Jan Holub

Biological activity of cytokinins derived from Ortho- and Meta-Hydroxybenzyladenine

To determine the structure-activity relationships of natural aromatic cytokinins, the activity of 6-benzylaminopurine (BAP) and its hydroxylated derivatives was compared in three bioassays based on stimulation of tobacco callus growth, retention of chlorophyll in excised wheat leaves, and dark induction of betacyanin synthesis in Amaranthus cotyledons. The aromatic cytokinins 6-(2-hydroxybenzylamino)purine (ortho-topolin) and 6-(3-hydroxybenzylamino)purine (meta-topolin), their 9-ribosides and 9-glucosides, were synthesized by the condensation of 6-chloropurine and its 9-glycosides with the appropriate hydroxybenzylamine. The activity of free bases, 9-ribosides and 9-glucosides was compared with that of BAP, trans-zeatin and their 9-glycosides. Hydroxylation of the benzyl ring in the meta position increased the activity of BAP and its riboside in tobacco callus and chlorophyll retention bioassays, whereas ortho-hydroxylation decreased the activity. In contrast, in the Amaranthus bioassay meta-hydroxylation of BAP substantially decreased its activity. Ribosylation at position 9 had no significant effect on the activity of zeatin, BAP and both topolins. The activity of 9-glucosides of all cytokinins tested was near zero. The biological activity of meta-topolin and its riboside is comparable to that of the most active isoprenoid cytokinin, zeatin, in tobacco callus growth and senescence bioassays. The results establish the existence of a family of endogenous aromatic cytokinins centered around the highly active compound, meta-topolin. We also report here an improved chlorophyll retention bioassay based on incubation of 2.5 cm long detached wheat leaf segments in microtiter plate wells containing 150 µl of cytokinin solution. The consumption of cytokinin to be tested is 0.1 µmol per assay only. The amount as small as 1.5 pmol of substance can be estimated using this biotest.

Synthesis, characterization and biological activity of two nickel(II) complexes with 6-(2-chlorobenzylamino)purine

Compounds of composition [Ni(L)(H2O)2Cl] and [Ni(L)(H2O)(NO3)] · EtOH [HL = 6-(2-chlorobenzylamino)purine] have been synthesized and characterized by elemental analyses, i.r. and electronic spectra, magnetic measurements and mass spectroscopy as tetrahedral nickel(II) complexes. The geometry of both complexes has been optimized using molecular mechanics modelling. Although the monoanionic ligand L is potentially bidentate, we assume that the coordination to nickel is via the N9 atom only. Cytokinin and anti-cancer activities of the complexes were also tested in an Amaranthus cytokinin bioassay and in an in vitro MTT-based cytotoxicity assay, respectively. In human T-lymphoblastic leukemia cell line CCRF-CEM both complexes showed potent cytotoxic activity.

Identification of aromatic cytokinins in suspension cultured photoautotrophic cells of Chenopodium rubrum by capillary liquid chromatography/frit – fast atom bombardment mass spectrometry

Two previously unrecorded endogenous cytokinin metabolites,6-[2-(β-D-glucopyranosyloxy)benzylamino]purine and6-[2-(β-D-glucopyranosyloxy)benzylamino]-2-methylthiopurine, wereidentified, together with 6-benzylamino-9-β-D-glucopyranosylpurine(BAP9G),from Chenopodium rubrum cells, grown autotrophically insuspension culture. The new metabolites belong to the aromatic class ofcytokinins, in which an aromatic side chain is attached at theN6-position of the adenine species. The identification was performedby capillary-liquid chromatography/frit-fast atom bombardment - massspectrometry (LC/FAB MS) after pre-column derivatisation and the structuralelucidation was confirmed by organic synthesis. Cytokinin activity of thecompounds was tested in an Amaranthus bioassay. Theendogenous synthesis of the identified compounds was verified byin vivo deuterium labelling of the analysed cytokininspecies, thereby for the first time providing absolute evidence for theendogenous origin of these compounds.

Deciphering the growth pattern and phytohormonal content in Saskatoon berry ( Amelanchier alnifolia ) in response to in vitro cytokinin application

Clonal propagation plays a critical integral role in the growth and success of a global multi-billion dollar horticulture industry through a constant supply of healthy stock plants. The supply chain depends on continuously improving the micropropagation process, thus, understanding the physiology of in vitro plants remains a core component. We evaluated the influence of exogenously applied cytokinins (CKs, N6-benzyladenine = BA, isopentenyladenine = iP, meta-topolin = mT, 6-(3-hydroxybenzylamino)-9-(tetrahydropyran-2-yl)purine = mTTHP) in Murashige and Skoog (MS)-supplemented media on organogenic response and accumulation of endogenous CK and indole-3-acetic acid (IAA) metabolites. The highest shoot proliferation (30 shoots/explant) was obtained with 20 μM mT treatment. However, the best quality regenerants were produced in 10 μM mT treatment. Rooting of Amelanchier alnifolia in vitro plantlets was observed at the lowest CK concentrations, with the highest root proliferation (3 roots/explant) in 1 μM mTTHP regenerants. Similar to the organogenic response, high levels of endogenous bioactive CK metabolites (free bases, ribosides, and nucleotides) were detected in mT and mTTHP-derived regenerants. The level of O-glucosides was also comparatively high in these cultures. All CK-treated plants had high levels of endogenous free IAA compared to the control. This may suggest an influence of CKs on biosynthesis of IAA.