A revised medium for rapid growth and bio assays with tobacco tissue cultures

48 Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals 49 to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen 50 species (ROS), which can operate protein redox switches by oxidation of specific cysteine 51 residues. Redox buffers such as the highly reduced glutathione pool serve as reservoirs of 52 reducing power for several ROS scavenging and ROS-induced damage repair pathways. 53 Formation of glutathione disulfide (GSSG) and a shift of the glutathione redox potential 54 ( E GSH ) towards less negative values is considered a hallmark of several stress conditions. 55 Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of 56 intact Arabidopsis seedlings and recorded dynamic changes in E GSH and H 2 O 2 levels with the 57 genetically-encoded biosensors Grx1-roGFP2 (for E GSH ) and roGFP2-Orp1 (for H 2 O 2 ) 58 targeted to chloroplasts, the cytosol or mitochondria. Treatment of seedlings with MV caused 59 a rapid oxidation in chloroplasts and subsequently also in the cytosol and mitochondria. The 60 MV-induced oxidation was significantly boosted by illumination with actinic light and 61 largely abolished by inhibitors of photosynthetic electron transport. In addition, MV also 62 induced an autonomous oxidation in the mitochondrial matrix in an electron transport chain 63 activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the 64 combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics 65 of compartmental responses to local ROS generation and provide a basis for understanding how compartment-specific redox dynamics may operate in retrograde signaling and stress 67 acclimation in plants.