RIKEN Plant Hormone Research Network: Hormone Analysis Platform

1. Highly sensitive and high-throughput analysis system for cytokinins, auxins, abscisic acid, and gibberellins.

RIKEN PSC has developed a highly sensitive and high-throughput method for the simultaneous analysis of 43 molecular species of cytokinins, auxins, ABA and gibberellins (GAs). This method consists of an automatic liquid handling system for solid phase extraction and ultra-performance liquid chromatography coupled with a tandem quadrupole mass spectrometer equipped with an electrospray interface (UPLC-ESI-qMS/MS). In order to improve the detection limit of negatively charged compounds, we introduced a chemical derivatization, that we call MS-probe derivatization. It increased quantification limits of gibberellins increased up to 50-fold and permits all compounds of auxins, ABA and GAs, to be measured in a single LC run. Our current method needs 1 to 100 mg fresh weight of plant tissues to determine phytohormone profiles and enables us to analyze 180 plant samples simultaneously. Required tissue amount is totally dependent on the nature of the tissues and the target hormone species.

Reference

• Kojima, M. et al. (2009) Highly sensitive and high-throughput analysis of plant hormones using MS-probe modification and liquid chromatography-tandem mass spectrometry: an application for hormone profiling in Oryza sativa. Plant Cell Physiol. 50: 1201-1214. [PubMed]

2. Fine-tuned highly sensitive analysis for specific hormones

We have developed a fine-tuned analysis method of bioactive hormones and their related compounds. This method contains JA, JA-Ile and SA as bioactive hormones in addition to IAA, ABA, GA, CKs. High resolution LC system are connected to a tandem quadrupole and a quadruple time-of-flight mass spectrometer equipped with electrospray interface (LC-ESI-qMS/qMS and LC-ESI-qMS/tofMS). LC-ESI-qMS/qMS has been used for high-sensitive analysis and LC-ESI-qMS/tofMS has been used for high-resolution analysis. In order to analyze brassinosteroids and strigolactones we are developing specific purification methods which are optimized for these neutral hormones. At the same time we are optimizing purification methods for specific tissue and organ as root or meristem tips which are targeted on limited hormone species. One example, we could analyze IAA from 10 of Arabidopsis 2 mm root tips and ABA from Arabidopsis single dry seed. Because the sensitivities of each hormone are strongly affected by impurities contained in target material, we do the detection test at first to confirm required amount for specific purpose before starting quantification analysis.