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NADH and NAD + are a ubiquitous cellular redox couple. Although the central role of NAD in plant metabolism and its regulatory role have been investigated extensively at the biochemical level, analyzing the subcellular redox dynamics of NAD in living plant tissues has been challenging. Here, we established live monitoring of NADH/NAD + in plants using the genetically encoded fluorescent biosensor Peredox-mCherry. We established Peredox-mCherry lines of Arabidopsis ( Arabidopsis thaliana ) and validated the biophysical and biochemical properties of the sensor that are critical for in planta measurements, including specificity, pH stability, and reversibility. We generated an NAD redox atlas of the cytosol of living Arabidopsis seedlings that revealed pronounced differences in NAD redox status between different organs and tissues. Manipulating the metabolic status through dark-to-light transitions, respiratory inhibition, sugar supplementation, and elicitor exposure revealed a remarkable degree of plasticity of the cytosolic NAD redox status and demonstrated metabolic redox coupling between cell compartments in leaves. Finally, we used protein engineering to generate a sensor variant that expands the resolvable NAD redox range. In summary, we established a technique for in planta NAD redox monitoring to deliver important insight into the in vivo dynamics of plant cytosolic redox metabolism., (© 2020 American Society of Plant Biologists. All rights reserved.)

Hypoxia regularly occurs during plant development and can be induced by the environment through, for example, flooding. To understand how plant tissue physiology responds to progressing oxygen restriction, we aimed to monitor subcellular physiology in real time and in vivo. We establish a fluorescent protein sensor-based system for multiparametric monitoring of dynamic changes in subcellular physiology of living Arabidopsis thaliana leaves and exemplify its applicability for hypoxia stress. By monitoring cytosolic dynamics of magnesium adenosine 5'-triphosphate, free calcium ion concentration, pH, NAD redox status, and glutathione redox status in parallel, linked to transcriptional and metabolic responses, we generate an integrated picture of the physiological response to progressing hypoxia. We show that the physiological changes are surprisingly robust, even when plant carbon status is modified, as achieved by sucrose feeding or extended night. Inhibition of the mitochondrial respiratory chain causes dynamics of cytosolic physiology that are remarkably similar to those under oxygen depletion, highlighting mitochondrial electron transport as a key determinant of the cellular consequences of hypoxia beyond the organelle. A broadly applicable system for parallel in vivo sensing of plant stress physiology is established to map out the physiological context under which both mitochondrial retrograde signalling and low oxygen signalling occur, indicating shared upstream stimuli., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Summary: Hydrogen peroxide (H2O2) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H2O2 notoriously hard to detect dynamically, specifically and at high resolution. Genetically encoded sensors overcome persistent shortcomings, but pH sensitivity, silencing of expression and a limited concept of sensor behaviour in vivo have hampered any meaningful H2O2 sensing in living plants.We established H2O2 monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein roGFP2‐Orp1 using confocal microscopy and multiwell fluorimetry.We confirmed sensor oxidation by H2O2, show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H2O2, pharmacologically‐induced H2O2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H2O2 dynamics in response to elicitor exposure reveals the late and prolonged impact of the oxidative burst in the cytosol that is modified in redox mutants.We provided a well defined toolkit for H2O2 monitoring in planta and showed that intracellular H2O2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H2O2 dynamics and redox regulation, including intracellular NADPH oxidase‐mediated ROS signalling. See also the Commentary on this article by McLachlan, 221: 1180–1181. [ABSTRACT FROM AUTHOR]