Water deficiency compromises plant performance and yield in many habitats and in agriculture. In addition to survival of the acute drought stress period which depends on plant-genotype-specific characteristics, stress intensity and duration, also the speed and efficiency of recovery determine plant performance. Drought-induced deregulation of metabolism enhances generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) which in turn affect the redox regulatory state of the cell. Strong correlative and analytical evidence assigns a major role in drought tolerance to the redox regulatory and antioxidant system. This review compiles current knowledge on the response and function of superoxide, hydrogen peroxide and nitric oxide under drought stress in various species and drought stress regimes. The meta-analysis of reported changes in transcript and protein amounts, and activities of components of the antioxidant and redox network support the tentative conclusion that drought tolerance is more tightly linked to up-regulated ascorbate-dependent antioxidant activity than to the response of the thiol-redox regulatory network. The significance of the antioxidant system in surviving severe phases of dehydration is further supported by the strong antioxidant system usually encountered in resurrection plants.

Thiol-dependent redox regulation controls central processes in plant cells including photosynthesis. Thioredoxins reductively activate, for example, Calvin-Benson cycle enzymes. However, the mechanism of oxidative inactivation is unknown despite its importance for efficient regulation. Here, the abundant 2-cysteine peroxiredoxin (2-CysPrx), but not its site-directed variants, mediates rapid inactivation of reductively activated fructose-1,6-bisphosphatase and NADPH-dependent malate dehydrogenase (MDH) in the presence of the proper thioredoxins. Deactivation of phosphoribulokinase (PRK) and MDH was compromised in 2cysprxAB mutant plants upon light/dark transition compared to wildtype. The decisive role of 2-CysPrx in regulating photosynthesis was evident from reoxidation kinetics of ferredoxin upon darkening of intact leaves since its half time decreased 3.5-times in 2cysprxAB. The disadvantage of inefficient deactivation turned into an advantage in fluctuating light. Physiological parameters like MDH and PRK inactivation, photosynthetic kinetics and response to fluctuating light fully recovered in 2cysprxAB mutants complemented with 2-CysPrxA underlining the significance of 2-CysPrx. The results show that the 2-CysPrx serves as electron sink in the thiol network important to oxidize reductively activated proteins and represents the missing link in the reversal of thioredoxin-dependent regulation.

Protein oligomerization is central to biological function and regulation, yet its experimental quantification and measurement of dynamic transitions in solution remain challenging. Here, we show that single molecule mass photometry quantifies affinity and polydispersity of heterogeneous protein complexes in solution. We demonstrate these capabilities by studying the functionally relevant oligomeric equilibria of 2-cysteine peroxiredoxins (2CPs). Comparison of the polydispersity of plant and human 2CPs as a function of concentration and redox state revealed features conserved among all 2CPs. In addition, we also find species-specific differences in oligomeric transitions, the occurrence of intermediates and the formation of high molecular weight complexes, which are associated with chaperone activity or act as a storage pool for more efficient dimers outlining the functional differentiation of human 2CPs. Our results point to a diversified functionality of oligomerization for 2CPs and illustrate the power of mass photometry for characterizing heterogeneous oligomeric protein distributions in near native conditions.

2-Cysteine peroxiredoxins (2-CysPRX) are highly abundant thiol peroxidases in chloroplasts and play key roles in reactive oxygen species (ROS) defense and redox signaling. Peroxide-dependent oxidation of cysteines induces conformational changes that alter the ability for protein–protein interactions. For regeneration, 2-CysPRXs withdraw electrons from thioredoxins (TRXs) and participate in redox-dependent regulation by affecting the redox state of TRX-dependent targets, for example, in chloroplast metabolism. This work explores the redox conformation-specific 2-CysPRX interactome using an affinity-based pull down with recombinant variants arrested in specific quaternary conformations. This allowed us to address a critical and poorly explored aspect of the redox-regulatory network and showed that the interaction of TRXs, their interaction partners, and 2-CysPRX occur under contrasting redox conditions. A set of 178 chloroplast proteins were identified from leaf proteins and included proteins with functions in photosynthesis, carbohydrate, fatty acid and amino acid metabolism, and defense. These processes are known to be deregulated in plants devoid of 2-CysPRX. Selected enzymes like LIPOXYGENASE 2, CHLOROPLAST PROTEIN 12-1, CHORISMATE SYNTHASE, ß-CARBONIC ANHYDRASE, and FERREDOXIN-dependent GLUTAMATE SYNTHASE 1 were subjected to far Western, isothermal titration calorimetry, and enzyme assays for validation. The pull down fractions frequently contained TRXs as well as their target proteins, for example, FRUCTOSE-1,6-BISPHOSPHATASE and MALATE DEHYDROGENASE. The difference between TRX-dependent indirect interactions of TRX targets and 2-CysPRX and direct 2-CysPRX binding is hypothesized to be related to quaternary structure formation, where 2-CysPRX oligomers function as scaffold for complex formation, whereas TRX oxidase activity of 2-CysPRX controls the redox state of TRX-related enzyme activity.

The chloroplast primary metabolism is of central importance for plant growth and performance. Therefore, it is tightly regulated in order to adequately respond to multiple environmental conditions. A major fluctuation that plants experience each day is the change between day and night, i.e., the change between assimilation and dissimilation. Among other mechanisms, thioredoxin-mediated redox regulation is an important component of the regulation of plastid-localized metabolic enzymes. While assimilatory processes such as the Calvin–Benson cycle are activated under illumination, i.e., under reducing conditions, carbohydrate degradation is switched off during the day. Previous analyses have identified enzymes of the oxidative pentose phosphate pathway to be inactivated by reduction through thioredoxins. In this work, we present evidence that an enzyme of the plastidic glycolysis, the phosphofructokinase isoform AtPFK5, is also inactivated through reduction by thioredoxins, namely by thioredoxin-f. With the help of chemical oxidation, mutant analyses and further experiments, the highly conserved motif CXDXXC in AtPFK5 was identified as the target sequence for this regulatory mechanism. However, knocking out this isoform in plants had only very mild effects on plant growth and performance, indicating that the complex primary metabolism in plants can overcome a lack in AtPFK5 activity.