Distinct metabolism of N-glucosides of isopentenyladenine and trans-zeatin determines cytokinin metabolic spectrum in Arabidopsis.

Diversity of cytokinin (CK) metabolites suggests their interconversions to be the predominant regulatory mechanism of CK action. Despite that, little is still known about their directionality and kinetics in planta. CK metabolite levels were measured in two-week-old Arabidopsis thaliana plants at several time points up to 100 min following exogenous application of selected CKs. The data were then evaluated qualitatively and by mathematical modeling. Apart from elevated levels of trans-zeatin (tZ) metabolites upon application of N⁶ -(Δ² -isopentenyl)adenine (iP), we observed no conversions between the individual CK-types - iP, tZ, dihydrozeatin (DHZ), cis-zeatin (cZ). In particular, there was no sign of isomerization between tZ and cZ families. Also, no increase of DHZ-type CKs was observed after the application of tZ, thus suggesting low baseline activity of zeatin reductase. Among N-glucosides, those of iP were not converted back to iP while tZ N-glucosides were cleaved to tZ bases, thus affecting the whole metabolic spectrum. We present the first large-scale study of short-term CK metabolism kinetics and show that tZ N7- and N9-glucosides are metabolized in vivo. We thus refute the generally accepted hypothesis that N-glucosylation irreversibly inactivates CKs. The subsequently constructed mathematical model provides estimates of the metabolic conversion rates.