With or without a Ca2+ signal?: a proteomics approach toward Ca2+-dependent and -independent changes in response to oxidative stress in Arabidopsis thaliana

Our work identified Ca²⁺-dependent and -independent changes in protein contents upon oxidative stress, showing that Ca²⁺ signaling shapes the early oxidative stress response and identifying potential targets for stress resilience research. Calcium (Ca²⁺) and reactive oxygen species (ROS) are key secondary messengers in plant stress signaling, yet their interplay in regulating proteome-wide responses remains poorly understood. We employed label-free quantitative (LFQ) proteomics to investigate Ca²⁺-dependent and -independent proteome changes in Arabidopsis thaliana leaves upon oxidative stress induced by hydrogen peroxide (H₂O₂). To dissect the role of Ca²⁺ signaling, we inhibited H₂O₂-induced Ca²⁺ transients by pre-treatment with the Ca²⁺ influx blocker LaCl₃. Throughout all four treatment samples - control, H₂O₂-treated, LaCl₃-treated, H₂O₂- and LaCl₃-treated - we identified a total of 3724 and 3757 proteins after 10 and 30 min, respectively. Of these, 581 proteins showed significant changes in abundance between the 10 min and 909 proteins between the 30 min sample groups. The combined LaCl₃ and H₂O₂ treatment resulted in the highest number of differentially abundant proteins (DAPs), indicating a strong attenuating effect of Ca²⁺ signaling on the oxidative stress response. By contrast, only 37 and 57 proteins responded to H₂O₂ alone with distinct subsets of strictly Ca²⁺-dependent, partially Ca²⁺-dependent, and Ca²⁺-independent proteins. Ca²⁺-independent H₂O₂-responsive proteins predominantly showed increased abundance, while strictly Ca²⁺-dependent proteins exhibited decreased abundance, suggesting a role for Ca²⁺ signaling in protein degradation. Furthermore, three proteins-WLIM1, CYP97C1, and AGAP1-underwent shifts in Ca²⁺-dependency between the two time points, pointing to a dynamic Ca²⁺-regulation. This study provides insight into short-term Ca²⁺-dependent and independent regulation of the Arabidopsis leaf proteome in response to oxidative stress, thereby identifying potential new targets for research on plant stress resilience mechanisms.