Effect of exogenous cytokinins, auxins and adenine on cytokinin N-glucosylation and cytokinin oxidase/dehydrogenase activity in de-rooted radish seedlings

Abstract

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 α-naphthaleneacetic acid [NAA]) and cytokinins (N6- 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 μM concentration increased N-glucosylation of exogenously applied [ 3H]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 μM 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 μM concentration stimulated 7N-glucosylation of [3H]DHZ (by up to 40%). BA both at 1 and 10 μM, 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 μM 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-zeaun 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 μM) was preferentially 7N-glucosylated inhibiting competitively 7N-glucosylation of [3H]DHZ. At high concentrations (100-500 μM) 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.