Analysis of cytokinin metabolism in ipt transgenic tobacco by liquid chromatography-tandem mass spectrometry

• Published in: Plant physiology (Bethesda) Vol. 112; no. 1; pp. 141 - 148
• Main Authors: Redig, P. (Univ. of Antwerp, Tubingen, Germany.), Schmulling, T, Onckelen, H. van
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01.09.1996
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ADENINA; ADENINE; Agronomy. Soil science and plant productions; alkyl (aryl) transferases; Biological and medical sciences; CAL; CALLO; Callus; CITOQUININAS; CYTOKININE; Cytokinins; Development and Growth Regulation; Economic plant physiology; enzyme activity; Enzymes; EXPRESION GENICA; EXPRESSION DES GENES; Fundamental and applied biological sciences. Psychology; GENE; gene expression; GENES; genetic transformation; Growth and development; Growth regulators; HOMEOSTASIE; HOMEOSTASIS; isopentenyladenine; Metabolism; METABOLISME; METABOLISMO; NICOTIANA TABACUM; Nucleotides; Plant physiology and development; Plant tissues; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; Plants; Radioactive decay; ribulose-bisphosphate carboxylase; RUBISCO; Solvents; structural genes; Subcultures; SUBSTANCE DE CROISSANCE VEGETALE; SUSTANCIAS DE CRECIMIENTO VEGETAL; TRANSFERASAS; TRANSFERASE; TRANSFORMACION GENETICA; TRANSFORMATION GENETIQUE; transgenic plants; zeatin; ZEATINA; ZEATINE; Adenine derivatives; Enzyme; Transferases; Gene expression; Metabolism; Nicotiana tabacum; Cytokinin; Dicotyledones; Angiospermae; Plant growth substance; Spermatophyta; Qualitative analysis; Transgenic plant; Solanaceae; Experimental plant; Biological accumulation; Quantitative analysis
• ISSN: 0032-0889; 1532-2548; 1532-2548
• DOI: 10.1104/pp.112.1.141

The endogenous levels of the major, naturally occurring cytokinins in Pisum sativum ribulose-1,5-bisphosphate carboxylase small subunit promoter-isopentenyl transferase gene (Pssu-ipt)-transformed tobacco (Nicotiana tabacum L.) callus were quantified using electrospray-liquid chromatography-tandem mass spectrometry during a 6-week subcultivation period. An ipt gene was expressed under control of a tetracycline-inducible promoter for a more detailed study of cytokinin accumulation and metabolism. Activation of the ipt in both expression systems resulted in the production of mainly zeatin-type cytokinins. No accumulation of isopentenyladenine or isopentenyladenosine was observed. In Pssu-ipt-transformed calli, as well as in the tetracycline-inducible ipt leaves, metabolic inactivation occurred through O-glucoside conjugation. No significant elevation of cytokinin N-glucosides levels was observed. Side-chain reduction to dihydrozeatin-type cytokinins was observed in both systems. The levels of the endogenous cytokinins varied in time and were subject to homeostatic regulatory mechanisms. Feeding experiments of ipt transgenic callus with [3H]isopentenyladenine and [3H]isopentenyladenosine mainly led to labeled adenine-like compounds, which are degradation products from cytokinin-oxidase activity. Incorporation of radioactivity in zeatin riboside was observed, although to a much lesser extent