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.
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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).
Figure 1. Schematic representation of the hydroperoxide lyase (HPL) pathway in plants and the generation of derivatives of 12-oxo-(9Z)-dodecenoic acid ((9Z)-traumatin). In leaves, 18:2 and 18:3 fatty acids are released from membranes and dioxygenated by 13-lipoxygenases (13-LOXs) to generate 13S-hydroperoxides (13-HPODE and 13-HPOTE, respectively). These molecules are substrates of HPL and are cleaved to produce C6 aldehydes and (9Z)-traumatin (compound 1a). This molecule can undergo several enzymatic and nonenzymatic modifications to be converted into the products (10E)-traumatin (compound 1b), (3Z)-traumatic acid (compound 2a), (2E)-traumatic acid (compound 2b), 12-OH-(9Z)-dodecenoic acid (compound 3a), 12-OH-(10E)-dodecenoic acid (compound 3b) and 9-OH-traumatin (compound 4). The latter compound can be converted into 4-OH-traumatic acid (compound 6), 9,12-OH-(10E)-dodecenoic acid (compound 5) and 9,12-OH-(10E)-dodecanoic acid (compound 4) according to Mukhtarova et al. (2011). The formation of 9-OH-traumatin by LOX-mediated activity has been proposed by Gardner (1998), whereas its nonenzymatic formation has been proposed by Noordermeer et al. (2000).
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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.
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Fig. S1 Auto-oxidation of (9Z)-traumatin in vitro.
Fig. S2 Determination of the linearity and limit of detection of C12 metabolites.
Fig. S3 Accumulation of C12 derivatives in leaves of wild-type (WT), ir-lox2 and ir-hpl plants after wounding and fatty acid–amino acid conjugate (FAC) elicitation.
Fig. S4 Calibration curves for C12 standards.
Fig. S5 Quantification of Nicotiana attenuata hydroperoxide lyase (NaHPL) mRNA levels in wild-type (WT) and ir-hpl plants.
Fig. S6 Southern blot analysis of ir-hpl plants.
Table S1 Parameters used for detection of C12 molecules by liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS)
Table S2 Parameters used for quantification of C12 molecules by liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS)
Table S3 Complete list of genes changing expression in wounded leaves of ir-lox2/ir-lox3 plants after treatment with 9Z-traumatin : 9-OH-traumatin (1 : 1)
Table S4 Analysis of C6 volatiles emitted by wild-type (WT) and ir-hpl plants
Methods S1 Characterization of ir-hpl plants.
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