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

Plant resilience relies on detecting and responding to osmotic stress and pathogens. Calcium (Ca 2+ ) and hydrogen peroxide (H 2 O 2 ) 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 Ca 2+ channel MSL9, which may interact with kinases (CPK8), actins (ACT2, ACT7), and antioxidant enzymes (APX1–3, CAT1–3) to coordinate Ca 2+ 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 H 2 O 2 on early systemic signallling response of Ca 2+ and H 2 O 2 . Our results demonstrated directional Ca 2+ signals that propagated through stele tissues, with varying speeds depending on the stressor. In contrast, symplastic H 2 O 2 accumulation displayed distinct patterns from the observed Ca 2+ signals. This study integrated stress-specific Ca 2+ and symplastic H 2 O 2 signalling data with protein interaction networks, linking oxidative modifications induced by reactive oxygen species (ROS) with Ca 2+ influx to provide insights into early molecular signalling events in Arabidopsis roots during PAMP-triggered immunity and abiotic stress. • The bi-dfRC device enabled precise osmolyte/PAMP application to Arabidopsis roots, capturing early Ca 2+ signalling and H 2 O 2 accumulation. • 300 phosphorylated peptides in response to PEG and flg22 have been identified. • Early phosphorylation of MSL9 and PLDrp1 detected within 5 minutes of PEG and flg22 treatment. • PPIs involved in ROS detoxification, cytoskeletal dynamics, and membrane trafficking were identified.