Cytokinin Senescence Delay Is Shaped by Receptor Specificity and Metabolic Stability

Different cytokinin (CK) forms have distinct receptor affinities and metabolic rates, as seen in previous work with trans-Zeatin (tZ), isopentenyladenine (iP), dihydrozeatin (DHZ), and cis-Zeatin (cZ). However, it remains unclear how specific biochemical features of each form drives distinct tissue-specific physiological hormone output in response to application of these CK bases. Likewise, highly abundant N-glucoside CKs have also recently been attributed with having tissue-specific activities, yet their contribution to CK signaling output remains unclear. Here, we show that CK receptor preference and metabolic persistence together shape isoform-specific CK signaling strength, including tissue-dependent hormone responses in leaf versus root assays. We used physiological, genetic, and multi-omics integration in Arabidopsis to show that tZ and iP anti-senescence activity is matched by DHZ through a distinct receptor metabolic mechanism. DHZ requires AHK3[->]ARR2 signaling to be fully effective in Dark Induced Senescence (DIS) assay and overcomes its lower receptor affinity through higher metabolic persistence, accumulating at levels [~]2.5-fold above tZ and iP early in a senescence time course. Second, we demonstrate that N-glucosides, in a ratio-dependent manner, can act as modulators of CK response intensity. tZ N-glucoside co-applied with its base isoform tZ reduces CK signaling output by up to 41% as seen by pTCS::LUC in both protoplast transient expression and whole-leaf assays. Together, these findings provide a framework of how integration of receptor preference and metabolic stability determines CK isoform activity, in a model where abundant N-glucosides can modulate active CK signaling output.