The competitive interplay of 12‐oxophytodienoic acid ( OPDA ), protein thiols, and glutathione

Cis-(+)-12-oxophytodienoic acid (OPDA) is a bioactive oxylipin and phytohormone participating in regulation of plant stress responses, growth, and development. Due to its α, β-unsaturated carbonyl moiety, OPDA covalently binds to free thiol groups by Michael addition. This binding, termed OPDAylation, alters the activity of target proteins, such as cyclophilin 20-3 (EC:5.2.1.8) and thioredoxins, that are essential components of the cellular redox regulatory network. To function as a reversible redox regulatory mechanism, OPDAylation should be complemented by a process of de-OPDAylation allowing for fine-tuning of OPDA-dependent regulation. This study explored OPDAylation and de-OPDAylation in vitro with emphasis on the involvement of glutathione. OPDA can be transferred from protein to glutathione (GSH) and vice versa. In a competition experiment, OPDAylation of thioredoxins (TRX) occurred rapidly in the presence of GSH, while over extended incubation times, de-OPDAylation of TRX occurred due to the stoichiometric excess of GSH. These results support the hypothesis that the initial TRX-based OPDAylation is proceeding under kinetic control due to the higher reactivity of the more nucleophilic cysteine moiety in TRX compared to the one of GSH, while the OPDAylation of GSH observed at prolonged incubation time is then the result of a thermodynamically controlled process. De-OPDAylation depends on the protein's sensitivity towards OPDA, the pH and the concentration of excess thiol groups. This likely allows for precise modulation of OPDA amounts, as the rapid modification of protein activity enables subsequent induction of OPDA signaling, whereas de-OPDAylation, triggered by increasing glutathione, increasing cellular reduction or presumably enzymatically, reverses this effect.