ISOPENTENYL TRANSFERASE3 activation triggers root ammonium hypersensitivity via the cytokinin-ARR10/ARR12-CAP1 signaling pathway

High ammonium (NH₄⁺) levels inhibit primary root (PR) elongation in plants, but the underlying regulatory mechanisms remain poorly understood. In this study, we screened the Arabidopsis (Arabidopsis thaliana) PSKI015 activation-tagged mutant library and identified a dominant mutant, named Ammonium Sensitive 3D (amos3D), which shows increased sensitivity to high NH₄⁺ in terms of PR elongation. Gene cloning revealed that amos3D overexpresses IPT3, a gene involved in cytokinin biosynthesis. Pharmacological and genetic analyses revealed that the PR sensitivity to high NH₄⁺ in amos3D is due to elevated levels of the active cytokinins iP and tZ. Furthermore, we identified the type-B ARRs ARR10 and ARR12 as key transcription factors in the cytokinin-mediated inhibition of PR elongation under high-NH₄⁺ stress. Using CUT&RUN (Cleavage Under Targets & Release Using Nuclease), yeast one-hybrid, and dual-luciferase assays, we showed that ARR10 and ARR12 directly bind to the promoter of CAP1, a tonoplast-localized kinase, repressing its transcription. This repression reduces NH₄⁺ transport from the cytosol to the vacuole, leading to increased Gln/Glu ratios and enhanced NH₄⁺ toxicity. Collectively, our identification of AMOS3 as a key inhibitor of PR growth under high NH₄⁺ through the cytokinin-dependent ARR10/ARR12–CAP1 pathway not only reveals an NH₄⁺-sensing mechanism but also offers promising agronomic potential for optimizing root architecture and improving nitrogen-acquisition efficiency in crops under ammonium-based fertilization systems.