The PuHK2-PuHP5-PuRR9 cascade recruits PuZFP1 to regulate cytokinin and salt tolerance in poplar

Soil salinization severely inhibits plant growth, necessitating adaptive changes in root system architecture to enhance stress resilience. Cytokinin signaling plays a key role in regulating root plasticity under salt stress. Type-A response regulators, which lack DNA-binding domains, fine-tune cytokinin signaling through phosphorylation-dependent interactions or transcriptional repression. Although type-A response regulators are associated with stress adaptation, their specific mechanistic roles in salt tolerance remain unclear. Here, we identify PuRR9, a type-A response regulator in poplar, as a negative regulator of cytokinin signaling that enhances salt tolerance through a phosphorylation cascade (PuHK2-PuHP5-PuRR9). Salt stress promotes PuRR9 phosphorylation, strengthening its interaction with the transcriptional repressor PuZFP1. Phosphorylated PuRR9 recruits PuZFP1 to the promoter of PuIPT3, a key cytokinin biosynthesis gene, thereby suppressing its expression. This repression reduces cytokinin biosynthesis and promotes root growth under salt stress. We propose that the PuHK2-PuHP5-PuRR9-PuZFP1 module enhances salt tolerance by establishing a phosphorylation-dependent negative feedback loop within the cytokinin pathway. These findings reveal a novel mechanism by which a type-A response regulator modulates root system architecture and extend current understanding of cytokinin signaling.