Chloroplast-derived photo-oxidative stress causes changes in H2O2 and EGSH in other subcellular compartments

Abstract

Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen species (ROS), which can operate protein redox switches by oxidation of specific cysteine residues. Redox buffers, such as the highly reduced glutathione pool, serve as reservoirs of reducing power for several ROS-scavenging and ROS-induced damage repair pathways. Formation of glutathione disulfide and a shift of the glutathione redox potential (EGSH) toward less negative values is considered as hallmark of several stress conditions. Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of intact Arabidopsis (Arabidopsis thaliana) seedlings and recorded dynamic changes in EGSH and H2O2 levels with the genetically encoded biosensors Grx1-roGFP2 (for EGSH) and roGFP2-Orp1 (for H2O2) targeted to chloroplasts, the cytosol, or mitochondria. Treatment of seedlings with MV caused rapid oxidation in chloroplasts and, subsequently, in the cytosol and mitochondria. MV-induced oxidation was significantly boosted by illumination with actinic light, and largely abolished by inhibitors of photosynthetic electron transport. MV also induced autonomous oxidation in the mitochondrial matrix in an electron transport chain activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provides a basis for understanding how compartment-specific redox dynamics might operate in retrograde signaling and stress acclimation in plants.

Biosensors: A Sneak Peek into Plant Cell’s Immunity

“…They can be Förster resonance energy transfer (FRET) based, employing two FPs linked by calmodulin-sensing domain, e. g. Cameleons [3], or colourful GECOs (genetically encoded Ca 2+ indicators for optical imaging) [28], employing one, circularly permutated fluorescent protein. Prominent sensors of reactive oxygen species (ROS) are redox-sensitive GFPs in fusion with oxidant receptor peroxidase-1 (roGFP2-Orp1) [29] and glutaredoxin 1 (Grx1-roGFP2) [30], H2O2 and glutathione sensors, respectively, that were used to distinguish the patterns of these two molecular species in the cytosol, chloroplast and mitochondria upon illumination [31]. Main three hormones of immune response, salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), can be followed with direct degron-based sensor Jas9-VENUS in case of JA or with indirect transcriptional reporters based on defense genes' promoters, such as pathogen-responsive PR1, PR2 and PR5 for SA-and plant defensin 1.2 (PDF1.2) and vegetative storage protein (VSP) for JA-involving response.…”

“…As an example, roGFPs are mutated GFP molecules sensitive to redox levels in the cell [47], which were later fused to signal sequences to allow targeting to different subcellular organelles, such as mitochondria [48] and chloroplasts [49]. Fusion partners known to be targets of redox transitions by glutathione (Figure 1) [30] or H2O2 (Figure 1) [29] were also designed and recently used for time-resolved measurements of both molecular species in the cytosol, chloroplasts and mitochondria [31]. This allowed for better understanding the role of the mitochondria in sensing and signaling the cellular redox challenge in response to abiotic stress [50], deciphering the role of redox state in intercellular transport [49] or exploring the central role of glutathione in mediating redox signaling [51].…”

Live monitoring of ROS-induced cytosolic redox changes with roGFP2-based sensors in plants

“…3. Both sensors have also been targeted to mitochondria by fusion with the transit peptide sequence either from Serine Hydroxymethyltransferase (SHMT; for roGFP2-Grx1; [29]) or from the Nicotiana plumbaginifolia ATP synthase ß-subunit of (for roGFP2-Orp1; [21]), or to plastids by cloning the Transketolase transit peptide (TKTP) to the N-termini of the sensors [13]). All lines expressing these constructs are available upon request.…”

“…The unequal variance can be corrected for by log10 transformation, which will convert the skewed data distribution to a normal distribution. 13. Lower amounts of buffer can be also used, but the minimum recommended is 100 µL, to evenly cover the bottom of the well and to avoid evaporation during the run.…”

“…These modifications may alter the structural properties and activities of proteins [10,11]. Indirect H2O2-dependent signaling may also occur via the cellular glutathione redox buffer if significant fluxes of H2O2 are detoxified via the glutathione-ascorbate cycle, which can lead to a transient change in the glutathione redox potential (EGSH) [12,13]. The signaling function of H2O2 implies dynamic changes in H2O2 fluxes to ensure that activation or inactivation of downstream target proteins is only transient.…”