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Metabolism and Enzymology

Determination by Gas Chromatography-Mass Spectrometry of [¹⁵N₅]Adenine Incorporation into Endogenous Cytokinins and the Effect of Tissue Age on Cytokinin Biosynthesis in Datura innoxia Crown Gall Tissue

Research School of Biological Sciences, The Australian National University, Canberra, A.C.T. 2601, Australia, Research School of Chemistry, The Australian National University, Canberra, A.C.T. 2601, Australia

In this study gas chromatographic-mass spectrometric techniques have been used to identify and quantify the metabolic incorporation of [¹⁵N₅]adenine into zeatin and its metabolites by 3-week-old Datura innoxia Mill, crown gall tissue. In a parallel study the levels of endogenous cytokinins were also determined by the stable isotope dilution technique using deuterium (²H)-labeled internal standards. Incorporation levels of the [¹⁵N₅]adenine after 8 hours of incubation, expressed as a percentage of the endogenous cytokinins, were as follows: zeatin (1.0%), zeatin riboside (1.5%), and zeatin riboside 5'-phosphate (10.2%). These results are consistent with those observed in complementary experiments using [U-¹⁴C]adenine, and support the proposal that the cytokinin biosynthesis occurs primarily at the nucleotide level. The effect of tissue age on cytokinin biosynthesis, determined by [U-¹⁴C]adenine incorporation into cytokinins by tissues at varying growth stages, indicated a steady increase with time reaching maximal synthesis at five weeks following subculture after which the level of ¹⁴C incorporation into cytokinins declined.