Abscisic Acid Controlled Redox Proteome of Arabidopsis and its Regulation by Heterotrimeric G‐proteins

Abstract

Reactive oxygen species (ROS) are highly efficient, membrane permeable cell signals that are essential for cellular regulation. These signals can mark macromolecules for degradation, act as a retrograde signal for transcriptional regulation, as well as directly modulate enzymatic activity via the reversible oxidation of cysteine thiols. Although this reversible cysteine oxidation is an essential regulatory mechanism, the resulting modifications are labile and infrequent compared to the global proteome, making them challenging to maintain ex vivo and detect reproducibly. In our lab, we have optimized an approach for oxidized resin assisted capture (OxRAC) that enables thorough, reproducible analysis of the full reversible cysteine redoxome. By employing iodoacetamide as a blocking reagent during cell lysis, we ensure that cysteines reduced in vivoare excluded before non‐specifically reducing reversibly oxidized cysteines for enrichment and analysis via label free quantitative LC‐MS/MS.In this work, our optimized OxRAC platform was implemented to probe the dependence of heterotrimeric G‐proteins on reversible cysteine oxidation for the mediation of abscisic acid (ABA) signaling pathways in Arabidopsis thaliana. By cross comparing the wildtype Col0plants with the Gβ protein (AGB1) null mutant, agb1, we were able to quantify 6,891 unique oxidized cysteine‐containing peptides and reveal 923 significant changes in oxidation following ABA treatment. Divergent pathways, including primary metabolism, ROS response, translation, and photosynthesis, exhibited both ABA and G‐protein dependent redox changes, many of which occurred on proteins not previously linked to either ABA or G‐proteins. Together, these data uncover a complex network of reversible oxidations that allow ABA and G‐proteins to rapidly adjust cellular signaling to adapt to changing environments and suggest that a functional G‐protein complex is required to maintain intracellular redox homeostasis and fully execute plant stress responses.