Publikationen - Molekulare Signalverarbeitung

Cis-(+)-12-oxophytodienoic acid (cis-(+)-OPDA) is a bioactive jasmonate, a precursor of jasmonic acid, which also displays signaling activity on its own. Modulation of cis-(+)-OPDA actions may be carried out via biotransformation leading to metabolites of various functions. This work introduces a methodology for the synthesis of racemic cis-OPDA conjugates with amino acids (OPDA-aa) and their deuterium-labeled analogs, which enables the unambiguous identification and accurate quantification of these compounds in plants. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry-based method for the reliable determination of seven OPDA-aa (OPDA-Alanine, OPDA-Aspartate, OPDA-Glutamate, OPDA-Glycine, OPDA-Isoleucine, OPDA-Phenylalanine, and OPDA-Valine) from minute amount of plant material. The extraction from 10 mg of fresh plant tissue by 10% aqueous methanol followed by single-step sample clean-up on hydrophilic–lipophilic balanced columns prior to final analysis was optimized. The method was validated in terms of accuracy and precision, and the method parameters such as process efficiency, recovery and matrix effects were evaluated. In mechanically wounded 30-day-old Arabidopsis thaliana leaves, five endogenous (+)-OPDA-aa were identified and their endogenous levels were estimated. The time-course accumulation revealed a peak 60 min after the wounding, roughly corresponding to the accumulation of cis-(+)-OPDA. Our synthetic and analytical methodologies will support studies on cis-(+)-OPDA conjugation with amino acids and research into the biological significance of these metabolites in plants.

Jasmonates (JAs) are plant hormones that integrate external stress stimuli with physiological responses. (+)-7-iso-JA-L-Ile is the natural JA ligand of COI1, a component of a known JA receptor. The upstream JA biosynthetic precursor cis-(+)-12-oxo-phytodienoic acid (cis-(+)-OPDA) has been reported to act independently of COI1 as an essential signal in several stress-induced and developmental processes. Wound-induced increases in the endogenous levels of JA/JA-Ile are accompanied by two to tenfold increases in the concentration of OPDA, but its means of perception and metabolism are unknown. To screen for putative OPDA metabolites, vegetative tissues of flowering Arabidopsis thaliana were extracted with 25% aqueous methanol (v/v), purified by single-step reversed-phase polymer-based solid-phase extraction, and analyzed by high throughput mass spectrometry. This enabled the detection and quantitation of a low abundant OPDA analog of the biologically active (+)-7-iso-JA-L-Ile in plant tissue samples. Levels of the newly identified compound and the related phytohormones JA, JA-Ile and cis-(+)-OPDA were monitored in wounded leaves of flowering Arabidopsis lines (Col-0 and Ws) and compared to the levels observed in Arabidopsis mutants deficient in the biosynthesis of JA (dde2-2, opr3) and JA-Ile (jar1). The observed cis-(+)-OPDA-Ile levels varied widely, raising questions concerning its role in Arabidopsis stress responses.

Stress-induced changes in phytohormone metabolite profiles have rapid effects on plant metabolic activity and growth. The jasmonates (JAs) are a group of fatty acid-derived stress response regulators with roles in numerous developmental processes. To elucidate their dual regulatory effects, which overlap with those of other important defence-signalling plant hormones such as salicylic acid (SA), abscisic acid (ABA) and indole-3-acetic acid (IAA), we have developed a highly efficient single-step clean-up procedure for their enrichment from complex plant matrices that enables their sensitive quantitative analysis using hyphenated mass spectrometry technique. The rapid extraction of minute quantities of plant material (less than 20 mg fresh weight, FW) into cold 10% methanol followed by one-step reversed-phase polymer-based solid phase extraction significantly reduced matrix effects and increased the recovery of labile JA analytes. This extraction and purification protocol was paired with a highly sensitive and validated ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method and used to simultaneously profile sixteen stress-induced phytohormones in minute plant material samples, including endogenous JA, several of its biosynthetic precursors and derivatives, as well as SA, ABA and IAA.