title: Identification of new aromatic cytokinins in Arabidopsis thaliana and Populus x canadensis leaves by LC-(+)ESI-MS and capillary liquid chromatography/frit-fast atom bombardment mass spectrometry.
abstract: A search for naturally occurring aromatic cytokinins (ARCKs) in Arabidopsis thaliana plants and Populus x canadensis leaves led to the discovery of four new plant hormone substances: 6-(2-methoxybenzylamino)purine (ortho-methoxytopolin, MeoT), 6-(3-methoxybenzylamino)purine (meta-methoxytopolin, MemT) (Fig. 1) and their 9-beta-D-ribofuranosyl derivatives. These substances were identified by liquid chromatography electrospray ionization mass spectrometry [LC (+)ESI-MS] and capillary-liquid chromatography/frit-fast atom bombardment-mass spectrometry [CapLC/frit-FAB-MS] after pre-column derivatization. The chemical structures were subsequently confirmed by chemical synthesis. Because of lack of heavy labelled internal standards, the endogenous levels of methoxytopolins in A. thaliana plants, Populus x canadensis leaves and samples derived from cultures of Agrobacterium tumefaciens strain GV3101 were determined by enzyme-linked immunosorbent assay (ELISA) of HPLC-fractionated extracts. While the levels of MeoT, MemT and their ribosides in A. thaliana shoots and Populus x canadensis leaves were relatively low (approximately 0.25-10 pmol g-1 FW for MeoT and MemT, respectively), the A. tumefaciens strain produced up to 600 times more of the newly identified substances. Cytokinin activity of methoxytopolines was demonstrated in three bioassays testing their ability to stimulate tobacco callus growth, to delay chlorophyll degradation in excised wheat leaves, and to induce betacyanin synthesis in Amaranthus caudatus var. atropurpurea cotyledons. Notably, their anti-senescing activity in the wheat leaf assay exceeded that of BAP and Z by almost 200%. Methoxytopolins are proposed to be new members of the biologically active aromatic cytokinin family, which might have specific physiological functions.
authors.affiliation: Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany AS CR, Slechtitelů 11, CZ-783 71 Olomouc, Czech Republic Institute of Nuclear Medicine, The First Faculty of Medicine, Charles University, CZ-212 08 Prague, Czech Republic Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, The Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden The Swedish Defense Research Agency, SE-901 82 Umeå, Sweden Free University of Berlin, Institute of Biology/Applied Genetics, Albrecht-Thaer-Weg 6, D-14195 Berlin, Germany.
authors.forename: Danuse
authors.initials: D
authors.lastname: Tarkowská
authors.forename: Karel
authors.initials: K
authors.lastname: Dolezal
authors.forename: Petr
authors.initials: P
authors.lastname: Tarkowski
authors.forename: Crister
authors.initials: C
authors.lastname: Astot
authors.forename: Jan
authors.initials: J
authors.lastname: Holub
authors.forename: Kvetoslava
authors.initials: K
authors.lastname: Fuksová
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmülling
authors.forename: Göran
authors.initials: G
authors.lastname: Sandberg
authors.forename: Miroslav
authors.initials: M
authors.lastname: Strnad