Using DCP-Rho1 as a fluorescent probe to visualize sulfenic acid-containing proteins in living plant cells

Among the biologically relevant reactive oxygen species (ROS), hydrogen peroxide (H₂O₂) has special properties. H₂O₂ can diffuse across membranes, has a low reactivity, and is very stable. Deprotonated cysteine residues in proteins can be oxidized by H₂O₂ into a highly reactive sulfenic acid derivative (-SOH), which can react with another cysteine to form a disulfide. Under higher oxidative stress the sulfenic acid undergo further oxidation to sulfinic acid (Cys-SO₂H), which can subsequently be reduced. The sulfinic acid can be hyperoxidized to sulfonic acid (Cys-SO₃H), whose reduction is irreversible. Formation of sulfenic acids can have a role in sensing oxidative stress, signal transduction, modulating localization and activity to regulate protein functions. Therefore, there is an emerging interest in trying to understand the pool of proteins that result in these sorts of modification in response to oxidative stress. This is known as the sulfenome and several approaches have been developed in animal and plant cells to analyze the sulfenome under different stress responses. These approaches can be proteomic, molecular, immunological (i.e., antibodies), or expressing genetically encoded probes that specifically react to sulfenic modifications. In this chapter, we describe an additional approach that allows visualization of sulfenic modification in vivo. This is newly developed fluorescent probe DCP-Rho1 can be implemented in any plant cell to analyze the sulfenic modification.