An Integrated Spatially Resolved Mechanistic Model of Hierarchical Auxin-Cytokinin-Ethylene Crosstalk Underlying Root Growth Inhibition in Arabidopsis

Decoding how plants integrate multiple hormone signals to coordinate growth requires tools capable of resolving pathway interactions at cellular resolution in living tissue. Here we present ACE (Auxin-Cytokinin-Ethylene) and ACE2, proof-of-concept single-locus reporters to simultaneously capture activity of multiple hormones. Deploying ACE alongside well-established reporters, exogenous hormone treatments, and reverse-genetic perturbations of hormone biosynthesis, signaling, and transport in three-day-old etiolated Arabidopsis seedlings, we dissect the spatio-temporal hierarchy governing primary root elongation and root apical meristem (RAM) size. We demonstrate that both ethylene- and cytokinin-triggered root growth inhibition involve a boost of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1)-mediated auxin biosynthesis and AUXIN RESISTANT1 (AUX1)-dependent auxin redistribution. Two spatially distinct auxin responses underlie the respective root growth effects: ethylene expands TAA1-dependent auxin biosynthesis from the root vasculature into the epidermis and promotes AUX1-mediated auxin import into the transition and elongation zones to inhibit cell elongation, while cytokinin confines ethylene-dependent TAA1-boosted activity to the vasculature and drives auxin accumulation in lateral root cap cells to reduce RAM size. Together, these data establish a reciprocal regulatory loop between these hormones, positioning ethylene as a convergence node in auxin-cytokinin crosstalk, and cytokinin as a modulator of the ethylene-auxin interaction. Critically, the changes in cross-activated reporter patterns described for different genetic backgrounds, alongside quantitative assessment of hormone-specific inhibition of the mutants' growth, were consistent with the multi-hormone network established over two decades of research, and added cell-type-resolved spatial detail and a proposed hierarchy for the etiolated seedling root. Finally, a second-generation reporter, ACE2, overcomes key technical limitations of ACE, expanding the platform's capacity toward a higher-order multi-hormone monitoring system. These resources expand the Arabidopsis genetic toolkit and provide a generalizable framework instrumental for dissecting multi-hormone signaling hierarchies at the cellular level.