Abbreviations: (10E )-traumatin, 12-oxo-(10E )-dodecenoic acid; (9Z )-traumatin, 12-oxo-(9Z )-dodecenoic acid; 13S-HPODE, 13S-hydroperoxy-(9Z,11E )-octadecadienoic acid; 13S-HPOTE, 13S-hydroperoxy-(9Z,11E )-octadecatrienoic acid; 4-OH-traumatic acid, 4-hydroxy-(2E )-dodecenedioic acid; 9,12-hydroxy-(10E )-dodecenoic acid, 9,12-OH-(10E )-dodecenoic acid; 9,12-OH-(10E )-dodecanoic acid, 9,12-hydroxy-(10E )-dodecanoic acid; 9-OH-traumatin, 9-hydroxy-12-oxo-(10E )-dodecenoic acid; traumatic acid, (2E )-dodecenedioic acid.
- •In response to diverse stresses, the hydroperoxide lyase (HPL) pathway produces C6 aldehydes and 12-oxo-(9Z )-dodecenoic acid ((9Z )-traumatin). Since the original characterization of (10E )-traumatin and traumatic acid, little has been added to our knowledge of the metabolism and fluxes associated with the conversion of (9Z )-traumatin into diverse products in response to wounding and herbivory.
- •A liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) method was developed to quantify C12 derivatives of the HPL pathway and to determine their metabolism after wounding and simulated herbivory in Nicotiana attenuata leaves.
- •Ninety-eight per cent of the (9Z )-traumatin produced was converted to 9-hydroxy-(10E )-traumatin (9-OH-traumatin); two-thirds by product recycling through lipoxygenase-2 (NaLOX2) activity and one-third by nonenzymatic oxidation. Thirty-eight per cent of the de novo produced 9-OH-traumatin was conjugated to glutathione, consistent with this oxylipin being a reactive electrophile species. 12-OH-(9Z )-dodecenoic and dodecenedioic acids also showed rapid increases after wounding and simulated herbivory and a role for C12 derivatives as signals in these processes was consistent with their ability to elicit substantial changes in gene expression.
- •These results underscore the importance of metabolite reflux through LOX2, an insight which creates new opportunities for a functional understanding of C12 derivatives of the HPL pathway in the regulation of stress responses.
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In plants, the production of oxylipins from polyunsaturated fatty acids (PUFAs) is immediately induced in response to diverse stresses including wounding, and insect and pathogen attacks (Turner et al., 2002; Farmer et al., 2003; Mueller, 2004; Taki et al., 2005; Matsui, 2006; Browse, 2009; Mosblech et al., 2009). Oxylipins are diverse in structure and they play essential roles as signaling molecules during the plant’s responses to these environmental stresses. For example, jasmonic acid is essential for the induction of defense responses against pathogens and insect herbivores (Farmer et al., 2003; Kessler et al., 2004; Browse, 2005), C6 aldehydes are important signals acting during pathogenesis and plant–insect communication (Croft et al., 1993; Matsui, 2006; Baldwin, 2010), C12 diacids and ω-oxo-acids were originally described as wound-associated hormones (Bonner & English, 1937; Zimmerman & Coudron, 1979), divinyl ethers inhibit mycelial growth and spore germination of some oomycete species (Prost et al., 2005) and phytoprostanes play diverse roles in biotic stress responses (Loeffler et al., 2005).
13-lipoxygenases (13-LOXs) initiate the enzymatic biosynthesis of oxylipins by di-oxygenating PUFAs such as linoleic (18:2Δ9,12; 18:2) and α-linolenic (18:3Δ9,12,15; 18:3) acids to generate 13S-hydroperoxy dienoic (13S-HPODE) and trienoic (13S-HPOTE) acids, respectively. Hydroperoxide lyase (HPL) cleaves 13S-HPODE and -HPOTE to generate the green leaf volatiles (GLVs) hexanal and (3Z )-hexenal, respectively, and 12-oxo-(9Z )-dodecenoic acid ((9Z )-traumatin; compound 1a in Fig. 1) (Vick & Zimmerman, 1976). In Nicotiana attenuata leaves, the supply of 13S-HPODE and 13S-HPOTE for the biosynthesis of hexanal and (3Z )-hexenal requires the activity of the lipoxygenase-2 gene (NaLOX2 ); plants with reduced expression of this gene have a greatly reduced production of GLVs (Allmann et al., 2010). Nicotiana attenuata plants with reduced expression of NaHPL have similarly reduced production of GLVs (Halitschke et al., 2004 and this study).
Hexanal, (3Z )-hexenal and (9Z )-traumatin undergo rapid modifications by diverse enzymatic and nonenzymatic reactions, generating multiple potential chemical signals. These modifications involve, among others, the isomerization of double bonds (Z to E ), the oxidation of the aldehyde groups to carboxyl groups, their reduction to alcohols and their esterification (Grechkin, 2002). These modifications change the physicochemical properties of the molecules, and in some cases their importance in biological processes has been demonstrated (Zimmerman & Coudron, 1979; Ivanova et al., 2001; Allmann & Baldwin, 2010). In the case of (9Z )-traumatin, the Z double bond isomerizes to E to form 12-oxo-(10E )-dodecenoic acid ((10E )-traumatin; compound 1b in Fig. 1), and the aldehyde group can auto-oxidize to form (3Z )- or (2E )-dodecenedioic acid ((3Z )- or (2E )-traumatic acid; compounds 2a and 2b in Fig. 1) (Zimmerman & Coudron, 1979) or be reduced to form (9Z )- or (10E )-12-hydroxy-dodecenoic acid (12-OH-dodecenoic acid; compounds 3a and 3b in Fig. 1) (Grechkin, 2002). (9Z )-traumatin can be oxidized to 9-hydroxy-12-oxo-(10E )-dodecenoic acid (9-OH-traumatin; compound 4 in Fig. 1) either enzymatically by a LOX-mediated mechanism (Gardner, 1998) or nonenzymatically (Noordermeer et al., 2000). A recent study showed that 9-OH-traumatin can be subsequently converted into 4-hydroxy-(2E )-dodecenedioic acid (4-OH-traumatic acid; compound 5 in Fig. 1), 9,12-hydroxy-(10E )-dodecenoic acid (9,12-OH-(10E )-dodecenoic acid; compound 6 in Fig. 1) and 9,12-hydroxy-(10E )-dodecanoic acid (9,12-OH-(10E )-dodecanoic acid; compound 7 in Fig. 1) in pea (Pisum sativum ) seedlings (Mukhtarova et al., 2011). 9-OH-traumatin belongs to a class of oxylipins defined as oxylipin-reactive electrophile species (RES) based on their chemical reactivity, which in turn results from an α-β unsaturated carbonyl group (Gardner, 1998; Farmer & Davoine, 2007; Mueller & Berger, 2009) (Fig. 1). Moreover, the presence of the hydroxyl group at C-4 of the α-β unsaturated carbonyl group increases the reactivity of C-3 to nucleophiles such as thiols and amines (Esterbauer et al., 1976; Uchida, 2003).
In the case of C12 derivatives of the HPL pathway, early experiments have shown that (10E )-traumatin and (2E )-traumatic acid have growth-stimulating and wound-healing activities in plants (Bonner & English, 1937, 1938; English & Bonner, 1937; Zimmerman & Coudron, 1979) and, more recently, 12-OH-(9Z )-dodecenoic acid has been shown to act as a potent stimulator of the mitotic cycle (Ivanova et al., 2001). In recent years, research has been focused primarily on the biochemical and functional characterization of GLVs during herbivore and pathogen attacks, and less attention has been paid to the metabolism and signal capacities of the C12 derivatives of the HPL pathway. As a result, the metabolic fluxes and fates of these molecules under these stress conditions are largely unknown in plants. These shortcomings hamper the development of new hypotheses concerning the potential roles of these molecules as signals. Hence, in this study we present a detailed analysis of the fluxes and metabolism of C12 derivatives of the HPL pathway in N. attenuata plants induced by wounding and simulated herbivory. Part of this analysis is based on a new liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) method developed for the quantification of these metabolites. We reveal new aspects of the biogenesis of C12 derivatives of the HPL pathway and open new perspectives for possible roles of these metabolites in the regulation of stress responses.
Materials and Methods
Plant growth and treatments
Seeds of Nicotiana attenuata Torr. ex S. Watson wild-type (WT), ir-lox2 (line 52-2) (Allmann et al., 2010), ir-lox2/ir-lox3 (Kallenbach et al., 2010) and ir-hpl (line 428-8; Supporting Information Methods S1) plants were germinated on agar plates containing Gamborg’s B5 medium as previously described (Krügel et al., 2002). Plates were maintained in a growth chamber (Snijders Scientific, Tilburg, the Netherlands) at 26°C : 16 h (155 μmol s−1 m−2 light), 24°C : 8 h dark for 10 d. Ten-day-old seedlings were transferred to TEKU pots (Pöppelmann GmbH & Co. KG, Lohne, Germany) with Klasmann plug soil (Klasmann-Deilmann GmbH, Geesten, Germany). After 10 d, seedlings were transferred to soil in 1-l pots and grown in the glasshouse under high-pressure sodium lamps (200–300 μmol s−1 m−2) with a day : night ratio of 16 h (26–28°C) : 8 h (22–24°C) and 45–55% humidity.
Leaf wounding was performed by rolling a fabric pattern wheel three times on each side of the midvein of the leaves of 40-d-old rosette-stage plants, and the wounds were immediately supplemented with water. For fatty acid–amino acid conjugate (FAC) elicitation, the wounds were immediately supplemented with 20 μl of synthetic N-linolenoyl-glutamic acid (18:3-Glu; 0.03 nmol μl−1). Leaf tissue was collected at different times after the treatments and was frozen immediately in liquid nitrogen (N) for subsequent analysis. For C12 treatments, leaves from ir-lox2/ir-lox3 plants were wounded as described above and a 1 : 1 mixture of (9Z)-traumatin:9-OH-traumatin (1 μg each per leaf dissolved in 0.02% aqueous Tween-20) or solvent (control treatment) was immediately applied to the wounds.
Purchased chemicals and synthesis of C12 derivatives of the HPL pathway
(9Z )-traumatin and (10E )-traumatin were purchased from Larodan (Malmö, Sweden); (2E )-traumatic acid was purchased from Sigma (Taufkirchen, Germany). 12-OH-(9Z )-dodecenoic acid and 12-OH-(10E )-dodecenoic acid were synthesized by reduction of 25 μg of (9Z )-traumatin or (10E )-traumatin, respectively, with excess NaBH4 in 1 ml of ethanol for 4 h at 0°C. After acidification with HCl to pH 4, the products were purified by reverse solid-phase extraction (SPE) on a C18 column (Vac C18 3cc; Waters, Eschborn, Germany) by washing with water and eluting with methanol. 9-OH-traumatin was generated in vitro by two different methods: nonenzymatic oxidation (Noordermeer et al., 2000); and processing of N. attenuata leaf extracts using the protocol described in the section ‘In vitro assays with leaf extracts’. 9-OH-traumatin was fractionated by reverse-phase HPLC on a Gemini-NX® column (C18; 3 μm, 150 × 2 mm; Phenomenex, Aschaffenburg, Germany) using methanol and water as running solvents in a gradient mode in a Dionex UPLC system (Dionex, Germering, Germany) equipped with a fraction collector (WPS3000-FC; Dionex). The structures of the synthetic 12-OH-dodecenoic acids and 9-OH-traumatin were confirmed by gas chromatography–mass spectrometry (GC-MS) analysis: the carboxyl group of 12-OH-dodecenoic acids was transmethylated with diazomethane and the terminal hydroxyl group was silylated (trimethylsilyloxy ether (OTMSi)) by N-methy-N-(trimethylsilyl) trifluoroacetamide (MSTFA) (Machery-Nagel, Düren, Germany); the aldehyde group in 9-OH-traumatin was reduced with excess NaBH4, the carboxyl group transmethylated with diazomethane and the hydroxyl groups finally silylated (OTMSi) with MSTFA. GC-MS analyses were performed on a Varian CP-4000 GC coupled to a Varian Saturn 4000 ion trap MS in electron ionization (EI; 70 eV) mode (Varian, Palo Alto, CA, USA). One microliter of the sample was injected using a 1 to 10 split ratio on a DB-5 column (30 m × 0.25 mm i.d.; 0.25-μm film thickness; Agilent, Boeblingen, Germany) with helium at a constant flow of 1 ml min−1 as the carrier gas. The injector was at 260°C. The oven temperature program was: 140°C for 2 min, 180°C at 6.0°C min−1, 20°C min−1 ramp to 240°C and hold for 1 min. Electron impact (EI) spectra were recorded in scan mode from 40 to 400 m/z.
The glutathione (GSH) adduct of 9-OH-traumatin was synthesized by mixing 10 μg of HPLC-purified 9-OH-traumatin with 13 μg of GSH in 1 ml of buffer (10 mM MOPS and 0.02% (v/v) Tween-20; pH 7.0) for 4 h at room temperature. The solution was reduced to c. 50 μl in an Eppendorf SpeedVac® (Eppendorf, Wesseling-Berzdorf, Germany) connected to a high-vacuum membrane pump (Varian SH100; Varian). Then 200 μl of methanol was added to the sample and the 9-OH-traumatin–GSH conjugate was purified by reverse-phase HPLC using a Dionex UPLC system as described for the purification of 9-OH-traumatin.
Analysis of synthetic standards, and detection and isomeric separation of C12 derivatives of the HPL pathway in leaves
Optimization of the multiple reaction monitoring (MRM) parameters for LC-(electrospray ionization (ESI))-MS/MS analysis was carried out by direct injection of single analytes (1 ng μl−1 in 70% (v/v) methanol/water) into the ESI–MS interface. Ionization was performed in the negative mode and the first quadrupole was set on their corresponding [M-H]− masses (Table S1). The third mass analyzer was set in the scan mode, ranging from 50 to 350 m/z. The MS/MS fragmentation patterns of the analytes were obtained by collision-induced dissociation (CID) with argon gas in the second quadrupole with increasing collision energies. The parent > daughter (m/z ) ion transitions that gave the highest intensities were used to optimize the separation and detection of the analytes, and the final parameters used are listed in Table S1.
For the chromatographic separation of the C12 analytes and their Z/E isomers, 10 μl of standard solutions (1 ng μl−1) was injected onto a Gemini-NX® column (C18; 3 μm, 50 × 2 mm; Phenomenex) connected to a pre-column (Gemini-NX; C18, 4 × 2 mm; Phenomenex). As mobile phases, 0.05% : 1% (v/v/v) formic acid : acetonitrile : water (solvent A) and methanol (solvent B) were used. The solvent gradient of the mobile phases was 5% of B for 2 min (pre-run), followed by a linear gradient to 45% of B to 6 min, 45% of solvent B to 22 min, a linear gradient to 98% of solvent B to 24 min, 98% of B to 27 min, a linear gradient to 5% of B to 28 min, and 5% of solvent B to 32 min. The total flow rates were 0.4 ml min−1 for 0.5 min, 0.2 ml min−1 from 0.5 to 26 min and 0.4 ml min−1 to 32 min.
Extraction and quantification of C12 derivatives of the HPL pathway in leaf extracts
For extraction, 0.3 g of frozen leaf material was homogenized in 2-ml tubes containing two steel beads (ASK, Korntal-Muenchingen, Germany) by shaking the tubes in a Genogrinder (SPEX Certi Prep, Metuchen, NJ, USA) for 30 s at 1300 strokes min−1. One milliliter 2 : 1 (v/v) methanol : chloroform spiked with 200 ng of 10-hydroxy-(2E )-decenoic acid (internal standard (IS)) and containing 1% (w/v) butylhydroxytoluene (BHT) as a radical scavenger was added and the samples were vortexed for 10 min. After centrifugation for 10 min at 4°C (16 100 g), the organic phase was collected and the residual plant material was re-extracted with 0.5 ml of 2 : 1 (v/v) methanol : chloroform. The organic phases were combined and the solvent was evaporated under reduced pressure avoiding complete dryness. The samples were reconstituted in 0.2 ml of 70% (v/v) methanol : water for analysis.
Analysis was performed on an LC-(ESI)-MS/MS system (Varian 1200 Triple-Quadrupole-LC-MS system; Varian). Ten microliters of the sample was injected onto a ProntoSIL® column (C18; 5 μm, 50 × 2 mm; Bischoff, Leonberg, Germany) connected to a precolumn (C18; 4 × 2 mm; Phenomenex). As mobile phases, 0.05% : 1% (v/v/v) formic acid : acetonitrile : water (solvent A) and methanol (solvent B) were used in a gradient mode with the following conditions: time : concentration (min:%) for B: 0.0 : 5; 1.0 : 5; 8.0 : 98; 16.0 : 98; 17.0 : 5; 20.0 : 5; time : flow (min:ml min−1): 0.0 : 0.4; 0.5 : 0.2; 15.5 : 0.2; 16.0 : 0.4; 20.0 : 0.4. Compounds were detected in the ESI negative mode and MRM (Table S2).
The response curves of the synthetic or purified C12 analytes vs the IS (10-hydroxy-(2E )-decenoic acid) were determined and used to calculate the response factors (Fig. S4). For quantification of endogenous C12 derivatives, the corresponding peak areas obtained in the LC chromatogram were integrated and divided by the peak area of the IS, and the values were corrected by the respective response factor. For determination of the linearity and the sensitivity of the method, the standards of all C12 analytes were dissolved at different concentrations in a leaf matrix derived from unelicited ir-lox2 plants. Linearity (as expressed by the correlation coefficient (r )) was calculated by the method of least squares and sensitivity was calculated by determining the limit of detection according to the calibration curve method (Fig. S2).
In vitro assays with leaf extracts
Per assay, one 50-mm² leaf disc was cut from rosette-stage N. attenuata plants with a cork borer and was gently and thoroughly crushed with a pestle in a 1.5-ml tube containing 0.2 ml of reaction buffer (10 mM MOPS and 15% (w/v) glycerol; pH 6.8). The sample was centrifuged at 4°C for 5 min at 2300 g to pull down tissue debris and the supernatant was kept on ice. The amount of protein in the extract was quantified using the Bio-Rad Protein Assay kit (Bio-Rad, München, Germany) and BSA as a standard. One microgram of either (9Z )-traumatin or (10E )-traumatin (dissolved in 10 μl of 0.02% (v/v) Tween-20 : water) was added to 200 μl of reaction buffer containing 100 μg of total protein and the reaction was carried out at room temperature for different times. The reaction was stopped by the addition of 1 ml of 2 : 1 (v/v) chloroform : methanol containing 1 μg of 10-hydroxy-(2E )-decenoic acid (IS) and 1% (w/v) BHT. After centrifugation (1 min at 16 100 g), the supernatant was collected and concentrated under a stream of N, avoiding complete dryness, and the samples were reconstituted in 1 ml of 70% (v/v) methanol : water for LC-MS/MS analysis as described previously in the section ‘Extraction and quantification of C12 derivatives of the HPL pathway in leaf extracts’.
For LOX activity inhibition, a final concentration of 10 mM 3,4-dihydroxy-phenylethanol (DHPE; Sigma) was used in the reaction. Leaf extracts were heat-inactivated by incubation at 95°C for 5 min.
Analysis of surface-deposited C12 derivatives of the HPL pathway
Unelicited and elicited leaves from rosette-stage N. attenuata plants were cut at the base of the petiole, weighed, and washed for 1 min in 8 ml of methanol. Then 500 ng of 10-hydroxy-(2E )-decenoic acid (IS) and 1% (w/v) BHT were added to the 8-ml wash. The remaining leaf material was extracted with 2 : 1 (v/v) methanol : chloroform containing 500 ng of 10-hydroxy-(2E )-decenoic acid (IS) and 1% (w/v) BHT. The solvent was evaporated under a stream of N, avoiding complete dryness, and both leaf wash and leaf samples were reconstituted in 0.5 ml of 70% (v/v) methanol : water for analysis by LC-MS/MS as described above in Extraction and quantification of C12 derivatives of the HPL pathway in leaf extracts.
Total RNA was extracted based on the method of Kistner & Matamoros (2005) and its quality checked by spectrophotometry (NanoDrop, Wilmington, DE, USA). Genomic DNA was removed by DNAse treatment following commercial instructions (Turbo DNase; Ambion), RNA was cleaned up using RNeasy MinElute columns (Qiagen, Hilden, Germany) and the RNA quality was checked with the RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA) using an Agilent 2100 Bioanalyzer. Total RNA was used to generate labeled cRNA with the Quick Amp labeling kit (Agilent) and the yield was determined spectrophotometrically (NanoDrop). Labeled cRNA was hybridized using the Gene Expression Hybridization kit (Agilent) onto 44 K custom-designed 60-mer N. attenuata Agilent microarrays (sequences available upon request) containing 43 533 sequences. Microarrays were hybridized overnight at 65°C and slides were washed with the Gene Expression Wash Buffer kit (Agilent) as outlined in the One-Color Microarray-Based Gene Expression Analysis manual (Agilent). Three biological replicates were used per treatment with a total of six arrays. Arrays were scanned with an Agilent G2565BA scanner and image data were acquired with the Agilent scan control software (version A.7.0.1 for the B scanner). Data were extracted using the Agilent feature extraction software (version 9.5) and analyzed with the significance analysis of microarrays (sam) software (Tusher et al., 2001). The q-values for each gene corresponded to a computed false discovery rate (FDR) of 3.3%. Significant changes in gene expression were considered when the log2(fold change; treatment vs control) was > 1 or < −1 and q-values < 0.035 (according to the FDR value calculated by sam).
Statistics were calculated using the spss software version 17.0 (SPSS, Chicago, IL, USA).
Development of a method for the analysis of C12 derivatives of the HPL pathway by LC-MS/MS
The standards (9Z )-traumatin, (10E )-traumatin, 12-hydroxy-(9Z )-dodecenoic acid, 12-hydroxy-(10E )-dodecenoic acid, 9-OH-traumatin, (2E )-traumatic acid, (3Z )-traumatic acid and 10-hydroxy-(2E )-decenoic acid (used as internal standard) were either purchased or synthesized (see the Materials and Methods section). These standards were first directly injected into the ESI–MS interface to determine their [M-H]− parent ion and their parent > daughter (m/z ) ion transitions by collision-induced dissociation (CID) using increasing voltage energies. The parent > daughter (m/z ) ion transitions giving the strongest intensity at fixed collision energies were selected (Tables S1,S2) for MRM during the chromatographic separation of the standards by LC. This separation was performed by reverse-phase LC and the method was first established for the separation of the E and Z stereoisomers of the standards (Fig. 2a).
Next, to determine the C12 derivatives of the HPL pathway that accumulated endogenously in leaves of N. attenuata plants, the method was applied to the analysis of leaf extracts from unelicited and wounded WT plants. (9Z )-traumatin, 9-OH-traumatin, (2E )-traumatic acid, (3Z )-traumatic acid, and 12-OH-(9Z )-dodecenoic acid were detected while (10E )-traumatin, 12-OH-(10E )-dodecenoic acid, 4-OH-traumatic acid, 9,12-OH-(10E )-dodecenoic acid and 9,12-OH-(10E )-dodecanoic acid were either absent or below the limit of detection (LoD; Table S2).
Based on these results, a shorter method that did not discriminate between the dodecenedioic acid isomers ((2E )- and (3Z )-traumatic acids) was developed for the quantification of C12 derivatives in a large number of samples. When this method was used, all C12 analytes eluted from the column within 13 min (Fig. 2b). The C12 analytes presented a linear response, with an r value of 0.99 and LoD values of c. 1 ng in the column (Fig. S2). The extraction method was validated by performing 10 biological replicates of N. attenuata unelicited and wounded leaves after 60 min of the treatment. For each replicate, 0.3 g of leaf tissue was extracted and analyzed by LC-MS/MS. The standard deviations of the calculated amounts were below 10% of the average values for all detectable compounds. To determine the extraction recovery rate, the residual leaf material obtained after the extraction protocol was re-extracted and analyzed for the presence of C12 derivatives. The recovery rates were > 98% for all the molecules tested.
To corroborate the endogenous production of 9-OH-traumatin, leaf extracts from N. attenuata plants were supplied with (9Z )-traumatin and the products of the reaction were analyzed by GC-MS. Fig. 2(c) shows a chromatogram and the MS spectrum corresponding to 9-OH-traumatin. Formation of 11-OH-traumatin could not be detected in leaf extracts; however, it was detected when (9Z )-traumatin was subjected to auto-oxidation in vitro (Fig. S1), as previously described (Noordermeer et al., 2000).
9-OH-traumatin is the most abundant C12 derivative accumulating after wounding and FAC elicitation in N. attenuata leaves
The endogenous accumulation of C12 molecules was first quantified in leaves of WT N. attenuata after wounding and simulated herbivory. For the latter treatment, wounds were supplemented with the fatty acid–amino acid conjugate (FAC) N-linoleyl-glutamic acid (18:3-Glu), a major elicitor of herbivore responses in this plant species (Halitschke et al., 2001). The accumulation of C12 molecules was quantified in both treated (local) and distal (systemic) leaves.
In unelicited leaf tissue of early rosette-stage N. attenuata plants (40 d old), 9-OH traumatin accumulated to c. 2 nmol g−1 FW while (9Z )-traumatin, 12-OH-(9Z )-dodecenoic acid and dodecenedioic acids accumulated to < 0.1 nmol g−1 FW (Fig. 3). Fifteen minutes after wounding and FAC elicitation, 9-OH-traumatin concentrations increased on average to 8 and 10 nmol g−1 FW, respectively, and then decreased to reach on average 3 nmol g−1 FW after 8 h (Fig. 3). The concentrations of dodecenedioic acids and 12-OH-(9Z )-dodecenoic acid also increased several-fold rapidly after wounding and FAC elicitation; however, the absolute amounts remained below 0.25 nmol g−1 FW (Fig. 3). Analysis of earlier time-points (within 5 min) failed to show a rapid burst of (9Z )-traumatin after induction by wounding and FAC elicitation (data not shown), suggesting that this HPL product is rapidly utilized after production. In systemic leaves, no significant changes in the concentrations of these molecules were detected (data not shown).
NaLOX2 and NaHPL supply the substrates for the biosynthesis of (9Z)-traumatin
To validate the analysis of the C12 derivatives of the HPL pathway in N. attenuata leaves, ir-lox2 plants with reduced expression of NaLOX2 (Allmann et al., 2010) and ir-hpl plants with reduced expression of NaHPL (Methods S1, Figs S5, S6 and Table S4) were also analyzed. In unelicited leaves of ir-lox2 plants, 9-OH-traumatin accumulated to < 0.2 nmol g−1 FW (Fig. 4). Moreover, after wounding and FAC elicitation, the concentrations of this C12 metabolite remained largely unchanged and the absolute concentrations were below 0.2 nmol g−1 FW (Fig. 4). (9Z )-traumatin, 12-OH-(9Z )-dodecenoic acid and dodecenedioic acids were either not detected or accumulated to very low concentrations in leaves of ir-lox2 plants (Fig. S3).
Analysis of ir-hpl plants showed that the concentrations of 9-OH-traumatin were reduced to c. 50% of WT concentrations in unelicited leaves and that they were increased twofold after wounding (Fig. 4a). After FAC elicitation, the concentrations of this metabolite increased but at lower rates than in WT, and attained WT concentrations after 2 h of treatment (Fig. 4b). These results were consistent with a 50% reduction in green leaf volatile production in ir-hpl plants (Table S4) and therefore with residual HPL activity in these plants. The concentrations of the remaining C12 derivatives were also reduced in leaves of ir-hpl plants compared with the WT (Fig. S3).
The majority of 9-OH-traumatin is formed from (9Z)-traumatin via NaLOX2 activity
The mechanism of 9-OH-traumatin formation from (9Z )-traumatin in plants has been a matter of controversy, with some results supporting the enzymatic (Gardner, 1998) and others the nonenzymatic biogenesis of this metabolite (Noordermeer et al., 2000). To investigate the mechanism of 9-OH-traumatin formation in N. attenuata, equal amounts of proteins from leaf extracts of WT and ir-lox2 plants were incubated with either (9Z )-traumatin or (10E )-traumatin and the reaction products were analyzed by LC-MS/MS.
Consistent with the nonenzymatic formation of 9-OH-traumatin from (9Z )-traumatin (Noordermeer et al., 2000), approximately one-third of de novo produced 9-OH-traumatin in WT leaf extracts was generated immediately (first phase: T0) after supplying the substrate; however, the remaining two-thirds required an incubation period of at least 15 min (second phase; Fig. 5a). When leaf extracts of ir-lox2 plants were supplied with (9Z )-traumatin, the amount of 9-OH-traumatin produced immediately was similar to that in WT leaf extracts; however, the second phase of accumulation was suppressed (Fig. 5a). Consistent with the formation of the major fraction of 9-OH-traumatin by enzymatic mechanisms, the heat inactivation of WT leaf extracts suppressed the accumulation of 9-OH-traumatin (Fig. 5b) and co-incubation of WT leaf extracts with the LOX inhibitor 3,4-dihydroxy-phenylethanol (DHPE) also suppressed its accumulation during the second phase of the reaction (Fig. 5b). When (10E )-traumatin was used as the substrate, de novo formation of 9-OH-traumatin in WT and ir-lox2 leaf extracts was 10 times lower compared with its formation from (9Z )-traumatin (Fig. 5c).
It has been previously shown that the co-incubation of soybean (Glycine max) LOX-1 in vitro with (3Z )-nonenal and C18 hydroperoxides stimulated the activity of this enzyme toward formation of 4-OH-(2E )-nonenal (4HNE) (Gardner & Grove, 1998). To evaluate whether the oxidation of (9Z )-traumatin by NaLOX2 was indirect and depended on the accumulation of C18-hydroperoxides, equimolar amounts of (9Z )-traumatin and C18-hydroperoxides were added to leaf extracts of WT and ir-lox2 plants. The accumulation of 9-OH-traumatin was not affected by the added C18-hydroperoxides (data not shown).
9-OH-traumatin forms conjugates with GSH during the wound and FAC elicitation response
9-OH-traumatin is an oxylipin-RES (Gardner, 1998) and as a soft electrophile it is liable to form Michael-type adducts with, for example, the sulfhydryl groups of abundant nucleophilic targets, such as reduced glutathione (GSH) (Farmer & Davoine, 2007; Mueller & Berger, 2009). GSH is an important intracellular redox buffer that accumulates at 1–5 mM in leaves and controls the concentrations of reactive molecules (Mueller & Berger, 2009). To first evaluate whether 9-OH-traumatin forms adducts with GSH, purified 9-OH-traumatin was incubated in vitro with commercial GSH and the reaction products were analyzed by LC-MS/MS. Formation of the 9-OH-traumatin–GSH conjugate occurred in vitro and, after 4 h of reaction at room temperature, > 50% of the initial amount of 9-OH-traumatin reacted with GSH (data not shown). To analyze the endogenous formation of this adduct, leaves of WT and ir-lox2 N. attenuata plants were wounded and the levels of 9-OH-traumatin-GSH accumulation were quantified (Fig. 6a). In WT plants, 9-OH-traumatin-GSH adducts were < 0.05 nmol g−1 FW in unelicited leaves; however, their concentrations increased rapidly after the treatments to reach c. 9 nmol g−1 FW within 1 h (Fig. 6a). In ir-lox2 plants, 9-OH-traumatin-GSH was undetected in unelicited leaves and accumulated at < 0.3 nmol g−1 FW after wounding (Fig. 6a).
9-OH-traumatin is not deposited on the leaf surfaces of N. attenuata plants
In addition to conjugation, cells can control the effects of reactive molecules by depositing them outside cells; for example, on the leaf surface. To investigate whether this was the case for 9-OH-traumatin, the surfaces from unelicited and wounded leaves were washed with methanol and both the washes and the surface-washed leaves were analyzed (Fig. 6b). In unelicited leaves, 9-OH-traumatin and 9-OH-traumatin-GSH accumulated primarily in the surface-washed leaf, with lower amounts in the wash (time 0; Fig. 6b), while the remaining C12 derivatives were detected at very low levels (< 0.1 nmol g−1 FW) in both fractions (Fig. 6b). At 15 min after wounding, 9-OH-traumatin was recovered from the wash and the surface-washed leaf extract in comparable amounts (c. 4 nmol g−1 FW) whereas 9-OH-traumatin-GSH was detected in higher amounts (6 vs 1 nmol g−1 FW) in the surface-washed leaf (Fig. 6b). The remaining C12 derivatives were c. 2 times more abundant in the leaf extract than in the wash (Fig. 6b). These results suggested that 9-OH-traumatin was retained in the cells and that after wounding it probably became accessible to methanol through the damaged tissue during the leaf wash.
Formation of 9-OH-traumatin by NaLOX2 activity establishes a major flux for the production of reactive C12 derivatives of the HPL pathway
Calculation of the rates of accumulation of the C12 derivatives of the HPL pathway within 15 min of the wound response showed that on average 1 g of leaf converted 850 pmol min−1 of (9Z )-traumatin to 9-OH-traumatin, with approximately two-thirds of this conversion being catalyzed by NaLOX2 activity and the remaining one-third nonenzymatically (Fig. 7). It was found that 380 pmol min−1 of the 9-OH-traumatin was conjugated to GSH and the remaining (9Z )-traumatin was converted to (2E )-traumatic acid (7.9 pmol min−1), (3Z )-traumatic acid (2.1 pmol min−1) and 12-OH-(9Z )-dodecenoic acid (1.2 pmol min−1; Fig. 7).
C12 derivatives of the HPL pathway induce changes in gene expression
To evaluate the signaling capacity of the C12 derivatives of the HPL pathway during the wound response, leaves of plants with reduced expression of NaLOX2 and NaLOX3 (ir-lox2/ir-lox3; Kallenbach et al., 2010) were used. In this case, doubly silenced lines were chosen to minimize the effects of endogenously produced C12 derivatives and jasmonates after wounding. Thus, ir-lox2/ir-lox3 plants were wounded with a fabric pattern wheel, and either a 1 : 1 mixture of (9Z)-traumatin : 9-OH-traumatin or solvent (control treatment) was immediately applied to the wounds. In this case, 1 μg of each component was applied per leaf; amounts that corresponded to the endogenous amounts produced by leaves after wounding (Fig. 3). Total RNA was extracted from leaves at 120 min after the treatments and used for analysis of gene expression by microarray hybridization (see the Materials and Methods section).
The results of the analysis showed that, after the treatment with C12 derivatives, 320 genes showed significant changes in expression (−1 > log2(fold change) > 1; q-value < 0.035; FDR 3.3%) compared with the control treatment, with five genes down-regulated and the remainder up-regulated. A subset of these genes is presented in Table 1 and the complete list of genes in Table S3. Among the genes with known function that were most strongly up-regulated were N. attenuata homologs of glutathione S-transferase (GST), serine incorporator 1 (SERC1), monodehydroascorbate reductase (MDAR), protein disulfide isomerase (PDI) and a thioredoxin domain-containing protein; genes associated with oxidative stress responses in plants and animals (Table 1; see the Discussion section). Among the up-regulated genes were also four homologs of basic-helix-loop-helix (bHLH)-containing domain transcription factors and homologs of the defense-associated genes PATHOGENESIS-RELATED PROTEIN 5 (PR5 ), proteinase inhibitor (PI ) and a disease resistance response gene (Table 1). Among the signaling components, homologs of ETHYLENE RECEPTOR 1 (ETR1 ), receptor-like protein kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1)-like 3 (BRL3 ) and MITOGEN-ACTIVATED KKK3 (MKKK3 ) were also up-regulated (Table 1). Among the down-regulated genes was BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1; Table S3).
|Gene ID||log2(FC)||SD||q-value||Gene annotation|
|Na_30516||3.3||0.92||< 0.001||Unknown function|
|Na_24874||3.1||0.84||< 0.001|||Q7REH6|GST Glutathione S-transferase (Plasmodium yoelii)|
|Na_21166||2.6||0.52||< 0.001|||Q8WW36.1|ZCH13 Zinc finger CCHC domain-containing protein 13 (Homo sapiens)|
|Na_11915||2.5||0.19||< 0.001|||P40561.1|SGN1 RNA-binding protein Salivary gland transcription factor 1 (SGN1) (Saccharomyces cerevisiae)|
|Na_28441||2.5||0.60||0.017|||Q3MHV9|SERC1 Serine incorporator 1 (Bos taurus)|
|Na_22608||2.3||0.48||0.005|||BAB13708.1| Elicitor inducible protein (Nicotiana tabacum)|
|Na_17639||2.2||0.30||< 0.001|||Q9FFT9.1|RH32 DEAD-box ATP-dependent RNA helicase 32 (Arabidopsis thaliana)|
|Na_09481||2.1||0.18||0.004|||Q9LK94|MDAR2 Monodehydroascorbate reductase (Arabidopsis thaliana)|
|Na_29882||2.1||0.41||0.017|||Q9FF55.1|PDI14 Protein disulfide isomerase-like 1-4 (Arabidopsis thaliana)|
|Na_11459||2.0||0.30||0.017|||Q9CQ79.1|TXND9 Thioredoxin domain-containing protein 9 (Mus musculus)|
|Na_15649||1.9||0.21||0.006||sp|Q9SEL7.3|DEGP5 Protease Do-like 5 (Arabidopsis thaliana)|
|Na_20636||1.9||0.16||0.004|||Q6J163.1|5NG4 Auxin-induced protein 5NG4 (Pinus taeda)|
|Na_05174||1.8||0.23||< 0.001|||Q9LJF3|BRL3 Receptor-like protein kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1)-like 3 (Arabidopsis thaliana)|
|Na_01467||1.5||0.15||0.013|||Q9C5M8.2|PEL18 Pectate lyase 18 (Arabidopsis thaliana)|
|Na_21450||1.4||0.04||0.017|||Q9C670| Transcription factor basic-helix-loop-helix (bHLH) 76 (Arabidopsis thaliana)|
|Na_09136||1.4||0.14||0.021|||P28493|PR5 Pathogenesis-related protein 5 (Arabidopsis thaliana)|
|Na_15178||1.4||0.09||< 0.001|||P83241|IP23 Proteinase inhibitor PSI-1.2 (Capsicum annuum)|
|Na_30583||1.4||0.04||0.006|||Q08080|HSP7S Stromal 70-kDa heat shock-related protein (Spinacia oleracea)|
|Na_09273||1.4||0.11||0.021|||Q9ZWL6|ETR1 Ethylene receptor (Pinus edulis)|
|Na_18991||1.3||0.13||< 0.001|||Q9FLI1| Transcription factor bHLH36 (Arabidopsis thaliana)|
|Na_09028||1.3||0.12||0.017|||P13240|DR206 Disease resistance response protein 206 (Pisum sativum)|
|Na_32575||1.3||0.05||0.006|||O81893.3|ITPK3 Inositol-tetrakisphosphate 1-kinase 3 (Arabidopsis thaliana)|
|Na_17734||1.3||0.03||0.013|||O22042|MKKK3 Mitogen-activated protein kinase kinase kinase 3 (Arabidopsis thaliana)|
|Na_13278||1.2||0.07||0.006|||Q9XEF0| Transcription factor bHLH51 (Arabidopsis thaliana)|
|Na_39624||1.1||0.05||0.031|||Q8S3D5|BH069 Transcription factor bHLH69 (Arabidopsis thaliana)|
Understanding the biosynthesis and function of the C12 derivatives of the HPL pathway has been a challenge in plant biology, and efforts initiated to this end a few decades ago have not been pursued with the same impetus as those associated with the C6 derivatives of this pathway. With the opportunity to use reverse genetic approaches in the unraveling of the biosynthesis and signaling capacities of these molecules, the requirement for a suitable method for their analysis is self-evident. Here, we have described an LC-MS/MS-based approach to quantify C12 derivatives of the HPL pathway and have shown its application in the detection of changes induced by wounding and FAC elicitation in WT plants and by genetic deficiencies of biosynthetic enzymes (i.e. NaLOX2 and NaHPL).
Generation of 9-OH-traumatin in N. attenuata leaves
9-OH-traumatin was initially isolated as a product of 13-HPODE in extracts of soybean and alfalfa (Medicago sativa ) seedlings, and it has been suggested that (9Z )-traumatin is converted to 9-OH-traumatin by LOX activity (Gardner, 1998). A subsequent study showed, however, that 9-OH-traumatin is formed preferentially nonenzymatically and perhaps indirectly via 13-HPODE formation and not directly by LOX activity towards (9Z )-traumatin (Noordermeer et al., 2000). We observed that approximately one-third of the de novo synthesized 9-OH-traumatin was formed nonenzymatically. However, consistent with the proposed LOX-mediated biogenesis of this oxylipin, the production of the remaining two-thirds depended either directly or indirectly on NaLOX2 activity. The reduced capacity of ir-lox2 leaf extracts to produce 9-OH-traumatin after exogenous addition of (9Z)-traumatin, the sensitivity of its production to heat inactivation and to the LOX inhibitor DHPE, and the independence of its accumulation to the presence of C18 hydroperoxides strongly suggested that NaLOX2 directly acts on (9Z )-traumatin to form 9-OH-traumatin. Although it is unlikely, we cannot completely rule out the possibility that the formation of 9-OH-traumatin by this enzyme is indirect via the generation of an oxidative metabolite other than C18 hydroperoxides. We have previously shown that NaLOX2 also oxidizes the fatty acid moiety of the FAC 18:3-Glu to form 13-OOH-18:3-Glu and its derivatives (Vandoorn et al., 2010). Thus, the range of substrates that this enzyme can use is not restricted to free 18:2 and 18:3 and may include (9Z )-traumatin to generate 9-OH-traumatin, as previously proposed in other systems (Gardner, 1998).
Generation of other low-abundance C12 derivatives of the NaHPL pathway and systemic accumulation
Alcohol dehydrogenase activity reduces the aldehyde groups of (9Z )-traumatin to form 12-OH-(9Z )-dodecenoic acid (Grechkin et al., 1990). The low amounts of this derivative accumulating in wounded and FAC-elicited N. attenuata leaves suggest that either this enzymatic activity is very low in this tissue or the rapid oxidation of (9Z )-traumatin to 9-OH-traumatin outcompetes its reduction. The low levels of accumulation of dodecenedioic acids in wounded and FAC-elicited leaves were consistent with the rapid exchange of (9Z )-traumatin from NaHPL to NaLOX2, as the accumulation of dodecenedioic acids remained below 0.02 nmol g−1 FW (Fig. S3).
Even though the concentrations of dodecenedioic acids and 12-OH-(9Z )-dodecenoic acid were much lower than those of 9-OH-traumatin, their accumulation increased rapidly and transiently upon wounding and FAC elicitation (Fig. 3b). Thus, a possible scenario is that, as previously demonstrated for different plant responses (English & Bonner, 1937; Bonner & English, 1938; Ivanova et al., 2001), these molecules may work as signals during the responses of N. attenuata to wounding and herbivory.
In distal leaves, the concentrations of the C12 metabolites did not change significantly within 2 h after wounding and FAC elicitation; however, we cannot exclude the possibility that some of these derivatives are vascularly transported in small amounts to distal leaves. It has been recently shown that azelaic acid, a saturated C9 dicarboxylic acid, accumulates in the sap of Arabidopsis thaliana and confers local and systemic resistance against Pseudomonas syringae (Jung et al., 2009).
The role of C12 derivatives as potential signaling molecules acting during the response to wounding and herbivory
9-OH-traumatin accumulated to c. 2 nmol g−1 FW in unelicited WT leaves, probably reflecting a basal flux of 18:2 and 18:3 through NaLOX2 and NaHPL in this tissue and the cellular tolerance to small amounts of this oxylipin RES. This basal flux was also reflected in the basal production of C6 volatiles in WT plants (Table S4). The effect of oxylipin RES in cellular processes has been well documented in both plants and animals. For example, they can negatively affect cellular metabolism by the chemical modification of enzymes or by reducing photosynthetic rates (Almeras et al., 2003; Mueller & Berger, 2009). At the level of gene expression, oxylipin RES can induce the transcription of genes related to detoxification, stress responses and secondary metabolism (Bate & Rothstein, 1998; Vollenweider et al., 2000; Almeras et al., 2003; Weber et al., 2004; Mueller et al., 2008). Thus, one possibility is that 9-OH-traumatin plays a role in the wound and herbivore responses through its RES properties; for example, by the activation of cell protection genes (Farmer & Davoine, 2007). After wounding, 38% of de novo synthesized 9-OH-traumatin conjugated to GSH, indicative of a strict control of its accumulation above certain levels, probably to prevent cytotoxic effects. The formation of oxylipin RES conjugates with GSH has been observed during the hypersensitive response (HR) induced by cryptogein elicitation in tobacco leaves (Davoine et al., 2006) and it has been proposed that the generation of these conjugates may play a direct role in signaling; for example, by the alteration of the cellular redox balance (Meyer & Hell, 2005; Mullineaux & Rausch, 2005; Ogawa, 2005; Davoine et al., 2006). Thus, an additional possibility is that the conjugation of 9-OH-traumatin to GSH plays a signaling role during the responses of N. attenuata to wounding and herbivory.
Consistent with the potential signaling roles of C12 derivatives of the HPL pathway during the wound response, gene expression analysis by microarrays showed that these derivatives have the capacity to exert changes in the expression of 320 genes. Among the top 12 most up-regulated transcripts (> 4-fold at 120 min) were several involved in responses to oxidative stress and protein chemical modification; a result consistent with 9-OH-traumatin being sensed as an RES by cells. These transcripts corresponded to GST (an enzyme that conjugates GSH to electrophilic centers on a wide variety of substrates, including peroxidized lipids and RES), MDAR (a component of the GSH-ascorbate antioxidant system; Dinakar et al., 2010), PDI (an enzyme involved in the formation of disulfide bonds in chloroplasts; Wittenberg & Danon, 2008) and SERC1 (an enzyme involved in the protein mis-folding response in the ER-Golgi system; Aoki et al., 2002). Interestingly, the expression of four transcription factors (TFs) corresponding to the bHLH family were induced by C12 derivatives (Table 1). This family of TFs contains the well-characterized Myelocytomatosis transcription factor (MYC)2, 3 and 4 which are involved in the regulation of defense responses in plants (Niu et al., 2011). These results suggested that these TFs could also participate in the mediation of responses induced by C12 derivatives. In summary, the effect of these derivatives on the expression of 320 genes encoding proteins involved in diverse cellular processes including defense (e.g. PR5 and PI), signaling (e.g. ETR1, MKKK3 and BRLK3) and redox homeostasis (e.g. GST, MDAR and PDI; Tables 1,S3) provided strong evidence for the contribution of C12 derivatives of the HPL pathway to the control of plant responses to wounding and probably herbivory in N. attenuata. Future work will focus on disentangling the potential specific roles of 9-OH-traumatin, 9-OH-traumatin-GSH, 12-OH-(9Z )-dodecenoic acid and dodecenedioic acids in the regulation of responses to wounding and herbivory and on the mechanisms underlying the formation of 9-OH-traumatin from (9Z)-traumatin by NaLOX2.
We acknowledge the Max Planck Society for financial support. We thank Anja Paschold for performing the Southern blot for ir-hpl plants and Wibke Kröber and Ivan Galis for assistance with microarray experiments and data analysis.