Application of a high-throughput HPLC-MS/MS assay to Arabidopsis mutant screening; evidence that threonine aldolase plays a role in seed nutritional quality

doi:10.1111/j.1365-313X.2004.02140.x

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Wiley InterScience

The Plant Journal

Volume 39 Issue 3, Pages 465 - 475

Published Online: 17 Jun 2004

Published in association with the Society for Experimental Biology

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TECHNICAL ADVANCE

Application of a high-throughput HPLC-MS/MS assay to Arabidopsis mutant screening; evidence that threonine aldolase plays a role in seed nutritional quality

Georg Jander ^1,2,*, Susan R. Norris ^1,† , Vijay Joshi ² , Michele Fraga ^1,‡ , Arthur Rugg ^1,§ , Shaoxia Yu ^1,¶ , Lily Li ^1,§ and Robert L. Last ^1,**

¹ Cereon Genomics, 45 Sidney Street, Cambridge, MA 02139, USA , and ² Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853, USA

Correspondence to ^*(fax +1 607 254 2958; e-mail gj32@cornell.edu).

^†Present address: Monsanto Company, 800 North Lindbergh Boulevard, St Louis, St Louis 63167, MO, USA

^‡Present address: Altana Research Institute, 610 Lincoln Street, Waltham, MA 02451, USA

^§Present address: Cantata Pharmaceuticals, 300 Technology Square, Cambridge, MA 02139, USA

^¶Present address: Millennium Pharmaceuticals, 45 Sidney Street, Cambridge, MA 02139, USA

^**Present address: National Science Foundation, Plant Genome Reserach Program, 4201 Wilson Boulevard, Room 615, Arlington, VA 22230, USA

Any views, opinions, or conclusions expressed in this article are those of the author, and do not represent the official views, opinions or policy of the National Science Foundation.

KEYWORDS

amino acid • threonine • Arabidopsis • HPLC-MS/MS • isoleucine • mutant screen

ABSTRACT

Beyond their essential function as the building blocks of proteins, amino acids contribute to many aspects of plant biochemistry and physiology. Despite this, there are relatively large gaps in our understanding of the biochemical pathways and regulation of amino acid synthesis in plants. A rapid (1.5 min versus 20–90 min for standard methods) HPLC-MS/MS assay for separating 19 amino acids was developed for quantifying levels of free amino acids in plant tissue. This assay was used to determine the free amino acid content in the seeds of 10,000 randomly mutagenized Arabidopsis lines, and 322 Arabidopsis lines with increased levels of one or more amino acids were identified. The heritability of the mutant phenotype was confirmed for 43 lines with increased seed levels of the aspartate-derived amino acids Ile, Lys, Thr, or Met. Genetic mapping and DNA sequencing identified a mutation in an Arabidopsis threonine aldolase (AT1G08630, EC 4.1.2.5) as the cause of increased seed Thr levels in one mutant. The assay that was developed for this project has broad applicability to Arabidopsis and other plant species.

Received 13 March 2004; revised 10 May 2004; accepted 14 May 2004.

DIGITAL OBJECT IDENTIFIER (DOI)

10.1111/j.1365-313X.2004.02140.x About DOI