The prolongation of the effect of exogenous cytokinins by inhibition of their inactivation was studied in Raphanus sativus L. cv. Rampouch. As in radish the main way of cytokinin inactivation is their N-glucosylation, inhibitors of this pathway, papaverine, theophylline and olomoucine, as well as two olomoucine analogues, bohemine and roscovitine, were tested. The latter ones, which function as potent inhibitors of cyclin-dependent kinases, have also been found to effectively inhibit cytokinin N-glucosylation. Incubation (24 h) of de-rooted radish seedlings with inhibitors resulted in c. 50% decrease of the conversion of model cytokinins, [(3)H]dihydrozeatin and [(3)H]N(6)-benzyladenine, to the corresponding 7N-glucosides. Simultaneously the level of the non-metabolized cytokinin bases was elevated. The activity of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) was suppressed in a dose dependent manner. The concentration of physiologically active endogenous cytokinins was not increased significantly by inhibitor application. The inhibition of N-glucosylation was in the case of olomoucine, bohemine and roscovitine accompanied by the accumulation of physiologically nonactive cis-zeatin derivatives. The impact of inhibitors on the endogenous cytokinin pool seems to be balanced by the mechanisms involved in the maintenance of cytokinin homeostasis.

Bioaccessibility of four essential (Fe, Cu, Mn, Zn) and two toxic (Cd, Pb) elements in kernels of four walnut and four hazelnut cultivars was investigated using sequential enzymolysis approach and atomic absorption spectrometry. It was found that the assimilable part of elements was not dependent on nut cultivar. The bioaccessible fraction of Cu, Mn, and Zn was definitely higher for hazelnuts (62% Cu, 39% Mn, 58% Zn) than for walnuts (14% Cu, 21% Mn, 15% Zn). Bioaccessible Fe was 20-24% from its total content for both nut types. Solubility in the simulated intestinal juice is affected by both formation of stable soluble complexes and back sorption of dissolved elements on nut solid residues. Lead shows strong insolubility due to the high sorption affinity of lead ions to the insoluble fraction of nuts. Thus, walnuts and hazelnuts could act as effective biosorbents for lead detoxication.

The role of cytokinin N-glucosylation and degradation by cytokinin oxidase/dehydrogenase (CKX, EC 1.5.99.12) in response to application of exogenous auxins (2,4-dichlorophenoxyacetic acid [2,4-D] and a-naphthaleneacetic acid [NAA]) and cytokinins (N 6 -benzyladenine [BA] and trans-zeatin [Z]) was investigated in de-rooted seedlings of Raphanus sativus L. cv. Rampouch. Both auxins applied for 24 h at 1 and 10 lM concentration increased N-glucosylation of exogenously applied [ 3 H]dihydrozeatin (DHZ) by up to 20%. The level of endogenous 7N-glucosides (of Z, isopentenyladenine [iP] and DHZ) was increased by 2,4-D and NAA at 10 lM concentration by 28 and 23%, respectively, the level of Z being decreased by 90 and 59%, respectively. 2,4-D and NAA suppressed CKX activity ca. by half. Exogenous cytokinins Z and BA applied at 1 and 10 lM concentration stimulated 7N-glucosylation of [ 3 H]DHZ (by up to 40%). BA both at 1 and 10 lM, increased the level of endogenous Z by up to 35% and that of 7N-glucosides by up to 27%. BA application also strongly stimulated CKX activity (by up to 180%). Feeding with 1 and 10 lM Z resulted in ca. 100-fold and 2000-fold increase of Z level, respectively. The main metabolite, Z7G, was increased ca. 6-fold and 60-fold, respectively. Levels of Z 9-glucoside (Z9G), trans-zeatin riboside (ZR) and Z O-glucoside (ZOG) were elevated to lesser extent. As compared to BA, Z had only negligible effect on CKX activity. Adenine (1-500 lM) was preferentially 7N-glucosylated inhibiting competitively 7N-glucosylation of [ 3 H]DHZ. At high concentrations (100-500 lM) it increased endogenous levels of active cytokinins, especially of Z, however, it had no effect on CKX activity. Cytokinin N-glucosylation proved to be involved in down-regulation of active cytokinins in response to auxin and in the re-establishment of cytokinin homeostasis following application of exogenous cytokinins.

Dynamics of the response of tobacco cells (line BY-2) to exogenous cytokinin, N6-benzyladenine, and cyclin-dependent kinase inhibitor, roscovitine, was followed using alginate-immobilized cells packed into a column. N6-Benzyladenine (1.25 microM) increased the synthesis of the physiologically-active endogenous cytokinin, isopentenyladenosine, in the effluent up to 0.1 nM. Simultaneously, conversion of the excess of endogenous cytokinins to biologically inactive derivatives of cis-zeatin occurred, up to 0.8 nM. Roscovitine (50 microM) further increased cis-cytokinins, up to 2.2 nM.