Biosensors have emerged as a valuable tool with high specificity and sensitivity for fast and reliable detection of hazardous substances in drinking water. Numerous substances have been addressed using synthetic biology approaches. However, many proposed biosensors are based on living, genetically modified organisms and are therefore limited in shelf life, usability and biosafety. We addressed these issues by the construction of an extensible, cell-free biosensor. Storage is possible through freeze drying on paper. Following the addition of an aqueous sample, a highly efficient cell-free protein synthesis (CFPS) reaction is initiated. Specific allosteric transcription factors modulate the expression of ‘superfolder’ green fluorescent protein (sfGFP) depending on the presence of the substance of interest. The resulting fluorescence intensities are analyzed with a conventional smartphone accompanied by simple and cheap light filters. An ordinary differential equitation (ODE) model of the biosensors was developed, which enabled prediction and optimization of performance. With an optimized cell-free biosensor based on the Shigella flexneri MerR transcriptional activator, detection of 6 μg/L Hg(II) ions in water was achieved. Furthermore, a completely new biosensor for the detection of gamma-hydroxybutyrate (GHB), a substance used as date-rape drug, was established by employing the naturally occurring transcriptional repressor BlcR from Agrobacterium tumefaciens .

Cold temperatures restrict plant growth, geographical extension of plant species, and agricultural practices. This review deals with cold stress above freezing temperatures often defined as chilling stress. It focuses on the redox regulatory network of the cell under cold temperature conditions. Reactive oxygen species (ROS) function as the final electron sink in this network which consists of redox input elements, transmitters, targets, and sensors. Following an introduction to the critical network components which include nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductases, thioredoxins, and peroxiredoxins, typical laboratory experiments for cold stress investigations will be described. Short term transcriptome and metabolome analyses allow for dissecting the early responses of network components and complement the vast data sets dealing with changes in the antioxidant system and ROS. This review gives examples of how such information may be integrated to advance our knowledge on the response and function of the redox regulatory network in cold stress acclimation. It will be exemplarily shown that targeting the redox network might be beneficial and supportive to improve cold stress acclimation and plant yield in cold climate.

Cys-based redox regulation was long regarded a major adjustment mechanism of photosynthesis and metabolism in plants, but in the recent years, its scope has broadened to most fundamental processes of plant life. Drivers of the recent surge in new insights into plant redox regulation have been the availability of the genome-scale information combined with technological advances such as quantitative redox proteomics and in vivo biosensing. Several unexpected findings have started to shift paradigms of redox regulation. Here, we elaborate on a selection of recent advancements, and pinpoint emerging areas and questions of redox biology in plants. We highlight the significance of (1) proactive H2O2 generation, (2) the chloroplast as a unique redox site, (3) specificity in thioredoxin complexity, (4) how to oxidize redox switches, (5) governance principles of the redox network, (6) glutathione peroxidase-like proteins, (7) ferroptosis, (8) oxidative protein folding in the ER for phytohormonal regulation, (9) the apoplast as an unchartered redox frontier, (10) redox regulation of respiration, (11) redox transitions in seed germination and (12) the mitochondria as potential new players in reductive stress safeguarding. Our emerging understanding in plants may serve as a blueprint to scrutinize principles of reactive oxygen and Cys-based redox regulation across organisms.

Retrograde signals from the chloroplast control expression of nuclear genes. A large fraction of these genes is affected rapidly upon light intensity shifts. This study was designed to address the interdependence of signaling pathways involved in the rapid high light response and redox and reactive oxygen species signaling by exploiting the glutathione and ascorbate deficient mutants pad2 and vtc1. In the first set of experiments the transcriptional response of the two transcription factors ERF6 and ERF105 that had previously been shown to rapidly respond to light was shown to be deregulated in the pad2 mutant but not in the vtc1 background. The transcriptional response after combining the low-to-high light transfer with methylviologen pretreatment further demonstrated the significance of glutathione in strongly modulating the retrograde response. Transcripts encoding small heat shock proteins (HSP17.4, HSP176a, HSP20-like1 and HSP20-like2) and the lipid transfer protein LTP3 were taken as markers responding to the combinatorial treatment in wild type, and most strongly in pad2 in high light or upon methylviologen treatment. A correlation with H O accumulation was not observed. It is concluded that glutathione-dependent processes participate in light-triggered rapid gene regulation independent on cellular H O .

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.