Quantitative analysis of seven plant hormones in Lotus japonicus using standard addition method

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.

Fig 1. The chemical structures of the seven plant hormones used in this study.

Fig 2. Experimental workflow for extraction of phytohormones.

Fifty mg of tissues was extracted with 50% acetonitrile; it was then partially purified by RP-SPE. Dried residue was dissolved in 30% acetonitrile and was used for LC-MS/MS analysis.

Fig 3. Comparison of standard addition method with stable isotope dilution method.

Plant hormone contents in the leaves (A), roots (B), and stems (C) were compared between standard addition and stable isotope addition methods. Significant differences were not observed.