Redox-sensitive target detection in gibberellic acid-induced barley aleurone layer

Reactive oxygen species (ROS) are involved in redox regulation by their capacity to reversibly oxidize cysteine residues. This regulation is used by cells to modulate and integrate different responses to extracellular stimuli. In the barley (Hordeum vulgare L.) aleurone layer, gibberellic acid (GA3) is perceived at the plasma membrane and induces the synthesis and secretion of α-amylase. All aleurone membrane systems participate in the elaboration of this response. During these events, ROS are generated as a by-product from intense lipid metabolism. Therefore, we hypothesized that redox regulation may be operating in the GA3-induced response. To test this hypothesis, we measured if GA3 treatment induced changes in the redox state of aleurone membrane-associated proteins. Membrane proteins with sulfhydryl and disulfide groups were isolated from reduced and in situ NEM-alkylated microsomal fractions, respectively. Each fraction was enriched by thiol-affinity chromatography and separated by two-dimensional electrophoresis. The in vivo redox state of each membrane protein present in GA3-treated and -untreated tissue was determined. Results showed that GA3 induced the reduced state in 17 constitutive proteins and the oxidized state in another 5. These data indicate that redox changes occur in membrane proteins after GA3 signaling in the aleurone layer.