Summary 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. Highlights The foundational plastid-localized redox core was established at the root of Viridiplantae . Novel regulatory switches were integrated into conserved machinery via amino acid replacement. A punctuated burst of redox innovation at Zygnematophyceae and Embryophyta last common ancestor preceded plant terrestrialization. Redox acquisition rates declined sharply following the successful colonization of land.

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.

Photosynthetic induction, characterized by the lag in CO2 assimilation rates during 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) suggested a role for oxidative signals in regulating photosynthetic rates, although the underlying molecular mechanism remains unclear. SPEAR, a redox proteomics approach, was used to systematically map redox changes occurring 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. 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.

Nitrate reduction requires reducing equivalents produced by the photosynthetic electron transport chain. Therefore, it has been suggested that nitrate assimilation provides a sink for electrons under high light conditions. We tested this hypothesis by monitoring photosynthetic efficiency and the chloroplastic glutathione redox potential (chl-EGSH) of plant lines with mutated glutamine synthetase 2 (GS2) and ferredoxin-dependent glutamate synthase 1 (GOGAT1). Mutant lines incorporated significantly less isotopically-labelled nitrate into amino acids than wild-type plants, demonstrating impaired nitrogen assimilation. When nitrate assimilation was compromised, photosystem II (PSII) proved more vulnerable to photodamage. The effect of the nitrate assimilation pathway on the chl- EGSH was monitored using the chloroplast-targeted roGFP2 biosensor (chl-roGFP2). Remarkably, while oxidation followed by reduction of chl-roGFP2 was detected in WT plants in response to high light, oxidation values were stable in the mutant lines, suggesting that chl-EGSH relaxation after high light-induced oxidation is achieved by diverting excess electrons to the nitrogen assimilation pathway. Importantly, similar ΦPSII and chl-roGFP2 patterns were observed at elevated CO2, suggesting that mutant phenotypes are not associated with photorespiration activity. 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.

Visual estimates of plant symptoms are traditionally used to quantify disease severity. Yet, the methodologies used to assess these phenotypes are often subjective and do not allow tracking of disease progression from very early stages. Here, we hypothesized that quantitative analysis of whole-plant physiological vital functions can be used to objectively determine plant health, providing a more sensitive way to detect disease. We studied the tomato wilt that is caused by Fusarium oxysporum f. sp. lycopersici. Physiological performance of infected and noninfected tomato plants was compared using a whole-plant pot-based lysimeter functional phenotyping system in a semi-environmentally controlled greenhouse. Water-balance traits of the plants were measured continuously and simultaneously in a quantitative manner. Infected plants exhibited early reductions in transpiration and biomass gain, which preceded visual disease symptoms. These changes in transpiration proved to be effective quantitative indicators for assessing both plant susceptibility to infection and virulence of the fungus. Physiological changes linked to fungal outgrowth and toxin release contributed to reduced hydraulic conductance during initial infection stages. The functional phenotyping method objectively captures early-stage disease progression, advancing plant disease research and management. This approach emphasizes the potential of quantitative whole-plant physiological analysis over traditional visual estimates for understanding and detecting plant diseases.

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. ### Competing Interest Statement The authors have declared no competing interest.

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- E GSH ) 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- E GSH . 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-E GSH 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.

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 global identification of redox-modified cysteine (Cys) sites and quantification of their reversible 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 reversibly 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 reversible oxidation state and protein abundance. Using SPEAR, we were able to quantify the in-vivo reversible 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 chloroplastic proteins. SPEAR provides a simple, straightforward, and cost-effective means of studying redox proteome dynamics. The presented data provide a global quantitative view of the reversible 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.

SUMMARYLate 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 days post‐inoculation (dpi) 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 the mislocalization of chl‐roGFP2 outside the chloroplasts. 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 with chlorophyll fluorescence imaging showed that infected leaf areas that exhibit mislocalized chl‐roGFP2 also showed reduced non‐photochemical quenching and enhanced quantum PSII yield (ΦPSII) compared with 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 non‐destructive monitoring of the disease biotrophic stage using whole‐plant redox imaging.

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 Summary A genetically encoded biosensor unmasked the dominant role of photosynthetically-derived oxidative signals under habitual conditions.

Environmental stresses are among the major factors that limit crop productivity and plant growth. Various nondestructive approaches for monitoring plant stress states have been developed. However, early sensing of the initial biochemical events during stress responses remains a significant challenge. In this work, we established whole-plant redox imaging using potato (Solanum tuberosum) plants expressing a chloroplast-targeted redox-sensitive green fluorescence protein 2 (roGFP2), which reports the glutathione redox potential (E-GSH). Ratiometric imaging analysis demonstrated the probe response to redox perturbations induced by H2O2, DTT, or a GSH biosynthesis inhibitor. We mapped alterations in the chloroplast E-GSH under several stress conditions including, high-light (HL), cold, and drought. An extremely high increase in chloroplast E-GSH was observed under the combination of HL and low temperatures, conditions that specifically induce PSI photoinhibition. Intriguingly, we noted a higher reduced state in newly developed compared with mature leaves under steady-state and stress conditions, suggesting a graded stress sensitivity as part of the plant strategies for coping with stress. The presented observations suggest that whole-plant redox imaging can serve as a powerful tool for the basic understanding of plant stress responses and applied agricultural research, such as toward improving phenotyping capabilities in breeding programs and early detection of stress responses in the field.

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 introduced a peroxiredoxin-based biosensor to monitor the dynamic changes in photosynthetically-derived oxidative signals. Systematic monitoring of probe oxidation demonstrated the induction of oxidative signals under habitual light intensities and their inverse relationship with chloroplastic NADPH levels, unraveling the combined activity of reducing and oxidizing signals in fine-tuning chloroplast activity. Considering that peroxiredoxins mediate oxidative inhibition of carbon assimilation, the measured light-dependent oxidative signals reflect the limitation to photosynthesis imposed by the redox regulatory system.A genetically encoded biosensor unmasked the dominant role of photosynthetically-derived oxidative signals under habitual conditions.

Programmed cell death (PCD) in marine microalgae was suggested to be one of the mechanisms that facilitates bloom demise, yet its molecular components in phytoplankton are unknown. Phytoplankton are completely lacking any of the canonical components of PCD, such as caspases, but possess metacaspases. Metacaspases were shown to regulate PCD in plants and some protists, but their roles in algae and other organisms are still elusive. Here, we identified and biochemically characterized a type III metacaspase from the model diatom Phaeodactylum tricornutum, termed PtMCA-IIIc. Through expression of recombinant PtMCA-IIIc in E. coli, we revealed that PtMCA-IIIc exhibits a calcium-dependent protease activity, including auto-processing and cleavage after arginine. Similar metacaspase activity was detected in P. tricornutum cell extracts. PtMCA-IIIc overexpressing cells exhibited higher metacaspase activity, while CRISPR/Cas9-mediated knockout cells had decreased metacaspase activity compared to WT cells. Site-directed mutagenesis of cysteines that were predicted to form a disulfide bond decreased recombinant PtMCA-IIIc activity, suggesting its enhancement under oxidizing conditions. One of those cysteines was oxidized, detected in redox proteomics, specifically in response to lethal concentrations of hydrogen peroxide and a diatom derived aldehyde. Phylogenetic analysis revealed that this cysteine-pair is unique and widespread among diatom type III metacaspases. The characterization of a cell death associated protein in diatoms provides insights into the evolutionary origins of PCD and its ecological significance in algal bloom dynamics.

Plants are subjected to fluctuations in light intensity, and this might cause unbalanced photosynthetic electron fluxes and overproduction of reactive oxygen species (ROS). Electrons needed for ROS detoxification are drawn, at least partially, from the cellular glutathione (GSH) pool via the ascorbate-glutathione cycle. Here, we explore the dynamics of the chloroplastic glutathione redox potential (chl-EGSH) using high-temporal-resolution monitoring of Arabidopsis (Arabidopsis thaliana) lines expressing the reduction-oxidation sensitive green fluorescent protein 2 (roGFP2) in chloroplasts. This was carried out over several days under dynamic environmental conditions and in correlation with PSII operating efficiency. Peaks in chl-EGSH oxidation during dark-to-light and light-to-dark transitions were observed. Increasing light intensities triggered a binary oxidation response, with a threshold around the light saturating point, suggesting two regulated oxidative states of the chl-EGSH. These patterns were not affected in npq1 plants, which are impaired in non-photochemical quenching. 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 regulating the chl-EGSH dynamics. Remarkably, pgr5 plants showed an increase in chl-EGSH oxidation during the nights following light stresses, linking daytime photoinhibition and nighttime GSH metabolism. This work provides a systematic view of the dynamics of the in vivo chloroplastic glutathione redox state during varying light conditions.

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.

The formation of coccoliths, intricate calcium carbonate scales that cover the cells of unicellular marine microalgae, is a highly regulated biological process. For decades, scientists have tried to elucidate the cellular, chemical, and structural mechanisms that control the precise mineralogy and shape of the inorganic crystals. Transmission electron microscopy was pivotal in characterizing some of the organelles that orchestrate this process. However, due to the difficulties in preserving soluble inorganic phases during sample preparation, only recently, new intracellular ion-pools were detected using state-of-the-art cryo X-ray and electron microscopy techniques. Here, we combine a completely non-aqueous sample preparation procedure and room temperature electron microscopy, to investigate the presence, cellular location, and composition, of mineral phases inside mineral forming microalga species. This methodology, which fully preserves the forming coccoliths and the recently identified Ca-P-rich bodies, allowed us to identify a new class of ion-rich compartments that have complex internal structure. In addition, we show that when carefully choosing heavy metal stains, elemental analysis of the mineral phases can give accurate chemical signatures of the inorganic phases. Applying this approach to mineral forming microalgae will bridge the gap between the low-preservation power for inorganic phases of conventional chemical-fixation based electron microscopy, and the low-yield of advanced cryo techniques.

The discovery of giant viruses infecting eukaryotes from diverse ecosystems has revolutionized our understanding of the evolution of viruses and their impact on protist biology, yet knowledge on their replication strategies and transcriptome regulation remains limited. Here, we profile single-cell transcriptomes of the globally distributed microalga Emiliania huxleyi and its specific giant virus during infection. We detected profound heterogeneity in viral transcript levels among individual cells. Clustering single cells based on viral expression profiles enabled reconstruction of the viral transcriptional trajectory. Reordering cells along this path unfolded highly resolved viral genetic programs composed of genes with distinct promoter elements that orchestrate sequential expression. Exploring host transcriptome dynamics across the viral infection states revealed rapid and selective shutdown of protein-encoding nuclear transcripts, while the plastid and mitochondrial transcriptomes persisted into later stages. Single-cell RNA-seq opens a new avenue to unravel the life cycle of giant viruses and their unique hijacking strategies.

AbstractPlants 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-expressingArabidopsislines, over several days, under dynamic environmental conditions and in correlation with PSII operating efficiency. Peaks in chl-EGSHoxidation 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 innpq1plants which are impaired in non-photochemical quenching. Frequency-dependent oscillations between the two oxidation states were observed under fluctuating light in WT andnpq1plants, but not inpgr5plants, suggesting a role for PSI photoinhibition in regulation of oxidant production. Remarkably,pgr5plants showed an increase in chl-EGSHoxidation 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.