Biology subjects

Lampl, N.

Publications and source records attributed to Lampl, N..

3 recordsLinked to original sources

bioRxiv2021

SPEAR: a proteomics approach for simultaneous protein expression and redox analysis

Oxidation and reduction of protein cysteinyl thiols serve as molecular switches, which is considered the most central mechanism for redox regulation of biological processes, altering protein structure, biochemical activity, subcellular localization, and binding affinity. Redox proteomics allows for the global identification of redox-modified cysteine (Cys) sites and quantification of their oxidation/reduction responses, serving as a hypothesis-generating platform to stimulate redox biology mechanistic research. Here, we developed Simultaneous Protein Expression and Redox (SPEAR) analysis, a new redox-proteomics approach based on differential labeling of oxidized and reduced cysteines with light and heavy isotopic forms of commercially available isotopically-labeled N-ethylmaleimide (NEM). The presented method does not require enrichment for labeled peptides, thus enabling simultaneous quantification of Cys oxidation state and protein abundance. Using SPEAR, we were able to quantify the in-vivo oxidation state of thousands of cysteines across the Arabidopsis proteome under steady-state and oxidative stress conditions. Functional assignment of the identified redox-sensitive proteins demonstrated the widespread effect of oxidative conditions on various cellular functions and highlighted the enrichment of chloroplast-targeted proteins. SPEAR provides a simple, straightforward, and cost-effective means of studying redox proteome dynamics. The presented data provide a global quantitative view of cysteine oxidation of well-known redox-regulated active sites and many novel redox-sensitive sites whose role in plant acclimation to stress conditions remains to be further explored.

biochemistry↗

bioRxiv2021

Systematic monitoring of photosynthetically-derived oxidative signals using a 2-Cys peroxiredoxin-based redox probe

Transmission of reductive cues from the photosynthetic electron transport chain to redox-regulated proteins plays a crucial role in activating chloroplast metabolism. However, deciphering the role of their counterbalanced oxidative signals is challenging due to monitoring difficulties. Here, we demonstrate the light-depended redox modification of chloroplast-targeted 2-Cys peroxiredoxins and introduce peroxiredoxin-based biosensors to monitor photosynthetically-derived oxidative signals. By employing a set of genetically encoded biosensors, we show the induction of oxidative signals under habitual light intensities and their inverse relationship with NADPH levels, unraveling the combined activity of reducing and oxidizing signals in fine-tuning chloroplast metabolism. A faster increase in carbon assimilation rates during photosynthesis induction phase was measured in plants deficient in 2-Cys peroxiredoxins compared to wild-type, suggesting the involvement of oxidative signals in attenuating photosynthesis under variable light environments. We suggest that oxidative signals measured by peroxiredoxin-based biosensors reflect the limitation to photosynthesis imposed by the redox regulatory system. One-Sentence SummaryA genetically encoded biosensor unmasked the dominant role of photosynthetically-derived oxidative signals under habitual conditions.

plant biology↗

bioRxiv2020

Sensing stress responses in potato with whole-plant redox imaging

Environmental stresses are among the major factors that limit crop productivity and plant growth. Plant exposure to various abiotic stress, such as drought, cold temperatures, or high light, results in overproduction of reactive oxygen species (ROS). To avoid oxidative damage, critical mechanisms for their detoxification have evolved, consisting of ROS-scavenging enzymes and small antioxidant molecules, such as glutathione (GSH) and ascorbate. Thus, monitoring redox changes with high spatial and temporal resolution is critical for understanding oxidative stress signaling and has the potential to enable early detection of stress responses in crop plants. In this work, potato plants ( Solanum tuberosum) expressing a chloroplast-targeted reduction-oxidation-sensitive green fluorescent protein2 (roGFP2) were generated to report the redox potential of the glutathione (EGSH) in the chloroplast stroma. By applying whole-plant fluorescence imaging, we mapped alteration in the chloroplast EGSH under several stress conditions including, high-light, cold and drought. Extremely high increase in chloroplast EGSH was observed under the combination of high-light and low temperatures, conditions that specifically induce PSI photoinhibition. Intriguingly, whole-plant ratiometric imaging analysis noted a higher reduced state in newly developed as compared to mature leaves, suggesting a graded stress sensitivity as part of the plant strategies for coping with stress conditions. The presented observations suggest that whole-plant redox imaging can serve as a powerful tool for the basic understanding of plant stress responses as well as for applied agricultural research, such as improving phenotyping capabilities in breeding programs and early detection of stress responses in the field.

plant biology↗