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Biochemical and Biophysical Research Communications xxx (2016) 1e7

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Biochemical and Biophysical Research Communications

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Fatty acid esters produced by Lasiodiplodia theobromae function asgrowth regulators in tobacco seedlings

Carla C. Uranga a, Joris Beld b, Anthony Mrse b, Iv�an C�ordova-Guerrero c,Michael D. Burkart b, Rufina Hern�andez-Martínez a, *

a Centro de Investigaci�on Científica y de Educaci�on Superior de Ensenada (CICESE), Carretera Ensenada-Tijuana 3918, Zona Playitas, 22860 Ensenada, B.C.,Mexicob University of California, San Diego, Department of Chemistry and Biochemistry, 9500 Gilman Dr., La Jolla, CA 92093-0358, USAc Universidad Aut�onoma de Baja California (UABC), Calzada Universidad 14418 Parque Industrial Internacional Tijuana, Tijuana, B.C. 22390, Mexico

a r t i c l e i n f o

Article history:Received 17 February 2016Accepted 23 February 2016Available online xxx

Keywords:Trunk disease fungiNMRGC-MS

Abbreviations: NMR, nuclear magnetic resonance;mass spectrometry; FA, fatty acids; FAE, fatty acid esesters; FAEE, fatty acid ethyl esters; LAEE, linoleateethyl ester; SAEE, stearate ethyl ester; OAEE, oleate eGA, gibberellic acid.* Corresponding author.

E-mail addresses: curanga@cicese.edu.mx (C.C. Uredu (J. Beld), amrse@ucsd.edu (A. Mrse), icordovGuerrero), mburkart@ucsd.edu (M.D. Burk(R. Hern�andez-Martínez).

http://dx.doi.org/10.1016/j.bbrc.2016.02.1040006-291X/© 2016 Elsevier Inc. All rights reserved.

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a b s t r a c t

The Botryosphaeriaceae are a family of trunk disease fungi that cause dieback and death of various planthosts. This work sought to characterize fatty acid derivatives in a highly virulent member of this family,Lasiodiplodia theobromae. Nuclear magnetic resonance and gas chromatography-mass spectrometry of anisolated compound revealed (Z, Z)-9,12-ethyl octadecadienoate, (trivial name ethyl linoleate), as one ofthe most abundant fatty acid esters produced by L. theobromae. A variety of naturally produced esters offatty acids were identified in Botryosphaeriaceae. In comparison, the production of fatty acid esters in thesoil-borne tomato pathogen Fusarium oxysporum, and the non-phytopathogenic fungus Trichodermaasperellum was found to be limited. Ethyl linoleate, ethyl hexadecanoate (trivial name ethyl palmitate),and ethyl octadecanoate, (trivial name ethyl stearate), significantly inhibited tobacco seed germinationand altered seedling leaf growth patterns and morphology at the highest concentration (0.2 mg/mL)tested, while ethyl linoleate and ethyl stearate significantly enhanced growth at low concentrations, withboth still inducing growth at 98 ng/mL. This work provides new insights into the role of naturallyesterified fatty acids from L. theobromae as plant growth regulators with similar activity to the well-known plant growth regulator gibberellic acid.

© 2016 Elsevier Inc. All rights reserved.

1. Introduction

The Botryosphaeriaceae are a family of fungi that have beenfound to affect several economically important woody plantsaround the world and are considered trunk disease fungi. Some ofthe symptoms these fungi cause include gummosis, wedge-shapednecrotic cankers in tree wood, and stunted growth [1]. Presently, inVitis vinifera, Lasiodiplodia theobromae (teleomorph Botryosphaeria

GC-MS, gas chromatography-ters; FAME, fatty acid methylethyl ester; PAEE, palmitatethyl ester; PA, free palmitate;

anga), joris.beld@drexelmed.a@uabc.edu.mx (I. C�ordova-art), ruhernan@cicese.mx

ga, et al., Fatty acid esters proical Research Communication

rhodina) has been found to be the most virulent [1,2]. However, itcan also be found as an endophyte or latent pathogen [3]. Manyother Botryosphaeriaceae, including Neofusicoccum parvum, havebeen isolated from V. vinifera and other plant species [4].

Characterization of the metabolites produced by L. theobromaeis critical for understanding the metabolic pathways involvedduring colonization, as well as for the discovery of novel or inter-esting compounds. Lipases have an important function in patho-genicity of fungi (triacylglycerol acyl-hydrolases, E.C. 3.1.1.3) [5].These enzymes are involved in the degradation of cell membranesand storage lipids, and esterification of these with alcohols [6]. Li-pases liberate free fatty acids, which are the starting material formany secondary metabolites such as oxylipins, studied in otherphytopathogenic fungi, [7e9]. Free fatty acids are also a source ofenergy and the acetyl CoA necessary for polyketide-type secondarymetabolites produced by Botryosphaeriaceae [10]. The objective ofthis work was to characterize compounds produced or bio-transformed by L. theobromae in natural substrates, and assess theireffects in plants.

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C.C. Uranga et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e72

2. Materials and methods

Two isolates were kindly provided by Dr. Douglas Gubler fromthe University of California at Davis, (USA), L. theobromaeUCD256Ma (isolated in Madera County, California, USA) [11]; andan isolate of N. parvum, (UCD646So, isolated in Sonoma County).Isolates used belonging to CICESE at Mexico are: L. theobromaeMXL28, Fusarium oxysporum f. sp. lycopersici, and Trichodermaasperellum isolated from grapevine, tomato and carnation plants,respectively.

2.1. Induction of secondary metabolism in L. theobromae

Media for induction of metabolism of both L. theobromae iso-lates consisted of 25 g ground oatmeal powder and 50 mL Vogel'ssalts solution, autoclaved twice. Isolates were inoculated with one1 cmmycelial disc of the fungi grown in potato dextrose agar (PDA).As a negative control, oatmeal without fungus was used. Threebiological replicates for the negative controls and the experimentalconditions were set. Samples were incubated at room temperature(RT) in the dark for a total of 60 days. To assess the production ofcompounds of interest in different carbon sources, fungal isolateswere incubated in 50 mL Vogel's minimal media supplementedwith 5% glucose, 5% grape seed oil, 5% glucoseþ5% grape seed oil, or5% fructose. These were incubated in triplicate for 20 days at 25 �Cin the dark.

2.2. Solvent extraction of fungal incubations

Before extraction, samples were frozen at �80 �C then lyophi-lized for 48 h. A modified Folch extraction [12] was done using asolvent mixture of 75 mL dichloromethane (DCM), 75 mL methanoland 0.01% butylated hydroxytoluene (as antioxidant), and extractedovernight at 4 �C. The samples were placed in a separating funnel toseparate and collect each phase. The organic phases (DCM) wereevaporated with a rotovapor (Buchi R-114) at 45 �C and theremaining oils aliquotted to Eppendorf tubes and stored at �20 �Cuntil analysis.

Thin layer chromatography (TLC) on silica gel sheets (Merck)with a fluorescence indicator was performed on the crude oil ex-tracts and developed using 5% ethyl acetate (v/v) in hexane withtwo sequential chromatographic developments. The chromato-grams were stained with vanillin/H2SO4. The oil extract was sepa-rated on silica gel (Unisil) using step elutions consisting of differentconcentrations of ethyl acetate (0, 5, and 10% EtOAc) in hexane.Fractions with the compounds of interest (eluted in 100% hexane)were evaporated and re-fractionated by preparative chromatog-raphy with a C-18 column (Phenomenex), using a gradient of 100%H2O containing 0.1% formic acid to 100% acetonitrile containing0.1% formic acid. The compound of interest eluted with 100%acetonitrile/0.1% formic acid, was collected, evaporated and re-purified using preparative TLC with a solvent system of 1% ethylacetate in hexane. Pure compound, monitored by TLC, was used formass spectrometry analysis, GC-MS, proton and carbon NMR.

2.3. Nuclear magnetic resonance (NMR) and mass spectrometry

In order to determine the molecular weight and formula of thepurified compound of interest, high resolution mass spectrometrywas performed on an Agilent 6230 ESI-TOF MS. Proton (1H) andcarbon (13C) NMR analyses were obtained from a Varian 500 MHzinstrument equipped with an XSens 2-channel NMR cold probeoptimized for direct observation of 13C. Data was analyzed with theprogram ACD/NMR processor Academic Edition [13].

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2.4. In vitro FAE production (Fischer-Speier esterification) of oatand grapeseed oil

Knowing the nature of the compound, a positive control con-sisted of an in vitro Fischer-Speier esterification [14] of the oat oilfraction extracted, and the grape seed oil used in the incubations.Briefly, 2.5 mL of oil was mixed with 1 mL of ethanol or methanol,to which five drops of H2SO4 were added as a catalyst. The sampleswere placed in sealed glass vials and heated to 100 �C for 30 min.Saturated sodium bicarbonatewas added to neutralize the acid, andthe phases containing FAE were collected and analyzed via GC-MS.

2.5. Gas chromatography-mass spectrometry of crude extracts

All samples, including the positive controls, were analyzed fornaturally produced fatty acid ethyl esters by GC-MS. A standardcurve was created from octadecadienoate (Z, Z) ethyl ester (LAEE)standard (Cayman Chemical) from which concentrations of un-knownswere calculated. The standardwas diluted from625mg/L to40 mg/L in hexanes. Ten mL of all unknowns were dissolved in 1 mLhexaneswithout esterification and analyzed by analytical GC-MS onan Agilent 7890A GC system, connected to a 5975C VL MSD quad-rupole MS (EI), using helium as the carrier gas and a 60 m DB23column, with a gradient of 110 �Ce200 �C at 15 �C/min followed by20 min at 200 �C and 20 min at 240 �C. All compounds were iden-tified viaNIST library searches, andwhere applicable, co-injection ofstandard and comparison with a 37 FAME mix (SigmaeAldrich).LAEE, OAEE ((Z)-9-oleate ethyl ester), SAEE (stearate ethyl ester) andPAEE were purchased as purified standards (Cayman Chemical).

2.6. Effects of FAE on tobacco seed germination and hypocotylgrowth

With the aim to test the effect of the isolated compounds inplanta, we chose tobacco (Nicotiana tabacum), a well-studied plantmodel [15]. Seeds were surface-sterilized in 50% household bleach(8.25% NaOCl) for 1min and rinsed 3 timeswith sterilewater beforeuse. Approximately 100 ml packed volume of seeds were placed inMurashige and Skoog salts (with Gamborg vitamins), 0.8% agar, 3%sucrose and the antifungal Plant Preservative Mix (PPM, Plant CellTechnology Inc.), containing 200 mg/mL of either LAEE, PA, PAEE,OAEE and SAEE emulsified in 0.08% kolliphor-188. All experiments,including negative controls were done in triplicate under naturallighting conditions. The length of the hypocotyl was measured after7e10 days post-sowing by pictures taken with a calibratedOlympus stereo microscope (SZX12) at 7x magnification, usingImage J software [16]. Seedling lengths (N ¼ 30) from cotyledon tipto root tip were measured for each experimental condition.Morphology was assessed and documented 45 days post-dosingand sowing. Concentration dependence was then studied in Mur-ashige and Skoog (MS)þ3% sucrose using a concentration range of3.1 mg/mLe 98 ng/mL for SAEE and LAEE, in triplicate, includingnegative controls. Finally, a germination experiment was done us-ing 1 mg/mL of each FAE, including the known plant growth regu-lator gibberellic acid (GA, “Supergrow” from ConsolidatedChemical) as a positive control, using MS without sucrose toresemble field conditions. A one-way ANOVA followed by a Tukey-HSD post-hoc analysis was performed on the data with pvalues < 0.05 considered significant, using XLSTAT statistical anal-ysis software. Graphs were generated with Graphpad Prism soft-ware. Time-lapse video (Lapse It 2.5 pro) during germination wastaken of a negative control and N. tabacum exposed to 98 ng/mLSAEE under continuous white light.

Supplementary video related to this article can be found athttp://dx.doi.org/10.1016/j.bbrc.2016.02.104.

duced by Lasiodiplodia theobromae function as growth regulators ins (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.104

C.C. Uranga et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7 3

3. Results

3.1. Metabolite identification

Thin layer chromatography (TLC) of the oil portion fromextraction of L. theobromae incubated in oatmeal revealed aprominent band that stained dark green/blue with vanillin/H2SO4.This compound was present in all three biological replicates incu-bated with L. theobromae and absent in the negative controls(Fig. 1A). High resolution mass spectrometry of the compounddetected anMþ H ion at 309.278m/z, the most probable molecularformula being C20H36O2 (Dataset A in Ref. [17]). Proton and carbonnuclear magnetic resonance (NMR) spectra (Fig. 1 in Ref. [17])identified this compound as LAEE. The relative chemical shiftsagree with those published in the literature for LAEE [18,19]. The (Z,Z)-configuration is evident by the average coupling constant ofmultiplet 5.27e5.46 (6.1 Hz), and confirmed to be within the rangefor cis hydrogen coupling in double bonds [20,21]. From L. theo-bromae incubated in 5% glucose þ5% grape seed oil, TLC resultsrevealed a compoundwith the same Rf as LAEE. This was confirmedusing GC-MS by co-injection of LAEE standard with the crudesamples. Further GC-MS analysis demonstrated the presence of avariety of ethyl esters (Table 1) in both isolates of L. theobromaeincubated in oatmeal, not detected in the negative controls. Chro-matograms may be found in Fig. 2,3,4,5 in Ref. [17].

A four-point standard curve using (Z, Z)-9, 12-octadecadienoateethyl ester standard with an R2 value of 1.00 was obtained with thelinear equation y ¼ 3.53E þ 08x � 1.43Eþ06, from which the un-knownswere calculated. Using oat as substrate, LAEEwas themajorFAE produced by L. theobromae. The incubation of L. theobromae for60 days yielded 20.1 ± 1.3 g/L in UCD256Ma and 28.7 ± 7.1 g/L inMXL28 for LAEE. LAEE was also detected in strain UCD256Ma

Fig. 1. Identification of the compound isolated and characterized from L. theobromae. A: TLC ocompound isolated from L. theobromae. C: NIST library standard match of the unknown tograpeseed oil, error bars represent standard error of the mean.

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incubated in 5% glucose þ5% grape seed oil, producing 2.4 ± 0.7 g/L(Fig. 2), as well as in other isolates (Table 1) after 20 days.

No FAE were produced by L. theobromae UCD256Ma whenincubated in 5% fructose. Ethyl palmitate (PAEE) and 1H-2-Benzopyran-1-one, 3, 4-dihydro-8 hydroxy-3-methyl- (mellein)were detected in 5% glucose as the sole carbon source (Table 1, Fig. 3in Ref. [17]) indicating de novo production of PAEE by the fungus.FAE identified by GC-MS, 37 FAME standard comparison and theNIST library are listed in Table 1, several of which have not beenreported previously to be produced by these fungi. In grapeseed oilonly, metabolism shifted towards the production of 9-octadece-noate methyl ester (OAME) (44%) and LAEE (40.9%) (Table 1 inRef. [17]) indicating ethanol production in the absence of glucose.SAEE and PAEE were also produced in the absence of glucose.

3.2. Tobacco seed germination and growth in FAE

Statistically significant inhibitory effects on seed germinationwere observed by all FAE compounds tested at 0.2 mg/mL ascompared to the negative controls except for free palmitate (PA)and PAEE in MS without sucrose (Fig. 2A and B). In MS withoutsucrose, LAEE, OAEE, SAEE and the crude oil caused seedling growthinhibition. In MSþ3% sucrose, LAEE, PAEE, OAEE, SAEE and thecrude extract inhibited growth. However, both LAEE and SAEEinduced growth at lower concentrations in MSþ3% sucrose (Fig. 2Cand D). The effect was clear for LAEE, which increased seedlinglength at 98 ng/mL. SAEE induced growth at the lowest concen-trations tested, with more variability between concentrations.Time-lapse video shows a faster germination rate in N. tabacumexposed to 98 ng/mL SAEE under continuous white light (Video file1), indicating light to be a factor in this process.

Leaf morphology of tobacco seedlings germinated in FAE in

f the negative control (C-) and the compound of interest (Cþ, black arrow). B: GC-MS ofLAEE. D: Graph of LAEE quantification from incubations in oatmeal or 5% glucoseþ 5%

duced by Lasiodiplodia theobromae function as growth regulators ins (2016), http://dx.doi.org/10.1016/j.bbrc.2016.02.104

Table 1Fatty acid esters (FAE) from isolates of Lasiodiplodia theobromae, Neofusicoccum parvum, Fusarium oxysporum and Trichoderma asperellum, identified via GC-MS, 37 FAMEstandard and NIST library comparison. Average areas under the curve values from triplicates are shown for the incubations with fungal isolates. ChEBI identifications are shownwhere applicable.

Compound Positivecontrol, fischeresterification

L. theobromae incubationin oatmeal 60 days

5%Glucose

5% grape-seedoil

Incubation in 5% glucoseþ5% grapeseed oil, 20 days

RT

Oatoil

Grape seedoil

UCD 256Ma

MXL28 UCD256Ma

UCD 256Ma UCD256Ma N. parvumUCD646So

F. ox T. as

Methyl hexadecanoate ChEBI:69187 6 � 106 1.4 � 106 1.4 � 106 2.6 � 106 N/D 1.7 � 105 N/D N/D N/D N/D 12.7Ethyl hexadecanoate (PAEE)

ChEBI:849323.2 � 107 1.9 � 106 5.2 � 107 6.6 � 107 9.1 � 104 1 � 105 2.6 � 106 2.3 � 106 7 � 105 9.2 � 104 13.3

Hexadecanoate, 2-methylpropyl ester N/D N/D 9.5 � 104 1.2 � 106 N/D N/D N/D N/D N/D N/D 15.79-Octadecenoate (Z)- methyl ester

ChEBI:275429.8 � 106 2.4 � 107 1.1 � 106 1.1 � 106 N/D 2.8 � 106 3.1 � 105 2 � 106 N/D 6.9 � 105 17.0

Octadecanoate ethyl ester (SAEE)ChEBI:84936

1.8 � 106 7.9 � 105 1.8 � 106 2.2 � 106 N/D 1.3 � 104 1.6 � 106 7.9 � 105 7.7 � 104 N/D 17.3

9-Octadecenoate (Z), ethyl ester(OAEE) ChEBI:84940

5.3 � 107 3.2 � 107 3.1 � 107 4.2 � 107 N/D N/D 3.0 � 107 2.3 � 107 2.4 � 105 2.3 � 106 18.1

9-Octadecenoate (E) ethyl ester 1.1 � 106 2.5 � 105 1.5 � 106 3.4 � 106 N/D N/D 3.9 � 105 3.3 � 105 N/D N/D 18.39,12-Octadecadienoate (Z,Z)-, methyl

ester ChEBI:690802.3 � 107 7 � 106 2 � 106 3.1 � 106 N/D 6 � 105 2.7 � 104 5.7 � 105 N/D N/D 18.4

9,12-Octadecadienoate (Z,Z) ethylester (LAEE) ChEBI:31572

1.1 � 108 8.9 � 106 7 � 107 1 � 108 N/D 2.6 � 106 6.9 � 106 9.1 � 106 5.5 � 105 2.3 � 105 19.6

9,12,15-Octadecatrienoate(Z,Z,Z)-ethyl ester)ChEBI:84851

2.0 � 106 1.7 � 105 1.3 � 106 2.2 � 104 N/D 7.6 � 104 7.4 � 105 2.8 � 105 N/D N/D 21.7

2H-1-Benzopyran, 3,4-dihydro-(R ± mellein)

N/D N/D N/D 6.9 � 105 4.5 � 105 N/D N/D N/D N/D N/D 23.6

N/D: not detected; F.ox: Fusarium oxysporum; T.as: Trichoderma asperellum; RT: Retention time. Negative controls did not yield FAE, therefore are not included in the table. SeeFig. 2A in Ref. [17].

Fig. 2. Concentration dependence of FAE on tobacco seed germination rates. Seedling length 7e8 days post-planting, N ¼ 30 for each condition, using a one-way ANOVA and a post-hoc Tukey-HSD analysis, p-value < 0.05. A; 0.2 mg/mL FAE or crude oil extract in MS only. B; 0.2 mg/mL FAE or crude oil extract in MSþ3% sucrose. C; LAEE 3.1 mg/mL�98 ng/mL inMS-3% sucrose. D; SAEE 3.1 mg/mL- 98 ng/mL in MSþ3% sucrose. Letters above graphs indicate statistically significant differences or similarities between experimental conditions.

C.C. Uranga et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e74

MSþ3% sucrose was also affected. Leaves showed abnormal elon-gation and bifurcation when exposed to the crude extract or0.2 mg/mL SAEE, and expanded cotyledons with abnormal elon-gation of the first true leaf at lower SAEE concentration (3.1 mg/mL).

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Tobacco seedlings exposed to LAEE, PAEE, SAEE and the crudeextract during germination showed stunted growth and chlorosis(Fig. 3). More examples of effects may be found in Fig. 8, 9 inRef. [17].

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Fig. 3. Morphology of N. tabacum germinated with LAEE, SAEE, PAEE or crude extract of L. theobromae 45 days post-sowing and dosing. A; Negative control. B; 0.2 mg/mL crudeextract from L. theobromae incubated in 5% glucoseþ5% grapeseed oil. C; 0.2 mg/mL LAEE: D; 0.2 mg/mL PAEE. E; 0.2 mg/mL SAEE. F; 3.1 mg/mL SAEE.

C.C. Uranga et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7 5

In the final germination experiment, all FA and FAE were foundto induce germination at 1 mg/mL to varying degrees (Fig. 4). SAEEand LAEE induced germination similarly to gibberellic acid.

Fig. 4. N. tabacum exposed to 1 mg/mL FAE during germination in MS without sucrose.Gibberellic acid (GA) serves as a positive control for comparison. Letters above graphsindicate statistically significant differences or similarities between experimentalconditions.

4. Discussion

Fatty acids and modified fatty acids are important moleculesduring colonization of plants by pathogenic fungi, serving diversefunctions such as energy-sources, signaling, and virulence factors[8]. L. theobromae naturally produces a variety of FAE in plant-derived triglycerides. This is the first report of their production inL. theobromae, and the other fungi studied.

Two Botryosphaeriaceae were found to be able to produce awider variety and higher quantities of FAE than the rest of thetested fungi. FAE production in T. asperellum was lower than L.theobromae and N. parvum. The FAE that affect growth regulation intobacco were produced in higher abundance by the trunk disease

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fungi, and barely by the other tested fungi. For example, SAEE wasnot produced by T. asperellum, and F. oxysporum produced morePAEE than T. asperellum. These differences in fatty acid metabolismmay be a factor that differentiates trunk disease fungi from otherphytopathogenic fungi, or from non-phytopathogens such as T.asperellum.

FAE may act in a variety of plant growth processes. FAE areknown to have various functions in eukaryotes, activating steroidhormone receptors in humans [22], or inducing apoptosis [23]. LAEEand SAEE have been extracted from plants such as Allium sativum(garlic) [18], the purple shamrock Oxalis triangularis [19], and themedicinal plant Moringa oleifera [24]. The biosynthetic machineryleading to these compounds in the plant may involve pyruvatedecarboxylase, alcohol dehydrogenase and lipases [25,26]. Plantsare able to ferment glucose for energy production during flower andpollen development [27e29], and oxygen levels at the ovary areknown to be at zero [30]. Hypoxia is part of dormancyanddormancyrelease in V. vinifera [25] and other fruiting trees, with the produc-tion of cyanogenic glucosides and starch breakdown linked to theonset of flowering [31,32]. Hypoxia is also induced in agriculture ingrapevines and other trees to artificially stimulate bud break andincrease agricultural yields with the use of hydrogen cyanamide[33]. Another cause of low oxygen levels in the plant is excesswatering of roots [34]. Since L. theobromae has an endophytic phasein the plant, both natural dormancy periods and chemically inducedhypoxia in V. vinifera or other treesmay provide the funguswith thehabitat that promotes anaerobic fermentation, resulting in theproduction of ethanol and other alcohols required for fungal lipasesto esterify these to free fatty acids.

Fungi may be using FAE to manipulate plant growth. The abilityof fungi to affect plant growth has been observed in the fungusGibberella fujikuroi, which is known to produce gibberellic acid, aswell as in the fungus Botrytis cinerea, which produces abscisic acid[35,36]. In this work it was shown that Botryosphaeriaceae are ableto produce higher quantities and a wider variety of FAE ascompared to the other fungi studied. LAEE, and SAEE were found tohave significant physiological effects in tobacco, acting as growthregulators during germination and early growth, on par with gib-berellic acid at 1 mg/mL. Althoughmuchwork remains to be done tounderstand the detailed physiological routes affected in the plant, itis proposed that fatty acid esters be considered plant growth reg-ulators due to their ability to affect tobacco germination and earlygrowth.

Acknowledgements

Thanks to CONACyT and UCMEXUS, who provided a doctoralstipend for Carla C. Uranga. Thanks to Dr. Yongxuan Su from thesmall molecule mass spectrometry department at UCSD, EduardoMorales and Dr. Manuel Segovia from CICESE, special thanks to Dr.Katrin Quester from the UNAM in Ensenada for instrumentationsupport. Thanks to Dr. James Nowick from the University of Cali-fornia, Irvine for his support with NMR analysis in this work, andspecial thanks to Claudio Espinosa de los Monteros for help withfigure graphic design.

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