Abstract
Plant hormones have been identified to be versatile signaling molecules essential for plant growth, development, and stress response. Their content levels vary depending on the species, and they also change in response to any external stimuli. Thus, simultaneous quantification of multiple plant hormones is required to understand plant physiology. Sensitive and quantitative analysis using liquid chromatography-linked mass spectrometry (LC-MS/MS) has been used in detecting plant hormones; however, quantification without stable isotopes is yet to be established. In this study, we quantified seven representative plant hormones of Lotus japonicus, which is a model legume for standard addition method. Accurate masses for monoisotopic ions of seven phytohormones were determined for high-resolution mass spectrometry (HR-MS). Selected ion monitoring (SIM) mode based on accurate masses was used in detecting phytohormones in the roots, stems, and leaves. Evaluation of matrix effects showed ion suppression ranging from 10.2% to 87.3%. Both stable isotope dilution and standard addition methods were able to detect plant hormones in the roots, stems, and leaves, with no significant differences in using both approaches and thus a standard addition method can be used to quantify phytohormones in L. japonicus. The method will be effective, especially when stable isotopes are not available to correct for matrix effects.
Highlights
Plant hormones are identified as essential small molecules implicated in a variety of fundamental biological processes, including growth, development, and stress response [1]
Five phytohormones were detected in negative mode, and indole-3-acetic acid (IAA) and BL were only detected in positive mode
Stable isotope-labelled phytohormones were analyzed in order to determine accurate mass and retention time; they were compared to non-labelled hormones (S1 Table and S3 Fig)
Summary
Plant hormones are identified as essential small molecules implicated in a variety of fundamental biological processes, including growth, development, and stress response [1]. These hormones are classified into nine groups: auxins, cytokinins, gibberellins, ethylene, abscisic acid, salicylic acid, jasmonic acids, strigolactones, and brassinosteroids. Each class shows bioactivities alone and in combination with other hormones, which is generally known as hormonal crosstalk [2]. Quantifying multiple plant hormones simultaneously is required to understand plant physiology. Detecting trace amounts of phytohormones in plant tissues employs two major analytical tools, that is, gas chromatography coupled with mass spectrometry (GC-MS) [3] and liquid chromatography coupled to electrospray tandem mass.
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