Phosphoproteomics uncovers rapid and specific transition from plant two-component system signaling to Ser/Thr phosphorylation by the intracellular redox sensor AHK5

The Arabidopsis thaliana histidine kinase 5 (AHK5) is proposed to function as a intracellular sensor of active oxygen species (ROS) and redox conditions, fine-tuning responses to diverse ROS-inducing stimuli through a multistep phosphorelay (MSP) system. To elucidate the mechanism of ROS perception by AHK5 and signaling through posttranslational modifications, a comparative phosphoproteomics analysis was conducted using ahk5 loss-of-function and wild type seedlings in response to exogenously applied H ₂ O ₂ . Under control and H ₂ O ₂ -related conditions, a rapid signaling transition from the MSP to Ser/Thr phosphorylation was observed. AHK5 appears to regulate ROS-responsive phosphorylation at the plasma membrane by the modification of nanodomain components such as remorins, aquaporins, IQDs, and patellins, positioning it as a central integrator of redox signaling during adaptation to diverse stresses. AHK5 drives a distinct, ABA-independent signaling pathway for H ₂ O ₂ -induced stomatal closure, likely by regulating phosphorylation of key components such as RBOHD, CAS, and HPCA1. Moreover, AHK5-fine-tuned root development involves ABA-dependent signaling and appears to act through modulation of auxin transport via phosphorylation of auxin carrier, highlighting a mechanistic divergence between ROS responses in the shoot and root. Our data further suggest that AHK5 orchestrates stress-responsive nanodomain-associated signaling hubs through regulation of membrane trafficking, cytoskeletal dynamics, and endocytosis to mediate stress responses.