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Aims: Protein disulfide isomerases (PDIs) are a family of chaperones resident in the endoplasmic reticulum (ER). In addition to holdase function, some members catalyze disulfide bond formation and isomerization, a crucial step for native folding and prevention of aggregation of misfolded proteins. PDIs are characterized by an arrangement of thioredoxin-like domains, with the canonical protein disulfide isomerase A1 (PDIA1) organized as four thioredoxin-like domains forming a horseshoe with two active sites, a and a ', at the extremities. We aimed to clarify important aspects underlying the catalytic cycle of PDIA1 in the context of the full pathways of oxidative protein folding operating in the ER. Results: Using two fluorescent redox sensors, redox green fluorescent protein 2 (roGFP2) and HyPer (circularly permutated yellow fluorescent protein containing the regulatory domain of the H 2 O 2 -sensing protein OxyR), either unfolded or native, as client substrates, we identified the N-terminal a active site of PDIA1 as the main oxidant of thiols. From there, electrons can flow to the C-terminal a ' active site, with the redox-dependent conformational flexibility of PDIA1 allowing the formation of an interdomain disulfide bond. The a ' active site then acts as a crossing point to redirect electrons to ER downstream oxidases or back to client proteins to reduce scrambled disulfide bonds. Innovation and Conclusions: The two active sites of PDIA1 work cooperatively as an interdomain redox relay mechanism that explains PDIA1 oxidative activity to form native disulfides and PDIA1 reductase activity to resolve scrambled disulfides. This mechanism suggests a new rationale for shutting down oxidative protein folding under ER redox imbalance. Whether it applies to physiological substrates in cells remains to be shown.

Significance: Redox homeostasis consists of an intricate network of reactions in which reactive molecular species, redox modifications, and redox proteins act in concert to allow both physiological responses and adaptation to stress conditions. Recent Advances: This review highlights established and novel thiol-based regulatory pathways underlying the functional facets and significance of redox biology in photosynthetic organisms. In the last decades, the field of redox regulation has largely expanded and this work is aimed at giving the right credit to the importance of thiol-based regulatory and signaling mechanisms in plants. Critical Issues: This cannot be all-encompassing, but is intended to provide a comprehensive overview on the structural/molecular mechanisms governing the most relevant thiol switching modifications with emphasis on the large genetic and functional diversity of redox controllers ( i.e. , redoxins). We also summarize the different proteomic-based approaches aimed at investigating the dynamics of redox modifications and the recent evidence that extends the possibility to monitor the cellular redox state in vivo . The physiological relevance of redox transitions is discussed based on reverse genetic studies confirming the importance of redox homeostasis in plant growth, development, and stress responses. Future Directions: In conclusion, we can firmly assume that redox biology has acquired an established significance that virtually infiltrates all aspects of plant physiology.

Significance: Cellular reactive oxygen species (ROS) mediate redox signaling cascades that are critical to numerous physiological and pathological processes. Analytical methods to monitor cellular ROS levels and proteomic platforms to identify oxidative post-translational modifications (PTMs) of proteins are critical to understanding the triggers and consequences of redox signaling. Recent Advances: The prevalence and significance of redox signaling has recently been illuminated through the use of chemical probes that allow for sensitive detection of cellular ROS levels and proteomic dissection of oxidative PTMs directly in living cells., Critical Issues: In this review, we provide a comprehensive overview of chemical probes that are available for monitoring ROS and oxidative PTMs, and we highlight the advantages and limitations of these methods., Future Directions: Despite significant advances in chemical probes, the low levels of cellular ROS and low stoichiometry of oxidative PTMs present challenges for accurately measuring the extent and dynamics of ROS generation and redox signaling. Further improvements in sensitivity and ability to spatially and temporally control readouts are essential to fully illuminate cellular redox signaling.

Significance: Genetically encoded hydrogen peroxide (H 2 O 2 ) sensors, based on fusions between thiol peroxidases and redox-sensitive green fluorescent protein 2 (roGFP2), have dramatically broadened the available "toolbox" for monitoring cellular H 2 O 2 changes. Recent Advances: Recently developed peroxiredoxin-based probes such as roGFP2-Tsa2ΔC R offer considerably improved H 2 O 2 sensitivity compared with previously available genetically encoded sensors and now permit dynamic, real-time, monitoring of changes in endogenous H 2 O 2 levels., Critical Issues: The correct understanding and interpretation of probe read-outs is crucial for their meaningful use. We discuss probe mechanisms, potential pitfalls, and best practices for application and interpretation of probe responses and highlight where gaps in our knowledge remain., Future Directions: The full potential of the newly available sensors remains far from being fully realized and exploited. We discuss how the ability to monitor basal H 2 O 2 levels in real time now allows us to re-visit long-held ideas in redox biology such as the response to ischemia-reperfusion and hypoxia-induced reactive oxygen species production. Further, recently proposed circadian cycles of peroxiredoxin hyperoxidation might now be rigorously tested. Beyond their application as H 2 O 2 probes, roGFP2-based H 2 O 2 sensors hold exciting potential for studying thiol peroxidase mechanisms, inactivation properties, and the impact of post-translational modifications, in vivo. Antioxid. Redox Signal. 29, 552-568.