Cytokinin receptor AHK3 modulates root-to-shoot transport of trans-zeatin type cytokinin

Trans -zeatin ( t Z)-type cytokinins are synthesized in roots in response to nitrogen availability, transported to shoot via xylem, and coordinate diverse physiological processes in aerial organs. Within this mechanism, the regulation of cytokinin biosynthesis by nitrate signaling via NIN-like protein 7 as well as the loading of t Z-type cytokinins into xylem by ATP-binding cassette transporter G14 have been well studied. However, the roles of other components remain unclear. Here, we show that cytokinin perception and degradation in roots, as mediated by Arabidopsis histidine kinase 3 (AHK3) and cytokinin oxidase/dehydrogenase 4 (CKX4), modulate root-to-shoot transport of t Z-type cytokinins. Grafting experiments demonstrated that root-specific AHK3 deficiency systemically increased leaf blade area through long-distance signals of root-derived t Z-type cytokinin, perceived by shoot-expressed AHK3. Transcriptome, hormonome, and reporter analyses revealed that root-specific AHK3 deficiency reduced CKX4 expression in roots, elevating t Z-type cytokinin levels in roots and xylem sap and thereby enhancing leaf cytokinin response. Transfer experiments manipulating root nitrate levels showed that root-specific AHK3 deficiency promoted leaf blade area in a manner dependent on both nitrate and root-derived t Z-type cytokinin signaling. Moreover, both nitrate signals and root-expressed AHK3 are required for maximal CKX4 induction in roots, and root-specific CKX4 deficiency enhanced leaf blade area in a nitrate-dependent manner. These findings reveal a novel mechanism in which an AHK3-CKX4 module governs root-to-shoot transport of t Z-type cytokinins, fine-tuning leaf size according to nitrogen availability in roots.