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

Abstract Main conclusion Our work identified Ca 2+ -dependent and -independent changes in protein contents upon oxidative stress, showing that Ca 2+ signaling shapes the early oxidative stress response and identifying potential targets for stress resilience research. Abstract Calcium (Ca 2+ ) 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 Ca 2+ -dependent and -independent proteome changes in Arabidopsis thaliana leaves upon oxidative stress induced by hydrogen peroxide (H 2 O 2 ). To dissect the role of Ca 2+ signaling, we inhibited H 2 O 2 -induced Ca 2+ transients by pre-treatment with the Ca 2+ influx blocker LaCl 3 . Throughout all four treatment samples - control, H 2 O 2 -treated, LaCl 3 -treated, H 2 O 2 - and LaCl 3 -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 LaCl 3 and H 2 O 2 treatment resulted in the highest number of differentially abundant proteins (DAPs), indicating a strong attenuating effect of Ca 2+ signaling on the oxidative stress response. By contrast, only 37 and 57 proteins responded to H 2 O 2 alone with distinct subsets of strictly Ca 2+ -dependent, partially Ca 2+ -dependent, and Ca 2+ -independent proteins. Ca 2+ -independent H 2 O 2 -responsive proteins predominantly showed increased abundance, while strictly Ca 2+ -dependent proteins exhibited decreased abundance, suggesting a role for Ca 2+ signaling in protein degradation. Furthermore, three proteins—WLIM1, CYP97C1, and AGAP1—underwent shifts in Ca 2+ -dependency between the two time points, pointing to a dynamic Ca 2+ -regulation. This study provides insight into short-term Ca 2+ -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.