Hasannin, O.; Petrik, I.; Strnad, M.; Novak, O.; Cerny, M.; Rashotte, A. M.

Cytokinin (CK) N-glucosides are the most abundant CK metabolites in Arabidopsis and most angiosperms, yet their role in cytokinin activity and response is unclear. Here, we examined metabolomic, transcriptomic, and proteomic profiles of seven CK N-glucoside conjugates in detached Arabidopsis leaves across a 144-hour dark-induced senescence (DIS) timecourse. All tested N-glucosides were found to undergo a slow conversion to their corresponding base forms at position-dependent rates, with N9-glucosides releasing base faster than their corresponding N7-glucosides. Conversion during DIS was strictly isoform-specific and not accompanied by coordinated induction of CK biosynthesis genes, arguing against de novo synthesis as the source of accumulated base. Despite progressive base accumulation, N-glucoside-treated leaves produced substantially fewer Differentially Expressed Genes than direct base application at comparable base concentrations, revealing a disconnect between hormone presence and transcriptional output. Unbiased model comparison identified the base:glucoside ratio as a stronger predictor of CK-Two Component Signaling (TCS) gene expression than absolute base concentration, though modulated by base-type-specific receptor affinities. Early proteomic profiling further revealed a coordinated response shared across N-glucosides but largely absent from base treatments. Together, these findings support that CK N-glucosides as kinetically slow, position-dependent reservoirs whose presence in abundance modulate activation of CK-TCS elicited by bioactive forms.