Stable isotope techniques for the analysis of indole auxin metabolism in normal and mutant plants

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Techniques for the analysis of indole-3-acetic acid (IAA) using stable isotope dilution are now well established. This basic technology, designed for the measurement of levels of phytohormone, can be extended to study the metabolic relationship of IAA to other indolic compounds and thus provide a more complete picture of hormone metabolism. We have developed methods for the analysis of the biosynthesis of IAA in plants and have applied these techniques to study IAA metabolism in normal and mutant plants as well as during embryogenesis, seed germination and root formation. The approaches we have recently used in our laboratory are: 1) To expand our analytical techniques to encompass additional indolic compounds and precursors. 2) To use D2O as a totally invasive label which can be incorporated into very early precursors in the aromatic biosynthetic pathway. Such experiments are able to answer the question “Is IAA being made de novo in this plant tissue?” 3) To use stable isotope labelled precursors to measure pool sizes and turnover of compounds important for the biosynthesis of tryptophan (Trp) and IAA, resulting in a quantitative approach to understanding carbon flow in these pathways. 4) To use stable isotope labelled compounds to track the interconversion of indolic compounds. 5) To develop techniques for the production and selection of mutant plants well suited for measurement of auxin metabolism in situ. In this paper we will discuss the methodology we use to study several plant systems which have been chosen to illustrate how to answer specific questions with regard to auxin metabolism. The plant systems we will discuss include carrot embryogenic cultures, seed germination in Phaseolus, precursor studies using normal and mutant Lemna and measurement of IAA and IBA levels in carrot transformed with Agrobacterium rhizo genes.