Cytokinin receptor AHK3 influences leaf size by modulating trans-zeatin-type cytokinin levels in xylem

Trans-zeatin (tZ)-type cytokinins (CKs) are synthesized in roots in response to nitrate, transported to shoot via xylem, and coordinate diverse physiological processes in aerial organs. Within this mechanism, the regulation of CK biosynthesis by nitrate signaling via NIN-like protein 7 as well as the loading of tZ-type CKs into xylem by ATP-binding cassette transporter G14 have been well studied. However, the roles of other components remain unclear. Here, we show that CK perception and degradation in roots, as mediated by Arabidopsis histidine kinase 3 (AHK3) and CK oxidase/dehydrogenase 4 (CKX4), modulate xylem tZ-type CKs transport and leaf CK status. Grafting experiments demonstrated that root-specific AHK3 deficiency systemically increased leaf blade area through long-distance signals of root-derived tZ-type CK, perceived by shoot-expressed AHK3. Transcriptome and hormonome analyses revealed that root-specific AHK3 deficiency reduced CKX4 expression in roots, elevating tZ-type CK levels in roots and xylem sap and thereby enhancing leaf CK 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 tZ-type CK 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 xylem transport of tZ-type CKs, fine-tuning leaf size according to nitrogen availability in roots. SIGNIFICANCE STATEMENTTo clarify a mechanism that attenuates trans-zeatin-type cytokinin transport from roots to shoots in response to nitrate signaling, this study examined the root-specific role of cytokinin receptors. Our results show that cytokinin perception and degradation, as mediated by Arabidopsis histidine kinase 3 and cytokinin oxidase/dehydrogenase 4, modulate xylem trans-zeatin-type cytokinin transport, thereby fine-tuning leaf cytokinin status and growth in a nitrate-dependent manner, providing new insights into long-distance cytokinin transport according to nitrogen availability.