Cytokinin represses NH4+ efflux by suppressing GH3.5 transcription through an ARR10/12-dependent pathway.
Ammonium (NH4+) is an essential nitrogen source for plants, yet its excessive accumulation triggers toxicity, a process typically coupled to futile NH4+ cycling in roots cells. While the cytokinin-ARR10/12 signaling pathway is known to regulate vacuolar NH4+ sequestration, it is not known whether it also coordinates NH4+ efflux across the root-cell plasma membrane to maintain intracellular homeostasis. Here, we identify the auxin-amido synthetase GH3.5 as a direct transcriptional target of ARR10, using a combination of yeast one-hybrid (Y1H) assays, dual-luciferase reporter assays, and CUT&RUN-qPCR. We demonstrate that ARR10 directly binds the GH3.5 promoter to repress its transcription, sustaining free IAA levels and inhibiting NH4+ efflux from root cells, whereas ARR12 acts synergistically to potentiate this ARR10-mediated repression through indirect binding to the promoter. Thus, our study reveals that cytokinin signaling, via the ARR10/12 module, represses GH3.5 to suppress NH4+ efflux. This work elucidates a novel regulatory pathway that integrates cytokinin signaling, auxin homeostasis, and NH4+ transport, providing valuable genetic resources for improving nitrogen-use efficiency and stress tolerance in crops.