Ca2+ and ROS signalling exhibit divergent root-specific dynamics in Arabidopsis responses to abiotic stress and PAMPs

Abstract

Plant resilience relies on detecting and responding to osmotic stress and pathogens. Calcium (Ca²⁺) and hydrogen peroxide (H₂O₂) are key signalling molecules in plant stress, yet their interplay with protein modifications remains unclear. This study utilises a label-free quantitative proteomic approach to elucidate proteins involved in early signalling events in Arabidopsis thaliana (A. thaliana) roots exposed to osmotic stress induced by polyethylene glycol (PEG) and pathogen associated molecular pattern (PAMP)-triggered immunity by flagellin 22 (flg22). We identified over 300 phosphoproteins that changed in abundance within five minutes of stress exposure, including 153 phosphorylation and 49 methionine oxidations. Key signalling proteins included the mechanosensitive Ca²⁺ channel MSL9, which may interact with kinases (CPK8), actins (ACT2, ACT7), and antioxidant enzymes (APX1–3, CAT1–3) to coordinate Ca²⁺ influx and ROS regulation during early stress responses. Using the bi-directional dual-flowRootChip (bi-dfRC) we tested the effect of solute gradients of PEG, NaCl, flg22, Pep-13 and H₂O₂ on early systemic signallling response of Ca²⁺ and H₂O₂. Our results demonstrated directional Ca²⁺ signals that propagated through stele tissues, with varying speeds depending on the stressor. In contrast, symplastic H₂O₂ accumulation displayed distinct patterns from the observed Ca²⁺ signals. This study integrated stress-specific Ca²⁺ and symplastic H₂O₂ signalling data with protein interaction networks, linking oxidative modifications induced by reactive oxygen species (ROS) with Ca²⁺ influx to provide insights into early molecular signalling events in Arabidopsis roots during PAMP-triggered immunity and abiotic stress.