Manipulation of Arabidopsis fatty acid amidehydrolase expression modifies plant growthand sensitivity to N-acylethanolaminesYuh-Shuh Wang*†, Rhidaya Shrestha†‡§, Aruna Kilaru‡, William Wiant‡, Barney J. Venables‡, Kent D. Chapman‡,and Elison B. Blancaflor*¶

*Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401; and ‡Center for Plant Lipid Research,Department of Biological Sciences, University of North Texas, Denton, TX 76203

Edited by Daniel J. Cosgrove, Pennsylvania State University, University Park, PA, and approved June 16, 2006 (received for review May 1, 2006)

In vertebrates, the endocannabinoid signaling pathway is animportant lipid regulatory pathway that modulates a variety ofphysiological and behavioral processes. N-Acylethanolamines(NAEs) comprise a group of fatty acid derivatives that functionwithin this pathway, and their signaling activity is terminated byan enzyme called fatty acid amide hydrolase (FAAH), which hy-drolyzes NAEs to ethanolamine and their corresponding free fattyacids. Bioinformatic approaches led to the identification of planthomologues of FAAH that are capable of hydrolyzing NAEs in vitro.To better understand the role of NAEs in plants, we identifiedT-DNA knockouts to Arabidopsis FAAH (AtFAAH; At5g64440) andgenerated plants overexpressing AtFAAH. Here we show thatseeds of AtFAAH knockouts had elevated levels of endogenousNAEs, and seedling growth was hypersensitive to exogenouslyapplied NAE. On the other hand, seeds and seedlings of AtFAAHoverexpressors had lower endogenous NAE content, and seedlingswere less sensitive to exogenous NAE. Moreover, AtFAAH over-expressors displayed enhanced seedling growth and increased cellsize. AtFAAH expression and FAAH catalytic activity increasedduring seed germination and seedling growth, consistent with thetiming of NAE depletion during seedling establishment. Collec-tively, our results show that AtFAAH is one, but not the only,modulator of endogenous NAE levels in plants, and that NAEdepletion likely participates in the regulation of plant growth.

endocannabinoids � lipids � seedling growth � signaling

The discovery of membrane receptors (cannabinoid receptors) inmammalian brain tissue that bind to marijuana’s principal

psychoactive compound, �9-tetrahydrocannabinol, led to the es-tablishment of the endocannabinoid signaling pathway as a keyregulator of important neurological processes in animals (1). Cen-tral to endocannabinoid signaling are the N-acylethanolamines(NAEs), a group of fatty acids with amide linkages to ethanolamine.N-arachidonoylethanolamine, or anandamide (NAE20:4), an NAEtype present at low concentrations in mammalian tissues, is anendogenous ligand of cannabinoid (CB) receptors (2). Binding ofanandamide to CB receptors triggers a series of events that facilitateneuronal signaling. Although anandamide is the most widely stud-ied NAE in animals, other NAE types have emerged as regulatorsof important physiological processes such as embryo development,cell proliferation, immune responses, and apoptosis (1).

There is accumulating evidence that plants also use NAEs toregulate important physiological processes. This notion is supportedby the identification of NAEs in a variety of plant tissues (3, 4) andthe observation that the levels of NAEs in plants, as in mammals,appear to change quite dramatically under certain growth andenvironmental conditions (5). For instance, NAEs are elevated indesiccated seeds of a variety of plant species (6), but, duringimbibition and germination, NAE levels drop significantly andremain at low concentrations during subsequent seedling growth (7,8). These observations suggest that the rapid metabolism of NAEsduring seed germination might be necessary for normal seedling

development. In fact, exogenous application of N-lauroylethano-lamine (NAE12:0), a naturally occurring plant NAE, inhibits seed-ling growth, alters cell shape, and influences cytoskeletal dynamicsin Arabidopsis (9, 10).

The likelihood that NAEs play a role in plant physiologicalprocesses is strongly supported by the fact that the enzymaticmachinery for the degradation of NAE is conserved between plantsand animals. For example, an enzyme that rapidly hydrolyzes NAEsinto ethanolamine and their corresponding free fatty acids has beencloned from mammals. This enzyme, called fatty acid amidehydrolase (FAAH), belongs to a large group of proteins containinga conserved amidase signature sequence (11, 12). The cloning ofmammalian FAAH has provided a powerful system by which toinvestigate the physiological function of NAEs. For instance, thetargeted disruption of FAAH in mice resulted in hypersensitivity toexogenous anandamide and a 10-fold elevation of endogenousbrain anandamide levels. FAAH knockout mice also displayedphysiological abnormalities that were consistent with disruptedendocannabinoid signaling, such as reduced sensation to pain (13).These studies have provided evidence that NAE signal terminationin mammals is facilitated by FAAH, and that the activity of FAAHis an important factor regulating endocannabinoid signaling.

We recently reported the molecular identification of a functionalhomologue of FAAH in Arabidopsis thaliana that converts a widerange of NAEs to their corresponding free fatty acids and etha-nolamine (14). Functional homologues of the Arabidopsis FAAH(AtFAAH) also were identified in Oryza sativa and Medicagotruncatula, supporting a common mechanism for the regulation ofNAE hydrolysis in diverse plant species (15). To begin to under-stand the in vivo role of NAEs in plants, we generated Arabidopsisplants with altered AtFAAH expression and analyzed their responseto an NAE type that we have previously shown to induce strongmorphological effects on seedlings (9, 10). Here, we demonstratethat the manipulation of AtFAAH activity alters the physiologicalresponses of Arabidopsis seedlings to exogenously applied NAEs.The enhanced seedling growth of AtFAAH overexpressors, thehypersensitivity of AtFAAH knockouts to exogenous NAE, and theincreased expression and enzymatic activity of AtFAAH, whichcoincides with NAE depletion during seed germination, all areconsistent with the notion that these fatty acid amides may benegative regulators of seedling growth. We propose that FAAHmodulates endogenous NAE levels in plants and functions as a

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: FAAH, fatty acid amide hydrolase; AtFAAH, Arabidopsis thaliana FAAH;NAE, N-acylethanolamine; GUS, �-glucuronidase.

†Y.-S.W. and R.S. contributed equally to this work.

§Present address: U.S. Department of Energy Plant Research Laboratory, Michigan StateUniversity, East Lansing, MI 48824.

¶To whom correspondence should be addressed. E-mail: eblancaflor@noble.org.

© 2006 by The National Academy of Sciences of the USA

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metabolic correlate to the endocannabinoid signaling enzymefound in animals.

ResultsFAAH Is Expressed in Different Organs, and Its Expression and ActivityAre Consistent with the Timing of NAE Depletion During Seed Ger-mination and Seedling Growth. Expression of the AtFAAH gene wasevaluated by quantitative RT-PCR (Fig. 1). AtFAAH expressionwas detected in various organs and at different developmental

stages. Transcript levels were plotted relative to those in inflores-cence stems (lowest transcript levels; Fig. 1A). Among the tissuesof mature plants, transcript levels were highest in siliques (18-foldhigher than those measured in the stem). AtFAAH transcript levelsin desiccated seeds were 2.5-fold higher than those in stems,whereas FAAH transcript levels were 4- and 19-fold higher inimbibed seeds and 4-d-old seedlings, respectively (Fig. 1A). Thehigher transcript levels in imbibed seeds and seedlings were con-sistent with the notion that NAE metabolism is activated duringseed germination and seedling development. The generally consti-tutive nature of endogenous AtFAAH expression was consistentwith information in publicly available microarray databases (ref. 16;www.weigelworld.org�resources�microarray�AtGenExpress) ascompiled for At5g64440 by Beisson et al. (ref. 17; www.plantbiology.msu.edu�lipids�genesurvey�FAAH.htm).

To visualize spatial patterns of AtFAAH expression within dif-ferent plant organs, we cloned a 1.8-kb DNA fragment upstream ofthe AtFAAH coding sequence. This fragment included a 1.3-kbpromoter region, 5�-UTR, and the first intron of the AtFAAH wasfused to a �-glucuronidase (GUS) reporter gene. Transgenic plantsharboring the AtFAAH::GUS reporter construct were generatedand their GUS expression patterns examined. Consistent with ourRT-PCR analysis, weak GUS expression was detected in embryosof seeds imbibed for 30 min, whereas strong GUS expression wasobserved in 4-d-old seedlings (Fig. 1B). AtFAAH::GUS expressionin mature plant organs also was consistent with the RT-PCR results.Only weak GUS expression was observed in inflorescence stems,leaves, and flowers (data not shown).

Importantly, the pattern of AtFAAH::GUS expression in seedsand seedlings mirrored the depletion of NAEs during seed germi-nation and seedling growth. In desiccated Arabidopsis seeds, totalNAE content was �2,000 ng per gram, and these levels declinedsignificantly 24–192 h after sowing (Fig. 1C). IncreasedAtFAAH::GUS expression in dissected embryos at 24 and 48 h afterimbibition and 4-d-old seedlings was consistent with the catabolismof total NAEs during germination and early postgerminative seed-ling growth (Fig. 1B). Moreover, the depletion of total seed NAEsduring germination and enhanced AtFAAH::GUS expression wasaccompanied by increased FAAH enzyme activity (Fig. 1D).

Manipulation of Arabidopsis FAAH Gene Expression. We obtainedmutants for AtFAAH (At5g64440) from the SALK T-DNA mutantcollection. Two T-DNA insertional mutants SALK�095108 andSALK�118043 were identified with a T-DNA insertion in the 13thintron and the 17th exon, respectively (Fig. 6, which is published assupporting information on the PNAS web site). OverexpressingFAAH lines were generated by placing the AtFAAH cDNA underthe control of the cauliflower mosaic virus (CaMV) 35S promoter(35S::AtFAAH; Fig. 6). Several independent overexpressing lineswere generated, and Southern blot analysis identified lines withsingle copy of the transgene (data not shown). Three independentT3 FAAH-overexpressing lines (FAAHoe-2, FAAHoe-7, andFAAHoe-11) were chosen for further studies. SemiquantitativeRT-PCR analysis revealed that the two SALK knockout lines hadno detectable FAAH transcripts, whereas overexpressors had ele-vated FAAH transcript levels compared with wild type (Fig. 6).

FAAH-Impaired Arabidopsis Seedlings Exhibit Reduced NAE Amidohy-drolase Activity and Enhanced Sensitivity to Exogenous NAE. We nextanalyzed the NAE hydrolyzing activity of the AtFAAH knockoutsto determine whether the lack of transcript caused a correspondingreduction in enzyme activity. Indeed, cell-free homogenates andmicrosomes isolated from AtFAAH knockouts had reduced NAEhydrolytic activity in vitro, compared with wild type when NAE18:2,NAE12:0 (Fig. 2A), or NAE16:0 (data not shown) was used as asubstrate, suggesting that the endogenous capacity for general NAEcatabolism had been altered in these knockout lines.

Fig. 1. FAAH expression in Arabidopsis. (A) AtFAAH mRNA transcript levelsquantified in seeds, seedlings, and different organs of 6-week-old Arabidopsisplants by quantitative real-time RT-PCR. ACT8 was used to normalize AtFAAHexpression levels and plotted relative to transcript levels in inflorescence stems.AtFAAHwasexpressed inallplantorganswithhighest levelsquantifiedin4-d-oldseedlings and siliques of 6-week-old plants. Data points represent mean � SD oftriplicates of an experiment. (B) AtFAAH::GUS is expressed strongly in embryos24–48 h after imbibition and in 4-d-old seedlings. The depletion of total NAEs ingerminating seeds (C) correlated strongly with increased FAAH enzyme activitytoward NAE12:0 and NAE18:2 (D).

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Despite the lower NAE amidohydrolase activity in these Arabi-dopsis plants, there were no gross changes in overall morphology,although seedling growth and rosette diameter were somewhatreduced in one line (Table 1, which is published as supportinginformation on the PNAS web site). To determine whether Ara-bidopsis lacking endogenous FAAH activity displayed shifts insensitivity toward exogenous NAEs, we germinated AtFAAHknockouts in a range of NAE12:0 concentrations. We showedpreviously that exogenous NAE12:0 resulted in severe inhibitoryeffects on Arabidopsis seedling growth, manifested as reducedcotyledon expansion and shorter hypocotyls and primary roots (9,10). Therefore, the distinct morphological effects induced byNAE12:0 provided us with a clear biological assay by which to testin vivo AtFAAH function and sensitivity to NAE. Wild type andAtFAAH knockouts germinated on media supplemented withNAE12:0 exhibited various seedling growth defects. In comparisonwith wild type, the effects of NAE12:0 on the seedling morphologyof AtFAAH knockouts were more severe as they displayed astronger dose-dependent reduction of cotyledon area (Fig. 2B).The differences between wild type and AtFAAH knockouts weremost pronounced at NAE12:0 concentrations between 20 and 30�M. Upon prolonged exposure (after 2 weeks) to 30 �M NAE12:0,wild-type seedlings normally recovered (9), most likely because ofendogenous AtFAAH activity. However, both AtFAAH knockoutlines were unable to resume growth on NAE12:0 and remainedseverely stunted (Fig. 2C). Collectively, these results confirm thatthe gene, At5g64440, encodes a functional NAE amidohydrolaseand clearly indicate that disruption of this gene leads to predictableenhanced sensitivity toward exogenous NAE12:0.

Overexpression of FAAH Leads to Increased NAE AmidohydrolaseActivity, Reduced Sensitivity to Exogenous NAE, and Enhanced Seed-ling�Plant Growth. Cell-free homogenates from AtFAAH overex-pressors had up to 6.5-fold higher levels of NAE amidohydrolaseactivity compared with wild type (Fig. 3A). We anticipated that

NAE12:0 would not inhibit growth of the AtFAAH overexpressorsto the same degree as the wild type or knockouts. Indeed, seedlingsof AtFAAH overexpressors displayed less sensitivity toward exog-enous NAE12:0 compared with the ‘‘empty-vector’’ controls orwild-type seedlings. Notably, cotyledon and hypocotyl expansion ofthe overexpressors was not significantly inhibited, despite theincreasing levels of exogenous NAE12:0 (Fig. 3 B and C). The mostobvious difference between AtFAAH overexpressors and vectorcontrols in terms of their response to exogenous NAE was duringextended exposures (�18 days) to high levels of NAE12:0 (up to 500�M). Under these conditions, empty vector control seedlings wereseverely stunted, whereas AtFAAH overexpressors proceeded todevelop significantly larger organs (Fig. 3D).

Interestingly, when compared with wild type and empty-vectorcontrols, AtFAAH overexpressors exhibited enhanced growth atdifferent stages of development (Table 1). Seedlings of AtFAAHoverexpressors had generally longer primary roots and hypocotyls,larger cotyledon areas (Fig. 4 A and B; Table 1), and greaterseedling fresh weight (Fig. 7, which is published as supportinginformation on the PNAS web site). In addition to an overallincrease in seedling growth, AtFAAH overexpressors displayedrobust vegetative growth under short or long day conditions (Fig.4C; Table 1) and bolted earlier (Fig. 4D; Table 1). The averageheights of the inflorescences of AtFAAH overexpressors weresubstantially greater compared with wild type (Table 1). Becausethe increased cotyledon area was a distinct phenotype of theAtFAAH overexpressors (Fig. 4B), we measured average epidermalcell size from cotyledons of 11-d-old AtFAAH overexpressors andvector controls using the membrane dye FM 4–64 to clearly markthe outline of the individual epidermal cells. The average epidermalcell size from AtFAAH overexpressors was �25% larger than thatof the vector controls (Fig. 4E). This was due to overexpressorshaving more cells with a larger area than cells from cotyledons of

Fig. 2. Characterization of NAE amidohydrolase activity and NAE sensitivityof AtFAAH knockouts. (A) Reduced NAE amidohydrolase activity in micro-somes of knockouts is indicated by the absence of free fatty acid (FFA) peak.The amount of FFA formation was determined by incubating syntheticNAE18:2 and NAE12:0 with microsomes from seedlings. (B) NAE12:0 induceda dose-dependent reduction in seedling development in Arabidopsis wild typeas indicated by reduced cotyledon area. The effects of NAE12:0 on Arabidopsiscotyledon area was more pronounced in AtFAAH knockouts. (C) Wild-typeseedlings are able to recover from exogenous NAE12:0 2 weeks after germi-nation, whereas AtFAAH knockouts remain severely stunted.

Fig. 3. Characterization of NAE amidohydrolase activity and NAE sensitivityof AtFAAH overexpressors. (A) Increased NAE amidohydrolase activity inmicrosomes of three AtFAAH-overexpressing lines. The amount of free fattyacid formation was determined by incubating synthetic NAE18:2, NAE16:0,and NAE12:0 with microsomes from seedlings. Three independent AtFAAHoverexpressors were able to sustain hypocotyl (B) and cotyledon (C) expansiondespite elevated levels of exogenous NAE12:0. (D) Extended exposure to 500�M NAE12:0 is strongly inhibitory to vector control and wild-type seedlings.AtFAAH overexpressors, on the other hand, display robust growth despite thehigh levels and extended exposure to exogenous NAE12:0.

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control seedlings (Fig. 4F). In summary, overexpression of AtFAAHappeared to have a positive impact on both growth and overallorgan size, suggesting that an increase in capacity for NAE catab-olism promoted plant growth.

FAAH Knockouts and Overexpressors Possess Altered Endogenous NAELevels. In plants, NAEs are readily quantifiable in desiccated seeds,but their levels change dramatically during germination and seed-ling growth (Fig. 1C; refs. 6 and 7). Therefore, we first quantifiedNAEs in desiccated seeds of AtFAAH-altered plants, because thequiescent nature of seeds provided an opportunity to capture the‘‘resting’’ levels of NAEs. We predicted that plants with alteredAtFAAH expression should have modified levels of NAEs in theirdesiccated seeds. Indeed, total NAE content was 30% higher inseeds of the AtFAAH knockout line SALK�095108 (Fig. 5A), andmost of this increase was attributable to higher levels of the majorunsaturated 18C NAE types (NAE18:1, NAE18:2, and NAE18:3)and one saturated NAE type (NAE16:0) compared with wild type(Fig. 5B). There was little difference in the minor saturated NAEspecies (NAE12:0, NAE14:0, and NAE18:0) between knockoutand wild-type seeds (Fig. 5B Inset). On the other hand, AtFAAHoverexpressors exhibited consistently lower levels of total NAEcontent in seeds compared with wild type. For example, seeds ofFAAHoe-11 had about one-half the total NAE content measuredin wild-type seeds (Fig. 5A). These substantial differences in totalNAE content were attributable to the major NAE types, NAE18:1,NAE18:2, NAE18:3, and NAE16:0 (Fig. 5B), whereas the minorsaturated NAE types showed little change (Fig. 5B Inset).

Endogenous NAE levels were also quantified in 8-d-old seed-lings, because the differences in sensitivity to exogenous NAE andenhanced growth of AtFAAH overexpressors become clearly ap-

parent at this stage (Figs. 3 and 4). All genotypes showed asignificant reduction in endogenous NAE levels during germina-tion, as evident from the lower total NAE content of 8-d-oldseedlings (Fig. 5A). Differences among genotypes were not asobvious in seedlings as in desiccated seeds. Seedlings of AtFAAHoverexpressors had �15% lower total NAE content compared towild type, whereas knockout seedlings had total NAE levels thatwere �10% higher than wild type (Fig. 5A). However, on closerinspection of NAE profiles, a clear difference among overexpres-sors, knockouts, and wild type was evident (Fig. 5C). As in seeds,the difference was predominantly in levels of 18C polyunsaturatedNAEs. NAE18:3 and NAE18:2 levels in overexpressors were 40–60% lower than wild type or knockouts. The levels of other NAEtypes were relatively similar among genotypes. These results sug-gested that AtFAAH overexpression selectively lowered polyunsat-urated NAE levels in transgenic seeds and seedlings, and thatdifferences in seedling growth were likely associated with alteredendogenous NAE18:2 (and NAE18:3) levels. Earlier studies indica-ted that NAE16:0 had no effect on seedling growth (9). Here, we

Fig. 4. Enhanced growth of AtFAAH overexpressors. (A) Representativeimages of vector controls and AtFAAH-overexpressing seedlings 11 days aftergermination. Primary root and hypocotyl length is longer (A, arrows) andcotyledon area is larger (B) in the AtFAAH overexpressors. One-month-oldplants of the AtFAAH overexpressors are generally larger than vector controlswhen grown under short days (C) and generally bolt faster (D). The largercotyledon area in AtFAAH overexpressors is due to larger average epidermalcell size (E) and increased number of epidermal cells with a larger cell area (F).(Scale bar in C, 1 mm.)

Fig. 5. Comparison of NAE profiles in desiccated Arabidopsis seeds andseedlings of wild type, At5g64440 knockout (SALK�095108), and AtFAAHoverexpressors (OE11). NAE types were quantified by isotope-dilution massspectrometry and summed for total content (A) or plotted individually (B andC). Values represent mean � SD of three to six independent extractions fromseeds and seedlings of plants that were grown and harvested at the same time(within a 3-month period). Seeds were stored under identical conditions,whereas seedlings were grown under liquid culture for 8 days before NAEquantification. P values were obtained by Student’s t test.

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confirmed that, like NAE12:0, exogenous NAE18:2 indeed reducedseedling growth in a potent dose-dependent manner (Fig. 8, whichis published as supporting information on the PNAS web site).NAE18:2 reduced growth of wild-type and knockout seedlingssubstantially; and AtFAAH overexpressors, as might be predicted,displayed tolerance to the inhibitory effects of exogenous NAE18:2(Fig. 8).

DiscussionThe widespread occurrence of NAEs in plants and the manner bywhich their levels are regulated during seed germination andpostgerminative seedling growth (7) indicate that these fatty acidamides, which are known to have diverse physiological functions inmammals, might also have important roles in plant development(5). However, there has been little information on the function ofthese fatty acid amides in plant physiology. In animals, NAEfunction is terminated by the hydrolysis to free fatty acid andethanolamine, and this reaction is facilitated by FAAH (12). Thecloning of the mammalian FAAH led to a series of important studieson the in vivo consequences of reduced FAAH activity and con-stitutive elevation of endogenous NAEs in animal physiology (13,18). For example, the targeted disruption of FAAH in mice resultedin highly exaggerated behavioral responses to anandamide, whichtypically exerts only transient and weak effects when administeredexogenously (13). Here, the consequences of disrupted FAAH inArabidopsis showed a remarkable similarity to the scenario in mice.Two knockout lines to the AtFAAH gene lacked significant NAEhydrolase activity, and their seedlings displayed enhanced sensitiv-ity to exogenous NAE12:0. On the other hand, seedlings constitu-tively overexpressing AtFAAH exhibited elevated NAE hydrolaseactivity, which was coupled with the ability of these seedlings togrow in exogenous levels of NAE12:0 that would typically inhibitwild-type seedling development (9, 10). Moreover, AtFAAH knock-outs had �30% increase in total seed NAEs, whereas AtFAAH-overexpressing lines showed a 20–50% reduction in total seedNAE. In seedlings, evidence for FAAH-mediated hydrolysis ismore complex and appears to be combined with an alternativepathway for the depletion of NAEs. Nonetheless, our resultsprovide evidence that AtFAAH, like its mammalian counterpart,degrades NAEs in vivo.

Several key observations suggest that NAEs might function asnegative regulators of processes associated with cell expansion andseedling growth. First, seed germination and seedling establishmentdepend upon synchronized cell division and expansion, andAtFAAH expression and NAE hydrolase activity increased duringArabidopsis seed germination and early seedling development con-comitant with the depletion of endogenous NAEs (Fig. 1). Second,elevated levels of exogenous NAE12:0 profoundly disrupted nor-mal cell expansion in seedlings in a dose-dependent and selectivemanner (9, 10). Here we showed that NAE18:2, which is the NAEtype that exhibits the most dramatic decline during seed germina-tion, can also inhibit seedling growth (Fig. 8). Third, constitutiveoverexpression of AtFAAH led to plants with reduced NAE contentin their seeds and seedlings (Fig. 5), especially in terms of polyun-saturated 18C NAEs. Fourth, the reduction in seed and seedlingpolyunsaturated NAE content was accompanied by acceleratedseedling growth and organs that were significantly larger in size(Figs. 3 and 4). This accelerated growth phenotype was observed inmultiple overexpressing lines and persisted at later stages of plantdevelopment (Fig. 4; Table 1). Based on the experimental evidence,we conclude that the metabolism of NAE is associated with cellexpansion during early seedling growth and possibly overall plantgrowth. However, the precise mechanism underlying NAE metab-olism and plant growth may involve a more complex signalingnetwork, because there are multiple types of NAEs in plants, andthese may give rise to alternative products (such as NAE oxylipins;refs. 8 and 19), and�or FAAH may act on alternative substrates(e.g., sn-2 monoacylglycerols in mammals; ref. 20). Future work will

be necessary to determine the precise mechanism of FAAH actionin plants, but it is likely that this enzymatic pathway participates inthe regulation of seedling growth by modulating endogenous NAElevels.

An analysis of levels of individual NAE species revealed that theprincipal NAE types that were lower in seeds and seedlings ofAtFAAH overexpressors were the long-chain 18C unsaturatedspecies (Fig. 5C). NAE18:2, which like NAE12:0 induces stronginhibitory effects on seedling growth when applied exogenously(Fig. 8), was �50% lower in seedlings of AtFAAH overexpressors(Fig. 5C). The reduced NAE18:2 levels indicate that this NAE typemay be more relevant to the enhanced seedling growth phenotypeof AtFAAH overexpressors, and removal of the long-chain unsat-urated NAEs could in part be responsible for the larger size ofAtFAAH overexpressors. Indeed, AtFAAH overexpressors start outwith less overall NAE in their seeds and have lower levels ofunsaturated 18C NAEs during seedling development, suggestingthat the ability to maintain a reduced 18C NAE concentration maybe responsible for accelerated growth relative to wild type. How-ever, it is notable that AtFAAH overexpressors did not display thesame level of tolerance to exogenous NAE18:2 as they did toNAE12:0 (Fig. 8). The basis for these differences in sensitivity is notclear, but it is possible that breakdown�oxidation products for eachNAE type (e.g., lauric acid for NAE12:0 and linoleic acid orNAE-oxylipins for NAE18:2) might somehow influence the overallgrowth response of seedlings to exogenous NAE application and tochanging endogenous NAE levels resulting from AtFAAH overex-pression. Although additional studies will be required to clarifythese issues, our AtFAAH-altered plants, in combination withmetabolite measurements and enzyme activity assays, should allowan in-depth analysis of how specific NAE types influence variousaspects of plant development.

Despite the reduced NAE amidohydrolase activity in AtFAAHknockouts, we did not observe dramatic growth phenotypes in theselines. Because elevation of NAEs by exogenous application showeda dramatic retardation of seedling development (refs. 9 and 10; Figs.2 and 8), we expected that the AtFAAH knockouts would exhibitstunted seedling growth, presumably because of the accumulationof endogenous NAEs. Although total endogenous NAEs werehigher in seeds of AtFAAH knockouts relative to the wild type (Fig.5 A and B), it was surprising that AtFAAH knockout seedlingsappeared capable of depleting seed NAEs to levels similar to wildtype (Fig. 5C), particularly because no detectable NAE hydrolaseactivity was measured in seedling homogenates by using NAE12:0or NAE18:2 as substrates (Fig. 2). However, this does provide anexplanation as to why the growth of knockout seedlings was similarto that of wild type. It also suggests that an alternative pathwayexists for NAE depletion in seedlings. Interestingly, a competingoxidation pathway was identified in cotton seedlings that operatedby 13-lipoxygenase (and allene oxide synthase) to produce NAEoxylipins (8), and it is possible that flux through this pathway isconsiderable during seedling growth. It is also possible that a secondunidentified amidase is responsible for NAE hydrolysis in knock-outs. In this regard, it is worth mentioning that an acid amidase hasbeen identified in vertebrates capable of hydrolyzing NAEs (21),and perhaps a similar mechanism operates in AtFAAH knockoutplants. In any case, taken together, our results demonstrate thatAtFAAH expression influences NAE content in planta, but they alsopoint to additional factors that determine the overall NAE profilesduring seed germination and seedling growth. Additional studieswill be needed to understand the interaction between NAE oxi-dation by lipoxygenase and NAE hydrolysis by FAAH in regulatingNAE flux in seedlings, and the availability of AtFAAH knockoutsand overexpressors will help to tease apart the complexities of NAEmetabolism and the regulation of seedling growth.

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Materials and MethodsPlant Material and Growth Assays. Two T-DNA insertion mutantswere identified from the SALK collection (SALK�095108 andSALK�118043; ref. 22) and provided by the Arabidopsis BiologicalResource Stock Center (Ohio State University, Columbus, OH).The precise location and orientation of the T-DNA inserts wereconfirmed by DNA sequencing of PCR products amplified withT-DNA and gene-specific primers. To constitutively expressAtFAAH, an AtFAAH cDNA (without stop codon) was PCR-amplified from a previously reported expression construct inpTrcHis2 (16) using primers designed for cloning at the SalI�EcoRIsite. To generate the 35S::AtFAAH construct, the AtFAAH cDNAwas placed behind a cauliflower mosaic virus 35S promoter that hadbeen previously introduced into a pCAMBIA-1390 vector (23). Togenerate the AtFAAH::GUS reporter construct, a 1,834-bp DNAfragment that included a 1,364-bp putative promoter region, the5�-UTR and the first intron of AtFAAH was PCR-amplified fromArabidopsis genomic DNA. The PCR product was cloned into aGateway entry vector (pCR8�GW�TOPO TA vector; Invitrogen,Carlsbad, CA) and then introduced into the destination vector,pMDC165 (24). Transgenic Arabidopsis was generated by thefloral-dip method with Agrobacterium tumefaciens harboring thespecific construct.

Seeds of AtFAAH knockouts, AtFAAH overexpressors, wildtype, and empty vector controls were planted and grown ondifferent concentrations of NAE12:0, as described (9). Sevendays after planting, images of the hypocotyls, cotyledons, andprimary roots were captured by using a Nikon DMX 1200 digitalcamera coupled to a Nikon SMZ 1500 stereomicroscope(Melville, NY). In another set of experiments, Arabidopsisseedlings were germinated in liquid culture and after 3 days,NAE18:2 was added to the medium at a final concentration of20–40 �M. Hypocotyl length, primary root length, and cotyle-don area were measured by using Metamorph 4.6 (UniversalImaging, Downington, PA). Statistical analyses were conductedby using Sigma Plot, ver. 6.1, software (SPSS Inc., Chicago, IL).

AtFAAH Expression Analysis. Total RNA was isolated from differentorgans of the plants, seeds, imbibed seeds, and seedlings, asdescribed in ref. 25. Real-time RT-PCR was performed in a SmartCycler II (Cepheid, Sunnyvalle, CA) instrument with a real-timeone-step RNA PCR assay kit (Takara Bio, Tokyo, Japan) usingSYBR green I dye for quantification of PCR products. ACT8 wasused as the internal reference (26) for quantifying relative AtFAAHtranscript levels. The primers used for quantification of mRNAs ofinternal standard (ACT8) and the gene of interest (AtFAAH) were:ACT8 forward (F), 5�-GTTAAGGCTGGATTCGCTGG-3�;ACT8 reverse (R), 5�-GTTAAGAGGAGCCTCGGTAAG-3�;

AtFAAH-F, 5�-CCATCTCAAGAACCGGAGCATG-3�; andAtFAAH-R, 5�-GGTGTTGGAGGCTTGTCATAGC-3�. Theprimers spanned one intron of the genomic sequence, and meltingcurve analysis and agarose gel electrophoresis were used to opti-mize PCR conditions and verify results. The comparative cyclethreshold (CT) method was used to quantify the relative transcriptlevels of AtFAAH in different plant parts and stages (27).

Preparation of Microsomes and Analysis of FAAH Activity. Microso-mal fractions were prepared by differential centrifugation as de-scribed (8). Protein concentrations in cell fractions were estimatedby using BSA as standard according to Bradford (28). FAAHactivities were measured with equal amounts (400 �g) of microso-mal proteins isolated from 4- to 8-day-old seedlings using[1-14C]NAE12:0 or [1-14C]NAE18:2 as substrates. Enzyme reac-tions were carried out, and lipid products were extracted as de-scribed (8). FAAH-specific activity was quantified by radiometricscanning [Bioscan (Washington, DC) system 200 imaging scanner]of lipid-soluble enzyme reaction products separated by thin-layerchromatography (8).

Chemicals. [1-14C] linoleic acid (53 mCi�mmol�1 in ethanol; 1Ci �37 GBq) was purchased from PerkinElmer Life Sciences (Bos-ton, MA), and [1-14C] lauric acid (53 mCi�mmol�1 in ethanol)was from Amersham Pharmacia Biosciences (Piscataway, NJ).Specific types of NAEs were synthesized from respective radio-labeled free fatty acids as described (8). The purity of the NAEsubstrates was �99.5%.

Quantification of NAE Seeds and Seedlings. NAE quantification wasperformed by isotope dilution mass spectrometry from extracts ofdesiccated seeds and 8-d-old seedlings as described (6). Onehundred milligrams of desiccated Arabidopsis seeds (harvestedfrom plants grown at the same time under identical conditions) andseedlings grown in liquid culture were crushed in a ground-glasstissue homogenizer in the presence of hot 2-propanol (70°C) toinactivate endogenous phospholipases (7). A standard mix ofdeuterated NAEs (50 ng each; see ref. 6) was added for quantifi-cation purposes. Total lipids were extracted into chloroform, fil-tered, and fractionated by normal-phase HPLC with a lineargradient of 2-propanol in hexane, as described (7). EndogenousNAEs were quantified against the internal deuterated standards astetramethylsilane–ether derivatives by GC-MS (6).

We thank Mr. Matthew Cotter and Ms. Gia George for assistance withArabidopsis growth measurements. This work was supported by U.S.Department of Energy Grant DE-FG02–05ER15647 (to K.D.C. andE.B.B.).

1. De Petrocellis, L., Cascio, M. G. & Di Marzo, V. (2004) Br. J. Pharmacol. 141, 765–774.2. Devane, W. A., Hanus, L., Breuer, A., Pertwee, R. G., Stevenson, L. A., Griffin, G., Gibson,

D., Mandelbaum, A., Etinger, A. & Mechoulam, R. (1992) Science 258, 1946–1949.3. Chapman, K. D. (2000) Chem. Phys. Lipids 108, 221–230.4. Chapman, K. D. (2004) Prog. Lipid Res. 43, 302–327.5. Blancaflor, E. B. & Chapman, K. D. (2006) in Communication in Plants–Neuronal Aspects

of Plant Life, eds. Baluska, F., Mancuso, S. & Volkmann, D. (Springer, Heidelberg,Germany), pp. 205–219.

6. Venables, B. J., Waggoner, C. A. & Chapman, K. D. (2005) Phytochemistry 66, 1913–1918.7. Chapman, K. D., Venables, B., Blair, R. & Bettinger, C. (1999) Plant Physiol. 120, 1157–1164.8. Shrestha, R., Noordimeer, M., van der Stelt, M., Veldink, G. & Chapman, K. D. (2002) Plant

Physiol. 130, 391–401.9. Blancaflor, E. B., Hou, G. & Chapman, K. D. (2003) Planta 217, 206–217.

10. Motes, C. M., Pechter, P., Yoo, C.-M., Wang, Y.-S., Chapman, K. D. & Blancaflor, E. B.(2005) Protoplasma 226, 109–123.

11. Cravatt, B. F., Giang, D. K., Mayfield, S. P., Boger, D. L., Lerner, R. A. & Gilula, N. B. (1996) Nature384, 83–87.

12. McKinney, M. K. & Cravatt, B. F. (2005) Annu. Rev. Biochem. 74, 411–432.13. Cravatt, B. F., Demarest, K., Patricelli, M. P., Bracey, M. H., Giang, D. K., Martin, B. R.

& Lichtman, A. H. (2001) Proc. Natl. Acad. Sci. USA 98, 9371–9376.14. Shrestha, R., Dixon, R. A. & Chapman, K. D. (2003) J. Biol. Chem. 278, 34990–

34997.15. Shrestha, R., Kim, S.-C., Dyer, J. M., Dixon, R. A. & Chapman, K. D. (2006) Biochim.

Biophys. Acta 1761, 324–334.

16. Schmid, M., Davison, T. S., Henz, S. R., Pape, U. J., Demar, M., Vingron, M., Scholkopf,B., Weigel, D. & Lohmann, J. (2005) Nat. Genet. 37, 501–506.

17. Beisson, F., Koo, A. J. K., Ruuska, S., Schwender, J., Pollard, M., Thelen, J., Paddock, T.,Salas, J., Savage, L., Milcamps, A., et al. (2003) Plant Physiol. 132, 681–697.

18. Cravatt, B. F., Saghatelian, A., Hawkins, E. G., Clement, A. B., Bracey, M. H. & Lichtman,A. H. (2004) Proc. Natl. Acad. Sci. USA 101, 10821–10826.

19. van der Stelt, M., Noordermeer, M. A., Kiss, T., van Zadelhoff, G., Merghart, B., Veldink,G. A. & Vliegenthart, J. F. G. (2000) Eur. J. Biochem. 267, 2000–2007.

20. Bisogno, T., De Petrocellis, L. & Di Marzo, V. (2002) Curr. Pharm. Des. 8,533–547.

21. Tsuboi, K., Sun, Y. X., Okamoto, Y., Araki, N., Tonai, T. & Ueda, N. (2005) J. Biol. Chem. 280,11082–11092.

22. Alonso, J. M., Stepanova, A. N., Leisse, T. J., Kim, C. J., Chen, H., Shinn, P., Stevenson,D. K., Zimmerman, J., Barajas, P., Cheuk, R., et al. (2003) Science 301, 653–657.

23. Wang, Y.-S., Motes, C. M., Mohamalawari, D. R. & Blancaflor, E. B. (2004) Cell. Motil. Cytoskeleton59, 79–93.

24. Curtis, M. D. & Grossniklaus, U. (2003) Plant Physiol. 133, 462–469.25. Dunn, K., Dickstein, R., Feinbaum, R., Burnett, B. K., Peterman, T. K., Thoidis, G.,

Goodman, H. M. & Ausubel, F. M. (1988) Mol. Plant.–Microbe Interact. 2,66–74.

26. An, Y.-Q., McDowell, J. M., Huang, S., McKinney, E. C., Chambliss, S. & Meagher, R. B. (1996) PlantJ. 10, 107–121.

27. Livak, K. J. & Schmittgen, T. D. (2001) Methods 25, 402–408.28. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254.

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