Diurnal fluctuations in chloroplast GSH redox state regulate susceptibility to oxidative stress and cell fate in a bloom‐forming diatom

Diatoms are one of the key phytoplankton groups in the ocean, forming vast oceanic blooms and playing a significant part in global primary production. To shed light on the role of redox metabolism in diatom's acclimation to light–dark transition and its interplay with cell fate regulation, we generated transgenic lines of the diatom Thalassiosira pseudonana that express the redox‐sensitive green fluorescent protein targeted to various subcellular organelles. We detected organelle‐specific redox patterns in response to oxidative stress, indicating compartmentalized antioxidant capacities. Monitoring the GSH redox potential (E_GSH) in the chloroplast over diurnal cycles revealed distinct rhythmic patterns. Intriguingly, in the dark, cells exhibited reduced basal chloroplast E_GSH but higher sensitivity to oxidative stress than cells in the light. This dark‐dependent sensitivity to oxidative stress was a result of a depleted pool of reduced glutathione which accumulated during the light period. Interestingly, reduction in the chloroplast E_GSH was observed in the light phase prior to the transition to darkness, suggesting an anticipatory phase. Rapid chloroplast E_GSH re‐oxidation was observed upon re‐illumination, signifying an induction of an oxidative signaling during transition to light that may regulate downstream metabolic processes. Since light–dark transitions can dictate metabolic capabilities and susceptibility to a range of environmental stress conditions, deepening our understanding of the molecular components mediating the light‐dependent redox signals may provide novel insights into cell fate regulation and its impact on oceanic bloom successions.     

Crosstalk between chloroplast thioredoxin systems in regulation of photosynthesis

Thioredoxins (TRXs) mediate light‐dependent activation of primary photosynthetic reactions in plant chloroplasts by reducing disulphide bridges in redox‐regulated enzymes. Of the two plastid TRX systems, the ferredoxin‐TRX system consists of ferredoxin‐thioredoxin reductase (FTR) and multiple TRXs, while the NADPH‐dependent thioredoxin reductase (NTRC) contains a complete TRX system in a single polypeptide. Using Arabidopsis plants overexpressing or lacking a functional NTRC, we have investigated the redundancy and interaction between the NTRC and Fd‐TRX systems in regulation of photosynthesis in vivo. Overexpression of NTRC raised the CO₂ fixation rate and lowered non‐photochemical quenching and acceptor side limitation of PSI in low light conditions by enhancing the activation of chloroplast ATP synthase and TRX‐regulated enzymes in Calvin–Benson cycle (CBC). Overexpression of NTRC with an inactivated NTR or TRX domain partly recovered the phenotype of knockout plants, suggesting crosstalk between the plastid TRX systems. NTRC interacted in planta with fructose‐1,6‐bisphosphatase, phosphoribulokinase and CF₁γ subunit of the ATP synthase and with several chloroplast TRXs. These findings indicate that NTRC‐mediated regulation of the CBC and ATP synthesis occurs both directly and through interaction with the ferredoxin‐TRX system and is crucial when availability of light is limiting photosynthesis.     

Multi‐level regulation of the chloroplast ATP synthase: the chloroplast NADPH thioredoxin reductase C (NTRC) is required for redox modulation specifically under low irradiance

The chloroplast ATP synthase is known to be regulated by redox modulation of a disulfide bridge on the γ‐subunit through the ferredoxin–thioredoxin regulatory system. We show that a second enzyme, the recently identified chloroplast NADPH thioredoxin reductase C (NTRC), plays a role specifically at low irradiance. Arabidopsis mutants lacking NTRC (ntrc) displayed a striking photosynthetic phenotype in which feedback regulation of the light reactions was strongly activated at low light, but returned to wild‐type levels as irradiance was increased. This effect was caused by an altered redox state of the γ‐subunit under low, but not high, light. The low light‐specific decrease in ATP synthase activity in ntrc resulted in a buildup of the thylakoid proton motive force with subsequent activation of non‐photochemical quenching and downregulation of linear electron flow. We conclude that NTRC provides redox modulation at low light using the relatively oxidizing substrate NADPH, whereas the canonical ferredoxin–thioredoxin system can take over at higher light, when reduced ferredoxin can accumulate. Based on these results, we reassess previous models for ATP synthase regulation and propose that NTRC is most likely regulated by light. We also find that ntrc is highly sensitive to rapidly changing light intensities that probably do not involve the chloroplast ATP synthase, implicating this system in multiple photosynthetic processes, particularly under fluctuating environmental conditions.     

Changes in glutathione redox cycle during diapause determination and termination in the bivoltine silkworm,Bombyx moil

To explore whether glutathione regulates diapause determination and termina tion in the bivoltine silkworm Bombyx mori, we monitored the changes in glutathione redox cycle in the ovary of both diapanse and nondiapauseegg producers, as well as those in dia pause eggs incubated at different temperatures. The activity ofthioredoxin reductase （TrxR） was detected in ovaries but not in eggs, while neither ovaries nor eggs showed activity of glutathione peroxidase. A lower reduced glutathione/oxidized glutathione （GSH/GSSG） ratio was observed in the ovary of diapauseegg producers, due to weaker reduction of oxidized glutathione （GSSG） to the reduced glutathione （GSH） catalyzed by glutathione reductase （GR） and TrxR. This indicates an oxidative shift in the glutathione redox cy cle during diapause determination. Compared with the 25℃treated diapause eggs, the 5℃treated diapause eggs showed lower GSH/GSSG ratio, a result of stronger oxidation of GSH catalyzed by thioredoxin peroxidase and weaker reduction of GSSG catalyzed by GR. Our study demonstrated the important regulatory role of glutathione in diapause determination and termination of the bivoltine silkworm.     

Nrf2‐Mediated Resistance to Oxidant‐Induced Redox Disruption in Embryos

Events that control developmental changes occur during specific windows of gestation and if disrupted, can lead to dysmorphogenesis or embryolethality. One largely understudied aspect of developmental control is redox regulation, where the untimely disruption of intracellular redox potentials (E_h) may alter development, suggesting that tight control of developmental‐stage–specific redox states is necessary to support normal development. In this study, mouse gestational day 8.5 embryos in whole embryo culture were treated with 10 μM dithiole‐3‐thione (D3T), an inducer of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2). After 14 hr, D3T‐treated and ‐untreated conceptuses were challenged with 200 μM hydrogen peroxide (H₂O₂) to induce oxidant‐induced change to intracellular E_hs. Redox potentials of glutathione (GSH), thioredoxin‐1 (Trx1), and mitochondrial thioredoxin‐2 (Trx2) were then measured over a 2‐hr rebounding period following H₂O₂ treatment. D3T treatment increased embryonic expression of known Nrf2‐regulated genes, including those responsible for redox regulation of major intracellular redox couples. Exposure to H₂O₂ without prior D3T treatment produced significant oxidation of GSH, Trx1, and Trx2, based on E_h values, where GSH and Trx2 E_h recovered, reaching to pre‐H₂O₂ E_h ranges, but Trx1 E_h remained oxidized. Following H₂O₂ addition in culture to embryos that received D3T pretreatments, GSH, Trx1, and Trx2 were insulated from significant oxidation. These data show that Nrf2 activation may serve as a means to protect the embryo from chemically induced oxidative stress through the preservation of intracellular redox states during development, allowing normal morphogenesis to ensue.     

Plant responses to fungal volatiles involve global posttranslational thiol redox proteome changes that affect photosynthesis

Microorganisms produce volatile compounds (VCs) that promote plant growth and photosynthesis through complex mechanisms involving cytokinin (CK) and abscisic acid (ABA). We hypothesized that plants' responses to microbial VCs involve posttranslational modifications of the thiol redox proteome through action of plastidial NADPH‐dependent thioredoxin reductase C (NTRC), which regulates chloroplast redox status via its functional relationship with 2‐Cys peroxiredoxins. To test this hypothesis, we analysed developmental, metabolic, hormonal, genetic, and redox proteomic responses of wild‐type (WT) plants and a NTRC knockout mutant (ntrc) to VCs emitted by the phytopathogen Alternaria alternata. Fungal VC‐promoted growth, changes in root architecture, shifts in expression of VC‐responsive CK‐ and ABA‐regulated genes, and increases in photosynthetic capacity were substantially weaker in ntrc plants than in WT plants. As in WT plants, fungal VCs strongly promoted growth, chlorophyll accumulation, and photosynthesis in ntrc–Δ2cp plants with reduced 2‐Cys peroxiredoxin expression. OxiTRAQ‐based quantitative and site‐specific redox proteomic analyses revealed that VCs promote global reduction of the thiol redox proteome (especially of photosynthesis‐related proteins) of WT leaves but its oxidation in ntrc leaves. Our findings show that NTRC is an important mediator of plant responses to microbial VCs through mechanisms involving global thiol redox proteome changes that affect photosynthesis.     

Resolving diurnal dynamics of the chloroplastic glutathione redox state in Arabidopsis reveals its photosynthetically derived oxidation

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-E_GSH) 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-E_GSH 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-E_GSH. 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-E_GSH dynamics. Remarkably, pgr5 plants showed an increase in chl-E_GSH 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.

Monitoring the daily in vivo dynamics of the chloroplastic GSH redox state in light-stressed wild-type plants versus photoprotective mutants provides insight into the photosynthesis-dependent production of oxidants.     

Sensing stress responses in potato with whole-plant redox imaging

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.

Whole-plant imaging of potato plants expressing a genetically encoded biosensor allows for spatially resolved and nondestructive mapping of stress-induced redox perturbations.     

Antioxidant plasticity and thermal sensitivity in four types of Symbiodinium sp.

Warmer than average summer sea surface temperature is one of the main drivers for coral bleaching, which describes the loss of endosymbiotic dinoflagellates (genus: Symbiodinium) in reef‐building corals. Past research has established that oxidative stress in the symbiont plays an important part in the bleaching cascade. Corals hosting different genotypes of Symbiodinium may have varying thermal bleaching thresholds, but changes in the symbiont's antioxidant system that may accompany these differences have received less attention. This study shows that constitutive activity and up‐regulation of different parts of the antioxidant network under thermal stress differs between four Symbiodinium types in culture and that thermal susceptibility can be linked to glutathione redox homeostasis. In Symbiodinium B1, C1 and E, declining maximum quantum yield of PSII (F_v/F_m) and death at 33°C were generally associated with elevated superoxide dismutase (SOD) activity and a more oxidized glutathione pool. Symbiodinium F1 exhibited no decline in F_v/F_m or growth, but showed proportionally larger increases in ascorbate peroxidase (APX) activity and glutathione content (GSx), while maintaining GSx in a reduced state. Depressed growth in Symbiodinium B1 at a sublethal temperature of 29°C was associated with transiently increased APX activity and glutathione pool size, and an overall increase in glutathione reductase (GR) activity. The collapse of GR activity at 33°C, together with increased SOD, APX and glutathione S‐transferase activity, contributed to a strong oxidation of the glutathione pool with subsequent death. Integrating responses of multiple components of the antioxidant network highlights the importance of antioxidant plasticity in explaining type‐specific temperature responses in Symbiodinium.     

Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function

The light‐dependent regulation of stromal enzymes by thioredoxin (Trx)‐catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx‐mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol‐dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed Trx‐linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de‐epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox‐controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.     

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

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 (E_GSH) 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 E_GSH and H₂O₂ levels with the genetically encoded biosensors Grx1-roGFP2 (for E_GSH) 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.

Methyl viologen-induced photo-oxidative stress increases hydrogen peroxide and oxidation of glutathione in chloroplasts, cytosol, and mitochondria, as well as autonomous oxidation in mitochondria.     

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L.

Abstract

The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (E_{GSSG/2 GSH}) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (E_{CySS/2 Cys}). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a ‘thiol–disulphide redox environment’ (E_{thiol–disulphide}), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of E_{CySS/2 Cys} to E_{thiol–disulphide} in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.     

Changes in low-molecular-weight thiol-disulphide redox couples are part of bread wheat seed germination and early seedling growth

The tripeptide antioxidant glutathione (γ-l-glutamyl-l-cysteinyl-glycine; GSH) essentially contributes to thiol-disulphide conversions, which are involved in the control of seed development, germination, and seedling establishment. However, the relative contribution of GSH metabolism in different seed structures is not fully understood. We studied the GSH/glutathione disulphide (GSSG) redox couple and associated low-molecular-weight (LMW) thiols and disulphides related to GSH metabolism in bread wheat (Triticum aestivum L.) seeds, focussing on redox changes in the embryo and endosperm during germination. In dry seeds, GSH was the predominant LMW thiol and, 15?h after the onset of imbibition, embryos of non-germinated seeds contained 12 times more LMW thiols than the endosperm. In germinated seeds, the embryo contained 17 and 11 times more LMW thiols than the endosperm after 15 and 48?h, respectively. This resulted in the embryo having significantly more reducing half-cell reduction potentials of GSH/GSSG and cysteine (Cys)/cystine (CySS) redox couples (E_GSSG/2GSH and E_CySS/2Cys, respectively). Upon seed germination and early seedling growth, Cys and CySS concentrations significantly increased in both embryo and endosperm, progressively contributing to the cellular LMW thiol-disulphide redox environment (E_{thiol-disulphide}). The changes in E_CySS/2Cys could be related to the mobilisation of storage proteins in the endosperm during early seedling growth. We suggest that E_GSSG/2GSH and E_CySS/2Cys can be used as markers of the physiological and developmental stage of embryo and endosperm. We also present a model of interaction between LMW thiols and disulphides with hydrogen peroxide (H₂O₂) in redox regulation of bread wheat germination and early seedling growth.     

Mathematically combined half-cell reduction potentials of low-molecular-weight thiols as markers of seed ageing

Abstract

The half-cell reduction potential of the glutathione disulphide (GSSG)/glutathione (GSH) redox couple appears to correlate with cell viability and has been proposed to be a marker of seed viability and ageing. This study investigated the relationship between seed viability and the individual half-cell reduction potentials (E_is) of four low-molecular-weight (LMW) thiols in Lathyrus pratensis seeds subjected to artificial ageing: GSH, cysteine (Cys), cysteinyl-glycine (Cys-Gly) and γ-glutamyl-cysteine (γ-Glu-Cys). The standard redox potential of γ-Glu-Cys was previously unknown and was experimentally determined. The E_is were mathematically combined to define a LMW thiol-disulphide based redox environment (E_{thiol-disulphide}). Loss of seed viability correlated with a shift in E_{thiol-disulphide} towards more positive values, with a LD₅₀ value of ?0.90 ± 0.093 mV M (mean ± SD). The mathematical definition of E_{thiol-disulphide} is envisaged as a step towards the definition of the overall cellular redox environment, which will need to include all known redox-couples.     

Chloroplastic thioredoxin m functions as a major regulator of Calvin cycle enzymes during photosynthesis in vivo

Thioredoxins (Trxs) regulate the activity of various chloroplastic proteins in a light‐dependent manner. Five types of Trxs function in different physiological processes in the chloroplast of Arabidopsis thaliana. Previous in vitro experiments have suggested that the f‐type Trx (Trx f) is the main redox regulator of chloroplast enzymes, including Calvin cycle enzymes. To investigate the in vivo contribution of each Trx isoform to the redox regulatory system, we first quantified the protein concentration of each Trx isoform in the chloroplast stroma. The m‐type Trx (Trx m), which consists of four isoforms, was the most abundant type. Next, we analyzed several Arabidopsis Trx‐m‐deficient mutants to elucidate the physiological role of Trx m in vivo. Deficiency of Trx m impaired plant growth and decreased the CO₂ assimilation rate. We also determined the redox state of Trx target enzymes to examine their photo‐reduction, which is essential for enzyme activation. In the Trx‐m‐deficient mutants, the reduction level of fructose‐1,6‐bisphosphatase and sedoheptulose‐1,7‐bisphosphatase was lower than that in the wild type. Inconsistently with the historical view, our in vivo study suggested that Trx m plays a more important role than Trx f in the activation of Calvin cycle enzymes.     

Ferredoxin/thioredoxin system plays an important role in the chloroplastic NADP status of Arabidopsis

NADP is a key electron carrier for a broad spectrum of redox reactions, including photosynthesis. Hence, chloroplastic NADP status, as represented by redox status (ratio of NADPH to NADP⁺) and pool size (sum of NADPH and NADP⁺), is critical for homeostasis in photosynthetic cells. However, the mechanisms and molecules that regulate NADP status in chloroplasts remain largely unknown. We have now characterized an Arabidopsis mutant with imbalanced NADP status (inap1), which exhibits a high NADPH/NADP⁺ ratio and large NADP pool size. inap1 is a point mutation in At2g04700, which encodes the catalytic subunit of ferredoxin/thioredoxin reductase. Upon illumination, inap1 demonstrated earlier increases in NADP pool size than the wild type did. The mutated enzyme was also found in vitro to inefficiently reduce m‐type thioredoxin, which activates Calvin cycle enzymes, and NADP‐dependent malate dehydrogenase to export reducing power to the cytosol. Accordingly, Calvin cycle metabolites and amino acids diminished in inap1 plants. In addition, inap1 plants barely activate NADP‐malate dehydrogenase, and have an altered redox balance between the chloroplast and cytosol, resulting in inefficient nitrate reduction. Finally, mutants deficient in m‐type thioredoxin exhibited similar light‐dependent NADP dynamics as inap1. Collectively, the data suggest that defects in ferredoxin/thioredoxin reductase and m‐type thioredoxin decrease the consumption of NADPH, leading to a high NADPH/NADP⁺ ratio and large NADP pool size. The data also suggest that the fate of NADPH is an important influence on NADP pool size.