Identification of the 12-oxojasmonoyl-isoleucine, a new intermediate of jasmonate metabolism in Arabidopsis, by combining chemical derivatization and LC–MS/MS analysis
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The Arabidopsis cytochrome P450 CYP94C1 is involved in the metabolism of jasmonates, a family of phytohormones implicated in plant development and responses to bioagressors. It has been shown to down-regulate the level of the active hormone jasmonoyl-isoleucine (JA-Ile) by catalyzing its successive oxidation into hydroxyl (12OH-JA-Ile) and carboxyl (12COOH-JA-Ile) derivatives. The systematic investigation of this enzymatic reaction in vitro reported here revealed the formation of a third oxidized product: UPLC–MS/MS data matched a possible aldehyde, an intermediate form between 12OH-JA-Ile and 12COOH-JA-Ile. To confirm the presence of an aldehyde group, the extracted metabolite from incubations were derivatized with methoxyamine, an agent known to target carbonyl functions specifically. UPLC–MS/MS analysis after derivatization confirmed the presence of 12-oxojasmonoyl-isoleucine (12CHO-JA-Ile). Methanolic extracts from Arabidopsis leaves were then investigated after wounding, a treatment that triggers the accumulation of JA-Ile and its oxidized derivatives. The 12CHO-JA-Ile produced a significant signal with the same order of magnitude than the other oxidized forms. This study illustrates the underestimated potential of chemical derivatization for rapid metabolite identification in modern metabolomics and enriches our knowledge of the jasmonates family.
KeywordsLiquid chromatography–mass spectrometry (LC–MS) Chemical derivatization Jasmonates Cytochrome P450
This work was supported by a doctoral fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche. It was also partially funded by the Agence Nationale de la Recherche grant ANR-12-BSV8-005. The UPLC–MS/MS instrument was co-financed by the Centre National de la Recherche Scientifique, the Université de Strasbourg, the Région Alsace, the Institut National de la Recherche Agronomique, and the Tepral Company.
Conflict of interest
All authors declare that they have no conflict of interest.
Compliance with ethical guidelines
This article does not contain any studies with human or animal subjects.
- Gullberg, J., Jonsson, P., Nordström, A., Sjöström, M., & Moritz, T. (2004). Design of experiments: an efficient strategy to identify factors influencing extraction and derivatization of Arabidopsis thaliana samples in metabolomic studies with gas chromatography/mass spectrometry. Analytical Biochemistry, 331, 283–295.PubMedCrossRefGoogle Scholar
- Knapp, D. R. (1979). Handbook of analytical derivatization reactions. New York: Wiley.Google Scholar
- Kojima, M., Kamada-Nobusada, T., Komatsu, H., et al. (2009). Highly sensitive and high-throughput analysis of plant hormones using MS-probe modification and liquid chromatography-tandem mass spectrometry: An application for hormone profiling in Oryza sativa. Plant and Cell Physiology, 50, 1201–1214.PubMedCentralPubMedCrossRefGoogle Scholar
- Le Bouquin, R., Skrabs, M., Kahn, R., et al. (2001). CYP94A5, a new cytochrome P450 from Nicotiana tabacum is able to catalyze the oxidation of fatty acids to the v-alcohol and to the corresponding diacid. European Journal of Biochemistry, 268, 3083–3090.Google Scholar
- Miettinen, K., Dong, L., Navrot, N., et al. (2014). The seco-iridoid pathway from Catharanthus roseus. Nature Communications, 5, 1–11.Google Scholar
- Zaikin, V., & Kalket, J. M. (2009). A handbook of derivatives for mass spectrometry. West Sussex: IM Publications.Google Scholar