Biology subjects

Rosenwasser, S.

Publications and source records attributed to Rosenwasser, S..

16 recordsLinked to original sources

bioRxiv2026

ACHT2 deactivates carbon assimilation during light-dark transition

In chloroplasts, photosynthetic efficiency relies on a delicate balance between reductive and oxidative thiol-based signaling networks. Members of the high-midpoint-potential atypical thioredoxins (Trxs) were shown to oxidize photosynthetic enzymes by channeling reducing equivalents to H2O2 through 2-Cys-Prx activity. However, it remains unclear which atypical-Trx isoforms regulate Calvin-Benson cycle (CBC) inactivation, and whether they possess distinct functional specificities or operate redundantly in vivo. To resolve this, electron transport and carbon assimilation were continuously monitored during physiological dynamic light transitions in CRISPR-generated single, double and triple atypical Trx mutants. Notably, ACHT2 was identified as a primary determinant of CBC inactivation during dark-to-light transitions, as evidenced by the alleviation of redox-mediated bottlenecks in electron flow downstream of Fd and a lower CBC inactivation state in acht2 plants, resembling the phenotype observed in plants lacking 2-Cys Prxs A and B (2cpab). In contrast, plants mutated in ACHT1, ACHT4, or TrxL2 displayed CBC inactivation kinetics comparable to those of the wild type. Furthermore, mutation of ACHT2 did not compromise plant fitness. In contrast, growth retardation was observed in the acht1/acht4 double mutant, suggesting that the severe phenotype of 2cpab does not arise from impaired CBC inactivation, but rather from disruption of oxidative regulation of other metabolic pathways mediated by distinct atypical Trxs. Collectively, these findings reveal a high degree of regulatory specificity within the chloroplast oxidative network and provide a foundation for a deeper understanding of how activation-inactivation cycles contribute to plant adaptation to dynamic light environments.

plant biology↗

bioRxiv2026

Expansion of Plant Redox Protein Network Predated Plant Terrestrialization

The evolutionary transition of the green plant lineage (Viridiplantae) from aquatic environments to terrestrial habitats required unprecedented adaptations of cellular metabolism to severe environmental stressors, including desiccation, high irradiance, and rapid temperature fluctuations. Redox regulation, mediated by oxidation and reduction of reactive cysteine residues (RCys), plays a crucial role in translating environmental fluctuations into rapid cellular responses. Although comparative genomics has revealed expansions in multiple cellular systems preceding terrestrialization, the evolutionary history of redox-regulated protein networks remains elusive. This work integrated large-scale phylogenomic reconstructions across 37 Viridiplantae species with five independent Arabidopsis thaliana redox proteomics datasets to trace the evolutionary trajectory of RCys. The analysis showed that the ancestral core, consisting of plastid-localized regulatory cysteines, was already established at the base of the green lineage. Furthermore, an expansion driven by gains of RCys via amino acid replacements within pre-existing proteins occurred in the common ancestor of Zygnematophyceae and land plants. These findings suggest that a targeted incorporation of thiol-based regulatory switches provided early land plant ancestors with enhanced protein functional plasticity necessary to cope with the challenges of terrestrial environments. HighlightsO_LIThe foundational plastid-localized redox core was established at the root of Viridiplantae. C_LIO_LINovel regulatory switches were integrated into conserved machinery via amino acid replacement. C_LIO_LIA punctuated burst of redox innovation at Zygnematophyceae and Embryophyta last common ancestor preceded plant terrestrialization. C_LIO_LIRedox acquisition rates declined sharply following the successful colonization of land. C_LI

evolutionary biology↗

bioRxiv2026

Rainfall legacy effects on the rhizosphere bacterial diversity of Brachypodium ecotypes across an aridity gradient

Plants exhibit clinal trait variation along aridity gradients driven by strong selective pressures, yet whether plant-microbiome associations follow similar patterns remains unclear. Brachypodium spp., a model for temperate cereals spanning environments from hyper-arid to humid, provides an ideal system to test this hypothesis. Here, we compare rhizosphere bacterial communities of Brachypodium growing across arid, semi-arid, and dry sub-humid zones in Israel with those of ecotypes collected along the same precipitation gradient and grown under common-garden conditions. Rhizosphere bacterial diversity was highest in plants from mid-precipitation sites within the Mediterranean semi-arid transition zone, where annual precipitation decreases from 600 to 400 mm. Together with higher stochasticity and fewer significantly associated taxa in rhizosphere microbiomes from mid-precipitation plants suggest weaker plant-driven microbiome selection in this transition zone, a pattern that persisted under common-garden conditions. The results may represent a promising avenue to develop microbiome-based strategies for drought resilience by advancing our understanding of host filtering across aridity gradients.

ecology↗

bioRxiv2025

Two opposing redox signals mediated by 2-Cys peroxiredoxin shape the redox proteome during photosynthetic induction

Photosynthetic induction, characterized by the lag in CO2 assimilation rates typically observed upon plant transition from darkness to light, has traditionally been attributed to Rubisco activase activity and stomatal opening. Yet, the faster induction of photosynthesis in the 2-Cys peroxiredoxins (Prxs) mutant (2cpab) highlighted the critical role of chloroplast redox state in regulating photosynthetic rates during this phase. Since 2-Cys Prxs are involved in transmission of oxidative signals to target enzymes, it was hypothesized that it slows down photosynthesis during the induction phase. SPEAR, a redox proteomics approach for simultaneous protein expression and redox analysis, was used to systematically map redox changes occurring at the proteome level during photosynthesis induction and to unravel the role of 2-Cys Prxs in shaping these redox alterations. No significant difference was observed in protein expression levels between WT and 2cpab plants, suggesting that protein abundance does not account for the 2cpab phenotype. During the transition from dark to low light, 82 and 54 cysteine-containing peptides were reduced or oxidized, respectively, in WT plants. Most redox-regulated cysteines in photosynthetic proteins were found oxidized in the dark and became reduced in response to light, including ATP synthase gamma chain 1 (ATPC1) and glyceraldehyde-3-phosphate dehydrogenase (GAPB). A reverse pattern was observed among redox-regulated cysteines in proteins involved in starch degradation and chloroplast glycolysis, which shifted from a reduced to an oxidized state in response to light. These findings demonstrate the initiation of two opposing redox responses, affecting distinct sets of metabolic proteins during the induction phase. Remarkably, a significantly lower number of cysteines were reduced or oxidized in 2cpab plants, highlighting the crucial role 2-Cys Prxs play in shaping both signals. Taken together, rotational shifts between metabolic pathways during the photosynthesis induction phase are regulated by two opposing redox signals mediated by 2-Cys Prx activity.

plant biology↗

bioRxiv2024

Quantification of electron transport-related oxidative signals by time and wavelength-resolved redox biosensors and chlorophyll fluorescence

Reductive and oxidative signals transmitted from the photosynthetic electron chain to target proteins through the redox signaling network are key regulators of carbon assimilation and downstream metabolism. However, despite their crucial role in activating and inhibiting photosynthetic activity, their relation to photosynthetic efficiency is hardly quantified due to the methodological gap between traditional spectroscopic approaches for investigating photosynthesis and biochemical analyses used in the redox regulation field. Here, we simultaneously quantified redox signals and photosynthetic activity by exploring time and wavelength-resolved fluorescence spectra that capture biosensor and chlorophyll fluorescence signals. Using a set of potato plants expressing genetically encoded redox biosensors, we demonstrated how reductive and oxidative signals are amplified with elevated light intensities and revealed the tight connection between electron transport rate (ETR) and the generation of peroxiredoxin-related oxidative signals. These results demonstrate how full spectrum analysis can pave the way for the integration of genetically encoded biosensors in photosynthesis research and demonstrate light-dependent activation of inhibitory oxidative signals in major crop plants.

plant biology↗

bioRxiv2023

Nitrogen assimilation plays a role in balancing the chloroplastic glutathione redox state under high-light conditions

Nitrate reduction and subsequent ammonium assimilation require reducing equivalents directly produced by the photosynthetic electron transport chain. Therefore, it has been suggested that nitrate assimilation provides a valuable sink for excess electrons under high-light (HL) conditions, which protects the photosynthetic apparatus from excessive harmful reactive oxygen species. This work experimentally tested this hypothesis by monitoring photosynthetic efficiency and the chloroplastic glutathione redox state (chl-EGSH) of plant lines with mutated glutamine synthetase 2 (GS2) and ferredoxin-dependent glutamate synthase 1 (GOGAT1), two key enzymes of the nitrogen assimilation pathway. Unlike wild-type (WT) plants, mutant lines incorporated significantly less isotopically-labeled nitrate into amino acids, demonstrating impaired nitrogen assimilation. When nitrate assimilation was compromised, photosystem II (PSII) proved more vulnerable to photodamage, as shown by the low PSII quantum yields recorded in the mutant lines. High temporal resolution monitoring of the redox state of chloroplast-targeted reduction-oxidation sensitive green fluorescent protein 2 (chl-roGFP2), expressed in the background of the mutant lines, enabled assessment of the effect of the nitrate assimilation pathway on the chl-EGSH. Remarkably, while oxidation followed by reduction of chl-roGFP2 was detected in WT plants in response to HL, oxidation values were stable in the mutant lines, suggesting that the relaxation of chl-EGSH after HL-induced oxidation is achieved by diverting excess electrons to the nitrogen assimilation pathway. Together, these findings indicate that the nitrogen assimilation pathway serves as a sustainable energy dissipation route, ensuring efficient photosynthetic activity and fine-tuning redox metabolism under light-saturated conditions.

plant biology↗

bioRxiv2022

Early detection of late blight in potato by whole-plant redox imaging

Late blight caused by the oomycete Phytophthora infestans is a most devastating disease of potatoes (Solanum tuberosum). Its early detection is crucial for suppressing disease spread. Necrotic lesions are normally seen in leaves at 4 dpi (days post inoculation) when colonized cells are dead, but early detection of the initial biotrophic growth stage, when the pathogen feeds on living cells, is challenging. Here, the biotrophic growth phase of P. infestans was detected by whole-plant redox imaging of potato plants expressing chloroplast-targeted reduction-oxidation sensitive green fluorescent protein (chl-roGFP2). Clear spots on potato leaves with a lower chl-roGFP2 oxidation state were detected as early as 2 dpi, before any visual symptoms were recorded. These spots were particularly evident during light-to-dark transitions and reflected mislocalization of chl-roGFP2 outside the chloroplasts, demonstrating perturbation of the chloroplast import system by the pathogen. Image analysis based on machine learning enabled systematic identification and quantification of spots and unbiased classification of infected and uninfected leaves in inoculated plants. Comparing redox to chlorophyll fluorescence imaging showed that infected leaf areas which exhibit mislocalized chl-roGFP2 also showed reduced non-photochemical quenching (NPQ) and enhanced quantum PSII yield ({Phi}PSII) compared to the surrounding leaf areas. The data suggest that mislocalization of chloroplast-targeted proteins is an efficient marker of late blight infection and demonstrate how it can be utilized for nondestructive monitoring of the disease biotrophic stage using whole-plant redox imaging.

plant biology↗

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↗

bioRxiv2020

Resolving the dynamics of photosynthetically produced ROS by high-resolution monitoring of chloroplastic EGSH in Arabidopsis

Plants are naturally subjected to fluctuations in light intensity, causing unbalanced photosynthetic electron fluxes and overproduction of reactive oxygen species (ROS). While high rates of ROS production are harmful, moderate levels play a signaling role, coordinating photosynthetic activity and downstream metabolism. Here, we explore the dynamics of light-dependent oxidant production by high-temporal-resolution monitoring of chloroplastic glutathione redox potential (chl-EGSH) using chloroplast-targeted-roGFP2-expressing Arabidopsis lines, over several days, under dynamic environmental conditions and in correlation with PSII operating efficiency. Peaks in chl-EGSH oxidation during light-darkness transitions, when light harvesting is not balanced with downstream metabolism, were observed. Increasing light intensities triggered a binary oxidation response, with a threshold around the light saturating point, pointing for two regulated oxidative states of the chl-EGSH. These patterns were not affected in npq1 plants which are impaired in non-photochemical quenching. Frequency-dependent oscillations between the two oxidation states were observed under fluctuating light in WT and npq1 plants, but not in pgr5 plants, suggesting a role for PSI photoinhibition in regulation of oxidant production. Remarkably, pgr5 plants showed an increase in chl-EGSH oxidation during the nights following light stresses, linking between day photoinhibition and night redox metabolism. This work provides a comprehensive view on the balance between photosynthesis-dependent ROS production and antioxidant activity during light acclimation.

plant biology↗

bioRxiv2019

Tracking infection dynamics at single-cell level reveals highly resolved expression programs of a large virus infecting algal blooms

Nucleocytoplasmic large DNA viruses have the largest genomes among all viruses and infect diverse eukaryotes across various ecosystems, but their expression regulation and infection strategies are not well understood. We profiled single-cell transcriptomes of the worldwide-distributed microalga Emiliania huxleyi and its specific coccolithovirus responsible for massive bloom demise. Heterogeneity in viral transcript levels detected among single cells was used to reconstruct the viral transcriptional trajectory and to map cells along a continuum of infection states. This enabled identification of novel viral genetic programs, which are composed of five kinetic classes with distinct promoter elements. The infection substantially changed the host transcriptome, causing rapid shutdown of protein-encoding nuclear transcripts at the onset of infection, while the plastid and mitochondrial transcriptomes persisted to mid- and late stages, respectively. Single-cell transcriptomics thereby opens the way for tracking host-pathogen infection dynamics at high resolution within microbial communities in the marine environment.

microbiology↗

bioRxiv2018

A redox-regulated type III metacaspase controls cell death in a marine diatom

Programmed cell death (PCD) in marine phytoplankton was suggested as one of the mechanisms that facilitates large scale bloom demise. Yet, the molecular basis for algal PCD machinery is rudimentary. Metacaspases are considered ancestral proteases that regulate cell death, but their activity and role in algae are still elusive. Here we biochemically characterized a recombinant metacaspase 5 from the model diatom Phaeodactylum tricornutum (PtMC5), revealing calcium-dependent protease activity. This activity includes auto-processing and cleavage following arginine. PtMC5 overexpressing cells exhibited higher metacaspase activity and were more sensitive to a diatom-specific infochemical compared to WT cells. Mutagenesis of potential disulfide-forming cysteines decreased PtMC5 activity, suggesting redox regulation. This cysteine pair is widespread in diatom type III metacaspases, but was not found in any other taxa. The characterization of a cell death associated protein in marine phytoplankton will enable deeper understanding of the ecological significance of PCD in bloom dynamics.

microbiology↗

bioRxiv2018

Phenotypic variability in chloroplast redox state predicts cell fate in a marine diatom

Diatoms are photosynthetic microorganisms of great ecological and biogeochemical importance, forming vast blooms in diverse aquatic ecosystems. Current understanding of phytoplankton acclimation to stress is based on population-level analysis, masking cell-to-cell variability. Here we investigated heterogeneity within Phaeodactylum tricornutum populations in response to oxidative stress, which is induced by environmental stress conditions. We combined flow cytometry and a microfluidics system for live imaging to measure redox dynamics at the single-cell level using the roGFP sensor. Chloroplast-targeted roGFP exhibited a light-dependent, bi-stable oxidation pattern in response to H2O2, revealing distinct subpopulations of sensitive oxidized cells and resilient reduced cells. Subpopulation proportions depended on growth phase, linking the bi-stable phenotype to proliferation. Oxidation of chloroplast-targeted roGFP preceded commitment to cell death and was used as a novel cell fate predictor. We propose that light-dependent metabolic heterogeneity results in differential stress responses that regulate cell fate within diatom populations.

microbiology↗

bioRxiv2018

\"In plaque-mass spectrometry imaging\" reveals a major metabolic shift towards odd-chain fatty acid lipids induced by host-virus interactions

Tapping into the metabolic cross-talk between a host and its virus can reveal unique strategies employed during infection. Viral infection is a dynamic process that generates an evolving metabolic landscape. Gaining a continuous view into the infection process is highly challenging and is limited by current metabolomics approaches, which typically measure the average of the entire population at various stages of infection. Here, we took a novel approach to study the metabolic basis of host-virus interactions between the bloom-forming alga Emiliania huxleyi and its specific virus. We combined a classical method in virology, plaque assay, with advanced mass spectrometry imaging (MSI), an approach we termed in plaque-MSI. Taking advantage of the spatial characteristics of the plaque, we mapped the metabolic landscape induced during infection in a high spatiotemporal resolution, unfolding the infection process in a continuous manner. Further unsupervised spatially-aware clustering, combined with known lipid biomarkers, revealed a systematic metabolic shift towards lipids containing the odd-chain fatty acid pentadecanoic acid (C15:0) induced during infection. Applying in plaque-MSI might pave the way for the discovery of novel bioactive compounds that mediate the chemical arms race of host-virus interactions in diverse model systems.

microbiology↗

bioRxiv2017

Unmasking cellular response of a bloom-forming alga to virus infection by resolving expression profiling at a single-cell level

Marine viruses are major evolutionary and biogeochemical drivers of microbial life in the ocean. Host response to viral infection typically includes virus-induced rewiring of metabolic network to supply essential building blocks for viral assembly, as opposed to activation of anti-viral host defense. Nevertheless, there is a major bottleneck to accurately discern between viral hijacking strategies and host defense responses when averaging bulk population response. Here we use Emiliania huxleyi, a bloom-forming alga and its specific virus (EhV), as one of the most ecologically important host-virus model system in the ocean. Using automatic microfluidic setup to capture individual algal cells, we quantified host and virus gene expression on a single-cell resolution during the course of infection. We revealed high heterogeneity in viral gene expression among individual cells. Simultaneous measurements of expression profiles of host and virus genes at a single-cell level allowed mapping of infected cells into newly defined infection states and uncover a yet unrecognized early phase in host response that occurs prior to viral expression. Intriguingly, resistant cells emerged during viral infection, showed unique expression profiles of metabolic genes which can provide the basis for discerning between viral resistant and sensitive cells within heterogeneous populations in the marine environment. We propose that resolving host-virus arms race at a single-cell level will provide important mechanistic insights into viral life cycles and will uncover host defense strategies.

ecology↗