Palacký University Olomouc Faculty of Science Laboratory of Growth Regulators & Department of Botany

Indole-3-acetic acid (IAA) is the most important member of the group of phytohormones known as auxins, which regulate many aspects of plant growth and development. The concentration of IAA in cells and tissues is controlled by various mechanisms, including de novo biosynthesis, synthesis and hydrolysis of IAA conjugates, conversion of IAA to indole-3-butyric acid (IBA), and oxidative degradation. This thesis deals with those conjugates of indole-3-acetic acid in which IAA is coupled through its carboxyl group to an amino acid. The aim of this thesis was to develop an efficient, specific and sensitive analytical method suitable for the isolation and quantification of indole-3-acetic acid and its amino acid conjugates in diverse plant materials. To achieve this, a specific immunoaffinity extraction procedure was developed and implemented into an analytical protocol starting with a phosphate-buffer-based extraction followed by a Solid-Phase Extraction (SPE) and ending up with the final analysis done by High-Performance Liquid Chromatography coupled to tandem Mass Spectrometry (LCMS/MS). The protocol allows routine quantification of IAA and seven amino acid conjugates in samples as small as 20 mg fresh weight, with overall quantitative recovery ranging between 30 and 70 per cent. The limits of detection of the final analysis, based on the use of heavy-labeled internal standards, vary typically around 1 fmol per injection. The developed analytical protocol was successfully used to quantify IAA and seven amino acid conjugates in immature seeds of the Christmas rose (Helleborus niger L.). Three of these conjugates were isolated and identified for the first time in higher plants. Currently, the method is being routinely used for the study of IAA and its derivatives in a variety of plant materials, which is documented by the enclosed articles published in scientific journals.