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Identification of new aromatic cytokinins in Arabidopsis thaliana and Populus × canadensis leaves by LC‐(+)ESI‐MS and capillary liquid chromatography/frit–fast atom bombardment mass spectrometry
Author(s) -
Tarkowská Danuše,
Doležal Karel,
Tarkowski Petr,
Åstot Crister,
Holub Jan,
Fuksová Květoslava,
Schmülling Thomas,
Sandberg Göran,
Strnad Miroslav
Publication year - 2003
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1034/j.1399-3054.2003.00071.x
Subject(s) - chemistry , chromatography , cytokinin , fast atom bombardment , electrospray ionization , mass spectrometry , high performance liquid chromatography , botany , biochemistry , biology , auxin , gene
A search for naturally occurring aromatic cytokinins (ARCKs) in Arabidopsis thaliana plants and Populus × 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‐β‐ 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 [ Cap LC/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 × 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 × 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. 1The structures of 6‐(2‐methoxybenzylamino)purine ( ortho ‐methoxytopolin, MeoT) and 6‐(3‐methoxybenzylamino)purine ( meta ‐methoxytopolin, MemT).