Abstract
BackgroundPhytohormones are the key metabolites participating in the regulation of multiple functions of plant organism. Among them, jasmonates, as well as abscisic and salicylic acids are responsible for triggering and modulating plant reactions targeted against pathogens and herbivores, as well as resistance to abiotic stress (drought, UV-irradiation and mechanical wounding). These factors induce dramatic changes in phytohormone biosynthesis and transport leading to rapid local and systemic stress responses. Understanding of underlying mechanisms is of principle interest for scientists working in various areas of plant biology. However, highly sensitive, precise and high-throughput methods for quantification of these phytohormones in small samples of plant tissues are still missing.ResultsHere we present an LC-MS/MS method for fast and highly sensitive determination of jasmonates, abscisic and salicylic acids. A single-step sample preparation procedure based on mixed-mode solid phase extraction was efficiently combined with essential improvements in mobile phase composition yielding higher efficiency of chromatographic separation and MS-sensitivity. This strategy resulted in dramatic increase in overall sensitivity, allowing successful determination of phytohormones in small (less than 50 mg of fresh weight) tissue samples. The method was completely validated in terms of analyte recovery, sensitivity, linearity and precision. Additionally, it was cross-validated with a well-established GC-MS-based procedure and its applicability to a variety of plant species and organs was verified.ConclusionThe method can be applied for the analyses of target phytohormones in small tissue samples obtained from any plant species and/or plant part relying on any commercially available (even less sensitive) tandem mass spectrometry instrumentation.
Highlights
Phytohormones are the key metabolites participating in the regulation of multiple functions of plant organism
In order to correct phytohormone content values for effects related to the plant matrix and for losses during sample preparation, for all analytes we employed internal standardization using corresponding stable isotope-labeled counterparts amended to the extraction solvent
For these compounds (2H6-Salicylic acid (SA), 2H6-abscisic acid (ABA), 2H6-jasmonic acid (JA), 2H2-JA-Ile and 2H5-oxophytodienoic acid (OPDA)), the Q1 and Q3 mass ranges selected for authentic standards were corrected for the presence of deuterium atoms (Additional file 1: Table S1) while the same set of mass spectrometry (MS) parameters (CE, DP, CEP and CXP) was applied for their quantification as for the nonlabeled isotopologues
Summary
Phytohormones are the key metabolites participating in the regulation of multiple functions of plant organism. Jasmonates, as well as abscisic and salicylic acids are responsible for triggering and modulating plant reactions targeted against pathogens and herbivores, as well as resistance to abiotic stress (drought, UV-irradiation and mechanical wounding). These factors induce dramatic changes in phytohormone biosynthesis and transport leading to rapid local and systemic stress responses. The fine mechanisms underlying distribution of JA, JA-Ile and their precursor OPDA as well as their hydroxylated and carboxylated derivatives [9] within plant tissues are still unknown To fill this gap, highly-sensitive analytical approaches providing hormone content information for small (≤ 50 mg) tissue amounts are required
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