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BMC Plant Biology (1)
Plant Methods (1)
Baldwin, Ian T (2)
Bonaventure, Gustavo (2)
Kallenbach, Mario (2)
Borquez, Alejandro A (1)
VanDoorn, Arjen (1)
Year of Publication
Rapid modification of the insect elicitor N-linolenoyl-glutamate via a lipoxygenase-mediated mechanism on Nicotiana attenuata leaves
Borquez, Alejandro A
Baldwin, Ian T
BMC Plant Biology
Some plants distinguish mechanical wounding from herbivore attack by recognizing specific constituents of larval oral secretions (OS) which are introduced into plant wounds during feeding. Fatty acid-amino acid conjugates (FACs) are major constituents of Manduca sexta OS and strong elicitors of herbivore-induced defense responses in Nicotiana attenuata plants.
The metabolism of one of the major FACs in M. sexta OS, N-linolenoyl-glutamic acid (18:3-Glu), was analyzed on N. attenuata wounded leaf surfaces. Between 50 to 70% of the 18:3-Glu in the OS or of synthetic 18:3-Glu were metabolized within 30 seconds of application to leaf wounds. This heat-labile process did not result in free α-linolenic acid (18:3) and glutamate but in the biogenesis of metabolites both more and less polar than 18:3-Glu. Identification of the major modified forms of this FAC showed that they corresponded to 13-hydroxy-18:3-Glu, 13-hydroperoxy-18:3-Glu and 13-oxo-13:2-Glu. The formation of these metabolites occurred on the wounded leaf surface and it was dependent on lipoxygenase (LOX) activity; plants silenced in the expression of NaLOX2 and NaLOX3 genes showed more than 50% reduced rates of 18:3-Glu conversion and accumulated smaller amounts of the oxygenated derivatives compared to wild-type plants. Similar to 18:3-Glu, 13-oxo-13:2-Glu activated the enhanced accumulation of jasmonic acid (JA) in N. attenuata leaves whereas 13-hydroxy-18:3-Glu did not. Moreover, compared to 18:3-Glu elicitation, 13-oxo-13:2-Glu induced the differential emission of two monoterpene volatiles (β-pinene and an unidentified monoterpene) in irlox2 plants.
The metabolism of one of the major elicitors of herbivore-specific responses in N. attenuata plants, 18:3-Glu, results in the formation of oxidized forms of this FAC by a LOX-dependent mechanism. One of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of JA biosynthesis and differential monoterpene emission.
A rapid and sensitive method for the simultaneous analysis of aliphatic and polar molecules containing free carboxyl groups in plant extracts by LC-MS/MS
Baldwin, Ian T
Aliphatic molecules containing free carboxyl groups are important intermediates in many metabolic and signalling reactions, however, they accumulate to low levels in tissues and are not efficiently ionized by electrospray ionization (ESI) compared to more polar substances. Quantification of aliphatic molecules becomes therefore difficult when small amounts of tissue are available for analysis. Traditional methods for analysis of these molecules require purification or enrichment steps, which are onerous when multiple samples need to be analyzed. In contrast to aliphatic molecules, more polar substances containing free carboxyl groups such as some phytohormones are efficiently ionized by ESI and suitable for analysis by LC-MS/MS. Thus, the development of a method with which aliphatic and polar molecules -which their unmodified forms differ dramatically in their efficiencies of ionization by ESI- can be simultaneously detected with similar sensitivities would substantially simplify the analysis of complex biological matrices.
A simple, rapid, specific and sensitive method for the simultaneous detection and quantification of free aliphatic molecules (e.g., free fatty acids (FFA)) and small polar molecules (e.g., jasmonic acid (JA), salicylic acid (SA)) containing free carboxyl groups by direct derivatization of leaf extracts with Picolinyl reagent followed by LC-MS/MS analysis is presented. The presence of the N atom in the esterified pyridine moiety allowed the efficient ionization of 25 compounds tested irrespective of their chemical structure. The method was validated by comparing the results obtained after analysis of Nicotiana attenuata leaf material with previously described analytical methods.
The method presented was used to detect 16 compounds in leaf extracts of N. attenuata plants. Importantly, the method can be adapted based on the specific analytes of interest with the only consideration that the molecules must contain at least one free carboxyl group.
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