We describe a method combining fluorescence-activated cell sorting (FACS) with one-step miniaturized isolation and accurate quantification of cytokinins (CKs) using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to measure these phytohormones in specific cell types of Arabidopsis thaliana roots. The methodology provides information of unprecedented resolution about spatial distributions of CKs, and thus should facilitate attempts to elucidate regulatory networks involved in root developmental processes.
- MeSH
- Arabidopsis metabolismus MeSH
- cytokininy metabolismus MeSH
- genové regulační sítě fyziologie MeSH
- kořeny rostlin metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- průtoková cytometrie metody MeSH
- regulátory růstu rostlin metabolismus MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- Arabidopsis chemie MeSH
- cyklopentany chemie izolace a purifikace MeSH
- diazoniové sloučeniny MeSH
- isoleucin analogy a deriváty chemie MeSH
- květy chemie MeSH
- listy rostlin metabolismus MeSH
- nenasycené mastné kyseliny chemie izolace a purifikace metabolismus MeSH
- oxylipiny chemie izolace a purifikace MeSH
- pyridiny MeSH
- regulátory růstu rostlin metabolismus MeSH
- stereoizomerie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
