyme Substrate Vmax (lmol/h/mg protein) Km (lM) Vmax (Km)

13-LOX NAE 18:2 311 27 11.7NAE 18:3 312 15 20.8FFA 18:2 619 21 29.6FFA 18:3 712 32 22.4

9-LOX NAE 18:2 27 27 1.0NAE 18:3 11 3 3.2FFA 18:2 62 14 4.4FFA 18:3 81 30 2.7

2. Materials and methods

2.1. Chemicals

N-Linoleoylethanolamide (NAE 18:2), N-linolenoylethanola-mide (NAE 18:3), linoleic acid (FFA 18:2), FFA 18:3, 9-lipoxygenase(potato), 13-lipoxygenase (soybean) and lauric acid (FFA 12:0)were purchased from Cayman Chemicals (Michigan). Nordihydroguaiaretic acid (NDGA) was purchased from Fluka. NAE 12:0 wassynthesized from lauroylchloride and ethanolamine and purifiedby organic extraction as described previously [15]. Deuterated JA(D5-JA) was purchased from CDN Isotopes (Canada).

2.2. Plant materials

Arabidopsis seeds were surface-sterilized and then stratified for3 days at 4 �C in the dark for all experiments prior to sowing in li-quid or solid MS medium [17]. Germination and growth was main-tained in controlled conditions with 16-h-light/8-h-dark cycle(60 lmol/m�2/s�1) at 20 to 22 �C. Seedlings grown for four daysin liquid medium were used for LOX activity assays. Seedlingsgrown for eight days in liquid medium in the presence of 35 lMNAE 12:0, 25 lM NDGA or DMSO (0.1% final) were used for PU-NAE quantification. Growth of the seedlings was monitored on so-lid nutrient media.

2.3. Extraction and quantification of PU-NAEs

Total lipids were extracted from �50 mg of seedlings with 2-propanol/chloroform/water (2/1/0.45 [v/v/v]) and PU-NAEs wereseparated by normal phase HPLC (Econosphere™ silica, 5 lm,4.6 � 250 mm), as described previously [9]. Both NAE 18:2 andNAE 18:3 were quantified against deuterated NAE 20:4 internalstandard by GC–MS as their corresponding TMS ethers (derivatizedby BSTFA; Sigma–Aldrich).

2.4. LOX activity and inhibition assays

Enzyme assays for kinetic characterization of 13-LOX and 9-LOX were conducted in 0.1 M borate (pH 9.0) and 0.1 M Tris–HCl(pH 7.2) buffers, respectively, with substrate concentrations rang-ing from 10 lM to 300 lM. Commercially available soybean andpotato LOX enzymes (Cayman Chemicals) were chosen to repre-sent a 13-LOX and 9-LOX activity, respectively. Continual changesin absorbance at 234 nm (Spectronic Genesys 5; Fisher Scientific)due to the formation of conjugated double bond system in hydro-peroxides from PU-NAEs or FFAs (e = 25,000 M�1 cm�1) wererecorded. Absorbance changes at each substrate concentration

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without enzyme were subtracted to account for non-enzymaticoxidation. Auto-oxidation estimated in this way amounted to0.4–2.4% of the enzyme-dependent oxidation for all lipid sub-strates. Rates of reaction (lmol of product h�1 mg protein�1) weredetermined by saturation kinetics (Michaelis–Menten) or by dou-ble reciprocal plots using Graphpad (Prism) software. For verifica-tion of reaction stereochemistry, enzyme-generated lipid oxidationproducts were distinguished from auto-oxidative products by chi-ral phase-HPLC (CHIRALCEL™ OD-H, 2.1 � 150 mm, Diacel Chemi-cal Industries, Ltd, Osaka, Japan). For example, in the formation ofN-linolenoyl(13S-hydroxy)ethanolamine from NAE18:3 catalyzedby soybean 13-LOX, the products contained 97.9% of the S enantio-mer and 2.1% of the R enantiomer when the stereoisomers wereseparated by chiral-phase HPLC. Apparent Km, Ki and Vmax valueswere determined from the means of three replicates. For inhibitionstudies, inhibitors were pre-incubated with enzyme in a 1 ml cuv-ette for 10 min prior to substrate addition. Inhibition constants (Ki)were calculated from saturation-based enzyme assays at varyingconcentrations of either FFA12:0 or NAE 12:0.

Arabidopsis (At)LOX enzyme activities were assayed in crudeprotein extracts of four-day old faahKO and wild type (WT) Arabid-opsis seedlings grown in liquid media. Homogentates were pre-pared by grinding flash-frozen tissues in a mortar in 100 mM K-phosphate, pH 7.2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mMMgCl2, 0.2 mM dodecylmaltoside, and 400 mM sucrose. Crudehomogenates were clarified by centrifugation at 650�g in a SorvallSS-34 rotor (4 �C) and used directly for enzyme assays. Protein con-centration was determined against a bovine serum albumin stan-dard curve (Bradford Protein Assay; BIO-RAD). Inhibitors (orsolvent-only control) were pre-incubated with enzyme extractsin a 1 ml final volume for 10 min prior to substrate addition. Sub-strate-dependent AtLOX activity was determined by measuringproduction of hydroperoxides from appropriate polyunsaturatedlipid substrates indirectly at 500 nm (Lipid Hydroperoxide AssayKit; Cayman Chemicals). Assays with boiled enzyme extract wereused to correct for spontaneous oxidation.

2.5. NAE 12:0 affects JA formation

Eight-day old Arabidopsis seedlings grown in liquid media wereincubated with 35 lM NAE 12:0, 35 lM FFA 12:0 or comparable fi-nal concentration of DMSO only (0.1%) for 24 h prior to woundingby forceps. Subsequent to wounding, seedlings were transferred tomedium with NAE 12:0, FFA 12:0 or solvent (DMSO) for 3 h.Unwounded seedlings were maintained for all treatments as a con-trol. Following 3 h treatment, JA was extracted from the seedlingsby solid phase extraction (NH2-SPE columns, Grace Davison Dis-covery Science, IL) and reverse-phase HPLC (150 � 4.6 mm C18,Nucleosil 120-5, Macherey-Nagel, PA). JA was quantified againsta D5-JA standard by GC–MS as methyl ester (derivatized inethereal diazomethane) [25].

Eight-day old Arabidopsis seedlings grown in liquid media wereincubated with 35 lM NAE 12:0, 35 lM FFA 12:0 or comparablefinal concentration of DMSO only (0.1%) for 24 h prior to wounding

is a potent competitive inhibitor of lipoxygenase activity. FEBS Lett. (2010),

Fig. 2. Different concentrations of FFA 12:0 were incubated with 9- or 13-LOX enzymes to test inhibition of peroxidation of PU-FFA (18:2 and 18:3) and PU-NAE (NAE 18:2and NAE 18:3) substrates. Double-reciprocal plots of enzyme parameters (1/V and 1/[S]) were made using Graphpad Prism software. Competitive inhibition was evident andapparent Ki values were determined from the means of three replicates over four inhibitor concentrations; values are summarized in Table 2.

4 J. Keereetaweep et al. / FEBS Letters xxx (2010) xxx–xxx

by forceps. JA levels were quantified at 30 min, 1 h and 3 h afterwounding as described above.

3. Results and discussion

3.1. NAE 12:0 inhibits seedling growth and accumulates PU-NAEs

In plants, depletion of NAEs was shown to be important fornormal growth and development, which is achieved by FAAH-med-iated hydrolysis (saturated and unsaturated) and/or LOX-mediated

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oxidation [only PU species [6,11]]. At the cellular level, exogenousNAE 12:0 was shown to affect cytoskeletal organization, endo-membrane trafficking, cell wall and cell shape formation [26,27].At the physiological level, NAE action contributed to modulationof seedling growth, influenced responses of plants to biotic andabiotic stress, and interacted with salicylic acid (SA) and abscisicacid (ABA)-mediated signaling pathways [6,12,16,17]. Here, wetested if NAE 12:0 might act as an inhibitor of LOX-mediated oxi-dation of PU-NAEs and how NAE 12:0 compared with that ofNDGA, a potent but non-selective inhibitor of LOX [28].

is a potent competitive inhibitor of lipoxygenase activity. FEBS Lett. (2010),

Fig. 3. Different concentrations of NAE 12:0 were incubated with 9- or 13-LOX enzymes to test inhibition of peroxidation of PU-FFA (18:2 and 18:3) and PU-NAE (NAE 18:2and NAE 18:3) substrates. Double-reciprocal plots of enzyme parameters (1/V and 1/[S]) were made using Graphpad Prism software. Competitive inhibition was evident andapparent Ki values were determined from the means of three replicates over four inhibitor concentrations; values are summarized in Table 2.

J. Keereetaweep et al. / FEBS Letters xxx (2010) xxx–xxx 5

Arabidopsis seedlings that were grown in horizontally-orientedplates containing medium with either NAE 12:0 or NDGA showedsubstantial reduction in growth and abnormalities in seedlingdevelopment (Fig. 1A). Quantification of seedling growth by rootelongation in plates oriented vertically confirmed the reductionin seedling growth in the presence of NAE 12:0 and NDGA(Fig. 1B). The effect of NAE 12:0 (but not NDGA) was overcomeby ectopic overexpression of FAAH (OE7, OE11), and was exacer-bated by the loss of FAAH function (faah T-DNA knockout, KO), pre-

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sumably related to the inherent ability of seedlings to catabolizethe NAE 12:0 growth inhibitor and endogenous NAEs.

In seedlings where growth was inhibited by NAE 12:0 or NDGA,the PU-NAE content in tissues was elevated substantially com-pared with untreated seedlings (Fig. 1C and D). In faah KO seed-lings where hydrolysis activity was severely reduced, NAE 12:0treatment appeared to block the ability to metabolize exogenouslyprovided NAE 12:0 and to deplete endogenous PU-NAE levels. As aconsequence, PU-NAE (both NAE 18:2 and NAE 18:3) content in

is a potent competitive inhibitor of lipoxygenase activity. FEBS Lett. (2010),

Table 2Inhibition constants (Ki) (lM) of NAE 12:0 and FFA 12:0 for 13- and 9-LOX ondifferent substrates.

Enzyme Substrate Inhibitor

NAE 12:0 FFA 12:0

13-LOX NAE 18:2 0.6 ± 0.1 8.4 ± 0.9NAE 18:3 0.7 ± 0.1 14.6 ± 1.8FFA 18:2 0.5 ± 0.1 12.1 ± 1.7FFA 18:3 0.9 ± 0.1 15.0 ± 1.5

9-LOX NAE 18:2 1.2 ± 0.1 14.3 ± 1.6NAE 18:3 0.9 ± 0.1 9.5 ± 1.3FFA 18:2 0.9 ± 0.1 12.3 ± 1.2FFA 18:3 1.3 ± 0.2 11.9 ± 1.8

6 J. Keereetaweep et al. / FEBS Letters xxx (2010) xxx–xxx

NAE 12:0 treated faah KO seedlings was 10 times higher than thatin the untreated seedlings and they remained severely stunted (Fig1A and B), compared with WT and OEs. On the contrary, overex-pression of FAAH facilitated the increased capacity for NAE hydro-lysis, likely reducing the NAE 12:0 inhibitor concentration overtime as well as some endogenous PU-NAEs.

In the case of NDGA, all of the FAAH-altered genotypes were af-fected similar to WT, in terms of development and at the metabo-lite level (Fig. 1); there was a 2–4-fold increase in NAE 18:2 levelsand a 5–8-fold increase in NAE18:3 levels in NDGA-treated seed-lings regardless of capacity for NAE hydrolysis (Fig. 1C and D).Overexpression of FAAH was unable to reduce these PU-NAE levelsto levels in the untreated seedlings, suggesting no specific ability toovercome inhibition by NDGA. These results suggest that LOXactivity toward other PUFAs is important for normal seedlinggrowth and/or that the PU-NAE oxylipin metabolites may be re-quired for normal seedling establishment. In any case, there wasa clear elevation of PU-NAE species in NDGA-treated species andmost dramatically in NAE 12:0-treated faah KO seedlings, consis-tent with our hypothesis that NAE 12:0 is a potent inhibitor ofLOX activity. Moreover, these results support the concept of com-peting hydrolysis and oxidation pathways in the cooperativedepletion of PU-NAEs during normal seedling establishment [6,11].

3.2. LOX-mediated oxidation of PU-NAEs

To address the hypothesis that NAE 12:0 inhibits LOX-mediatedoxidation of PU-NAE, we first compared the enzymatic propertiesof representative 13-LOX (soybean) and 9-LOX (potato) enzymestoward both PU-NAE and FFA substrates to estimate the relative

Fig. 4. NAE 12:0 (10 lM), FFA 12:0 (10 lM) or DMSO (0.2%) were incubated with homogtest for inhibition of lipid hydroperoxide formation (hydroperoxide assay kit; Caymansubstrates. Assays each included 0.2 mg total protein and 100 lM substrate in 0.1 M Trissubstrate, and a further 10 min to assess oxidation. Pilot reactions were conducted to enminimal non-enzymatic oxidation. Quantitative data are the means ± S.D. of three biolo

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ability of each enzyme to contribute to NAE oxidation. Both en-zymes exhibited typical Michaelis–Menten kinetics when initialvelocity measurements were made at increasing PU-NAEs/PUFAsconcentrations. For the 13-LOX, the Km values were similar forNAE 18:2 and FFA 18:2 but were lower for NAE 18:3 (2-fold) whencompared with FFA 18:3 (Table 1). The apparent maximum rates ofreaction for 13-LOX was approximately 2-fold higher for FFA sub-strates, when compared with NAE substrates (Table 1). These re-sults were generally consistent with those reported by Van derStelt et al. [13] where the utilization of NAE 18:2 and NAE18:3by soybean 13-LOX was characterized; however, these earlier stud-ies did not examine whether a 9-LOX type enzyme activity wascapable of utilizing NAE substrates or not. Here we showed that in-deed 9-LOX from potato was able to oxidize PU-NAEs, albeit at alower maximal rate than for corresponding FFAs (Table 1). Perhapsmost notable was the affinity of 9-LOX for NAE 18:3, which wasconsiderably higher than that for FFA 18:3, but this differencewas not seen for the NAE 18:2 and FFA 18:2. Overall, the resultsfrom in vitro studies suggested that FA ethanolamides could serveas plausible substrates for either 9- or 13-type LOXs in planta ,although specific LOX isoforms may need to be examined in the fu-ture for additional enzymological or regulatory differences.

3.3. NAE 12:0 inhibits LOX activity

Following comparative kinetic studies, we tested the effect ofincluding either NAE 12:0 (lauroylethanolamide) or FFA 12:0 in en-zyme reactions to evaluate the influence of these non-substratelipids on LOX activities toward their NAE and FFA substrates. Dou-ble-reciprocal plots of saturation kinetic measurements made withincreasing concentrations of NAE 12:0 or FFA 12:0 showed thatboth types of lipids inhibited both 13- and 9-LOX activities in acompetitive and concentration-dependent manner (Figs. 2 and 3).Inhibition characteristics were summarized in Table 2. In general,LOX enzymes had much lower estimated inhibition constants (Ki)for NAE 12:0 than for FFA 12:0, indicating that NAE 12:0 is a morepotent inhibitor of LOX in vitro than FFA 12:0. This was evident forboth 13- and 9-LOX activities toward either NAE or FFA substrates.NAE 12:0 was approximately 10–20 times more potent than FFA12:0 in all cases, with inhibition in the low micromolar to sub-micromolar concentration range for NAE 12:0. The potent inhibi-tion of 13- and 9-LOX activities by NAE 12:0 was consistent withour hypothesis that NAE 12:0 treatment of seedlings interfereswith LOX-mediated oxidation of PU-NAEs in vivo.

enates of four-day old Arabidopsis seedlings of faah knockouts (A) or wild type (B) toChemicals) from PU-FFA (18:2 and 18:3) and PU-NAE (NAE 18:2 and NAE 18:3)

–HCl, pH 7.2. Reactions were pre-incubated for 10 min with inhibitor before addingsure conditions were saturating. Boiled enzyme controls were used to subtract for

gical replicates.

is a potent competitive inhibitor of lipoxygenase activity. FEBS Lett. (2010),

Table 3NAE 12:0, but not FFA 12:0, inhibits wound-induced JA synthesis in vivo.

Pre-incubation 24 h beforewounding

Post-incubation 3 h afterwounding

JA (ng/gFW)

Control (no wounding) 43 ± 4.4Media only Media only 597 ± 5735 lM NAE 12:0 35 lM NAE 12:0 219 ± 1435 lM NAE 12:0 Media only 230 ± 17Media only 35 lM NAE 12:0 518 ± 3235 lM FFA 12:0 35 lM FFA 12:0 625 ± 6435 lM FFA 12:0 Media only 572 ± 71Media only 35 lM FFA 12:0 659 ± 89

Fig. 5. Eight-day old Arabidopsis seedlings grown in liquid media were treated witheither 35 lM NAE 12:0, 35 lM FFA 12:0 or DMSO equivalent (0.1%) for 24 h prior tomechanical wounding. Jasmonic acid levels were quantified against a D5-JAstandard by GC–MS as methyl ester at 30 min, 1 h and 3 h after wounding.Inhibition by NAE 12:0 was evident at all time points compared with FFA 12:0treatment or control group. Quantitative data are the means ± S.D. of threebiological replicates.

J. Keereetaweep et al. / FEBS Letters xxx (2010) xxx–xxx 7

To examine directly the influence of NAE 12:0 and FFA 12:0 onArabidopsis (At)LOX activities toward PU-NAE and PU-FFA sub-strates, we used crude protein extracts from 4-day old Arabidopsisseedlings. Seedling homogenates likely possess both 13- and 9-LOXactivities since multiple LOX isoforms are expressed at these stages(per publicly available expression data). Indeed inhibition of AtLOXactivities by NAE 12:0 was observed in seedling homogenates(Fig. 4), but at these concentrations there was little or no inhibitionby FFA 12:0. In general, the inhibition of AtLOX activities by NAE12:0 in extracts of faahKO (Fig. 4A) was more pronounced than thatin WT (Fig. 4B) likely due to a greater capacity for NAE hydrolysisby FAAH in WT seedlings allowing for some depletion of inhibitorfrom the reaction. Also, it is likely that the higher levels of lipidhydroperoxide detected in reactions of WT homogenates withPU-NAE substrates (compared to that for faahKO) were due to acombination of hydrolysis of PU-NAEs into PU-FFAs and subse-quent oxidation by AtLOX activities.

The broad-based and potent inhibition of LOX activities by NAE12:0 suggested that this compound might be generally used toinfluence oxylipin metabolism in vivo and perhaps even manipu-late JA levels in plants. To address this possibility, we testedwhether NAE 12:0 treatment of Arabidopsis seedlings could miti-gate the characteristic wound-inducible formation of JA. When 8-day old seedlings were treated with NAE 12:0 (35 lM) for 24 hprior to the mechanical wounding, JA production 3 h after wound-ing was substantially lower than that without NAE treatment (Ta-ble 3). Inhibition was most effective with pre-incubation of tissuesin NAE 12:0, and was only modestly affected by incubation withNAE 12:0 following wounding (post-incubation). By comparison,

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the same concentrations of FFA12:0 were ineffective in modulatingJA levels, whether added pre- or post-wounding. Further, a timecourse of JA formation after wounding showed that NAE 12:0 treat-ment effectively reduced JA levels throughout the peak of accumu-lation (30–60 min; Fig. 5). Together, these results raise thepossibility that NAE 12:0 and/or derivatives thereof might be usedto manipulate plant processes that are dependent upon JA accumu-lation. In fact, NAE 12:0 was shown to delay lipid peroxidation,activity of LOX and superoxide anion production in carnations,thus improving the vase life of fresh-cut flowers [29]. Given themultitude of important processes in plants that depend upon accu-mulation of JA, lauroylethanolamide may find widespread com-mercial applications.

Caution should always be exercised when applying inhibitors toaffect biological processes since non-target effects of compoundsmay complicate interpretation of results. Nonetheless, these stud-ies provide a new, potent class of inhibitors for plant LOXs – theshort/medium chain acylethanolamides. Moreover they providefurther evidence for the cooperative metabolism of NAEs in plantseedlings and support the concept that PU-NAEs may be convertedinto ethanolamide oxylipins in planta. Future studies will be aimedat identification of new NAE–oxylipins and examination of theirphysiological relevance.

Acknowledgements

This work was supported by a grant from the Office of Science,U.S. Department of Energy, Basic Energy Sciences (DE-FG02-05ER15647).

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