Immunoaffinity purification using monoclonal antibodies for the isolation of indole auxins from elongation zones of epicotyls of red-light-grown Alaska peas

• Published in: Planta 1992-09, Vol.188 (2), p.182-189
• Main Authors: Ulvskov, P. (Danish Inst. of Plant and Soil Science, Lyngby (Denmark). Biotechnology Group), Marcussen, J, Seiden, P, Olsen, C.E
• Format: Article
• Language: English
• Subjects: ACIDE INDOLACETIQUE; ACIDO INDOLACETICO; Agronomy. Soil science and plant productions; ALARGAMIENTO DEL TALLO; ALLONGEMENT DE LA TIGE; ANTICORPS MONOCLONAL; ANTICUERPOS MONOCLONALES; Auxin; Biological and medical sciences; EPICOTILOS; EPICOTYLE; EPICOTYLS; Epikotyl; Erbse; Fundamental and applied biological sciences. Psychology; Growth regulators; IAA; IES; Immunverfahren; Lichtfarbe; LIGHT REGIMES; Metabolism; MONOCLONAL ANTIBODIES; monoklonale Antikoerper; PISUM SATIVUM; Plant physiology and development; REGIME LUMINEUX; REGIMENES DE LUZ; STEM ELONGATION; Streckungswachstum; Wachstumsregulator
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/bf00216812

The endogenous indole auxins of red-light grown pea (Pisum sativum L.) epicotyls were investigated. Immunoaffinity purification of indole-3-acetic acid (IAA) and its methylester was achieved using two monoclonal antibodies. Antibodies against free IAA were raised against IAA-C5-BSA, a hapten-carrier-conjugate giving rise to highly specific antibodies for indole auxins with a free acetic-acid group at position 3. Immunoaffinity adsorbents prepared with these antibodies were used for single-step purification of extracts of Alaska pea epicotylar tissue prior to quantification by high-performance liquid chromatography (HPLC) with on-line fluorescence detection. Monoclonal antibodies against a hapten-carrier-conjugate with IAA linked to bovine serum albumin through the carboxyl group (IAA-C1'-BSA) were used for the isolation of IAA esters. Indol-3-acetic acid was identified in the elongation zone of the third internode of red-light-grown Alaska pea. 4-Chloro-indole-3-acetic acid, a constituent of immature pea seeds which is considered to be a very active auxin, was absent from the elongation zone. Several compounds were retained by the column based on antibodies against IAA-C1'-BSA. Of these the methylester of IAA was identified by HPLC with on-line fluorescence detection, by co-migration in thin-layer chromatography and by gas chromatography-mass spectrometry. The methyl ester of IAA was very active in promoting elongation of pea third-internode segments. When fed to the epicotylar segments the IAA methylester was rapidly metabolized with IAA being the major metabolite. The methylester of IAA should therefore be classified as a labile auxin conjugate.