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The orientation of membrane proteins within the lipid bilayer is key to understanding their molecular function. Similarly, the proper topology of multispanning membrane proteins is crucial for their function. Although bioinformatics tools can predict these parameters assessing the presence of hydrophobic protein domains sufficiently long to span the membrane and other structural features, the predictions from different algorithms are often inconsistent. Therefore, experimental analysis becomes mandatory. Redox-based topology analysis exploits the steep gradient in the glutathione redox potential (E GSH ) across the ER membrane of about 80 mV to visualize the orientation of ER membrane proteins by fusing the E GSH biosensor roGFP2 to either the N- or the C-termini of the investigated protein sequence. Transient expression of these fusion proteins in tobacco leaves allows direct visualization of orientation and topology of ER membrane proteins in planta. The protocol outlined here is based on either a simple merge of the two excitation channels of roGFP2 or a colocalization analysis of the two channels and thus avoids ratiometric analysis of roGFP2 fluorescence., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. ER dynamics can be determined using optical flow techniques from computer vision or persistency analysis. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

To date, fluorescent protein biosensors are widely used in research. In vivo, they can be applied to dynamically monitor several physiological parameters in various subcellular compartments. Redox-sensitive green fluorescent protein 2 (roGFP2) senses the glutathione redox potential via a disulfide bridge formed between neighboring beta-strands of its beta-barrel structure. As changes in redox state affect both excitation maxima of roGFP2 oppositely, sensor responses are ratiometric. The reaction mechanism of roGFP2 is well characterized and involves an intermediate S-glutathionylation step. Thus, roGFP2 is also used in enzymatic in vitro assays, e.g., assessing glutaredoxin kinetics. In addition to the fluorescent read-out, the roGFP2 redox state can also be determined by differential migration on a non-reducing SDS-PAGE. This read-out mode may be beneficial in some applications, e.g., if mass-spectrometric analysis of posttranslational cysteine modifications is desired. Here, we describe a protocol for gel-based fluorescent read-out of the roGFP2 redox state, as well as modification of free cysteines by maleimide-based reagents., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Plant cells produce reactive oxygen species (ROS) as by-products of oxygen metabolism and for signal transduction. Depending on their concentration and their site of production, ROS can cause oxidative damage within the cell and must be effectively scavenged. Detoxification of the most stable ROS, hydrogen peroxide (H 2 O 2 ), via the glutathione-ascorbate pathway may transiently alter the glutathione redox potential (E GSH ). Changes in E GSH can thus be considered as an indicator of the oxidative load in the cell. Genetically encoded probes based on roGFP2 enable extended opportunities for in vivo monitoring of H 2 O 2 and E GSH dynamics. Here, we provide detailed protocols for live monitoring of both parameters in the cytosol with the probes Grx1-roGFP2 for E GSH and roGFP2-Orp1 for H 2 O 2 , respectively. The protocols have been adapted for live cell imaging with high lateral resolution on a confocal microscope and for multi-parallel measurements in whole organs or intact seedlings in a fluorescence microplate reader. Elicitor-induced ROS generation is used for illustration of the opportunities for dynamic ROS measurements that can be transferred to other research questions and model systems., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to a thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available.

Mitochondrial physiology sets the basis for function of the organelle and vice versa. While a limited range of in vivo parameters, such as oxygen consumption, has been classically accessible for measurement, a growing collection of fluorescent protein sensors can now give insights into the physiology of plant mitochondria. Nevertheless, the meaningful application of these sensors in mitochondria is technically challenging and requires rigorous experimental standards. Here we exemplify the application of three genetically encoded sensors to monitor glutathione redox potential, pH, and calcium in the matrix of mitochondria in intact plants. We describe current methods for quantitative imaging and analysis in living root tips by confocal microscopy and discuss methodological limitations.

Continuous control of metabolism and development is a key feature of life and is of particular importance under stress conditions. While under normal conditions most cellular compartments maintain a reducing environment, the cellular redox state can be influenced by external factors. Redox changes might in turn be employed as part of a signalling cascade leading to molecular responses to adverse situations. To enable dynamic measurements of the cellular redox poise in vivo, reduction-oxidation sensitive GFP (roGFP) can be expressed in plant cells and observed by confocal microscopy. When imaged by confocal microscopy this probe exhibits significant opposing shifts in the fluorescence intensities excited at 488 and 405 nm upon formation of an intramolecular disulfide bridge, which enables ratiometric analysis. The formation of the disulfide bridge is directly responsive to the redox state of the glutathione redox buffer within the subcellular compartment to which roGFP is targeted.