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Plant and Cell Physiology Advance Access published online on April 15, 2009
Plant and Cell Physiology, doi:10.1093/pcp/pcp057
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Mikiko Kojima1, Tomoe Kamada-Nobusada1, Hirokazu Komatsu2,5, Kentaro Takei1, Takeshi Kuroha1,6, Masaharu Mizutani3, Motoyuki Ashikari4, Miyako Ueguchi-Tanaka4, Makoto Matsuoka4, Koji Suzuki2 and Hitoshi Sakakibara1

1RIKEN Plant Science Center, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045, Japan
2Faculty of Science and Technology, Keio University, Kohoku, Yokohama 223-8522, Japan
3Graduate School of Agricultural Science, Kobe University, Rokkodai, Nada, Kobe 657-8501, Japan
4Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan

Corresponding author: Dr. Hitoshi Sakakibara. RIKEN Plant Science Center, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045, Japan, Tel: +81-45-503-9576; Fax: +81-45-503-9609; e-mail: sakaki{at}riken.jp


  Abstract

We have 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 (UPLC) coupled with a tandem quadrupole mass spectrometer (qMS/MS) equipped with an electrospray interface (ESI, UPLC-ESI-qMS/MS). In order to improve the detection limit of negatively charged compounds, such as GAs, we chemically derivatized fractions containing auxin, ABA, and GAs with bromocholine that has a quaternary ammonium functional group. This modification, that we call "MS-probe", makes these hormone-derivatives have a positive ion charge and permits all compounds to be measured in the positive ion mode with UPLC-ESI-qMS/MS in a single run. Consequently, quantification limits of GAs increased up to 50-fold. Our current method needs less than 100 mg (fresh weight) of plant tissues to determine phytohormone profiles and enables us to analyze simultaneously more than 180 plant samples. Application of this method to plant hormone profiling enabled us to draw organ-distribution maps of hormone species in rice and also to identify interactions among the 4 major hormones in the rice GA-signaling mutants, gid1-3, gid2-1, and slr1. Combining the results of hormone profiling data with transcriptome data in the GA signaling mutants allows us to analyze relationships between changes in gene expression and hormone metabolism.

Keywords: ABA - auxins - cytokinins - gibberellins - mass spectrometry - Oryza sativa

5Present address: Kyoto City Collaboration of Regional Entities for the Advancement of Technological Excellence, Kyoto, 615-8510, Japan

6Department of Biology, University of Washington, Seattle, WA 98195, USA

(Received March 8, 2009; Accepted April 12, 2009)
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