Half-cell Reduction Potential Research Articles

Reactive nitrogen species act as the enhancers of glutathione pool in embryonic axes of apple seeds subjected to accelerated ageing

Main conclusionReactive nitrogen species mitigate the deteriorative effect of accelerated seed ageing by affecting the glutathione concentration and activities of GR and GPX-like.The treatment of apple (Malus domestica Borkh.) embryos isolated from accelerated aged seeds with nitric oxide-derived compounds increases their vigour and is linked to the alleviation of the negative effect of excessive oxidation processes. Reduced form of glutathione (GSH) is involved in the maintenance of redox potential. Glutathione peroxidase-like (GPX-like) uses GSH and converts it to oxidised form (GSSG), while glutathione reductase (GR) reduces GSSG into GSH. The aim of this work was to investigate the impact of the short-time NOx treatment of embryos isolated from apple seeds subjected to accelerated ageing on glutathione-related parameters. Apple seeds were subjected to accelerated ageing for 7, 14 or 21 days. Isolated embryos were shortly treated with NOx and cultured for 48 h. During ageing, in the axes of apple embryos, GSH and GSSG levels as well as half-cell reduction potential remained stable, while GR and GPX-like activities decreased. However, the positive effect of NOx in the vigour preservation of embryos isolated from prolonged aged seeds is linked to the increased total glutathione pool, and above all, higher GSH content. Moreover, NOx increased the level of transcripts encoding GPX-like and stimulated enzymatic activity. The obtained results indicate that high seed vigour related to the mode of action of NO and its derivatives is closely linked to the maintenance of higher GSH levels.

Redox imbalance accompanies loss of viability in seeds of two cacti species buried in situ

Abstract This work provides insights into the deterioration of cacti seeds of Escontria chiotilla (F.A.C. Weber ex K. Schum) and Stenocereus pruinosus (Otto ex Pfeiff.) Buxbaum stored ex situ at 25 °C, under dry and dark conditions or buried in situ conditions in a xerophytic shrubland. Viability, germination speed, electrolyte leakage and indicators of the redox balance including glutathione content, glutathione half-cell reduction potential (EGSSG/2GSH) and malondialdehyde (MDA), oxidized protein content, together with water-soluble antioxidant enzyme activity were assessed. Over a period of two years of storage, viability was maintained when seeds were stored ex situ at 7–9% water content compared to seeds buried in the soil. A second burial experiment showed that seeds of E. chiotilla maintained viability during a year of storage that included a rainy season followed by a dry season. Thereafter, they died rapidly during the second rainy season. In contrast, those of S. pruinosus started to lose viability after 6 months of burial at the end of the rainy season and were mostly dead at the end of the dry season. This difference in persistence between species was related to a difference in the glutathione content and antioxidant enzyme activities. In both storage experiments, the loss of viability of both species was associated to a EGSSG/2GSH shift to a more oxidative state during burial. Yet, contents in MDA and oxidized soluble proteins were not related to redox imbalance and loss of viability, indicating that these compounds are not good markers of oxidative stress in cacti seeds during storage.

Mono-, di- and trimetallic coinage nanoparticles prepared via the Brust-Schiffrin method.

The Brust-Schiffrin two-phase method is a facile way to prepare thiolate-protected metal nanoparticles, but its mechanism remains controversial. In this work, we demonstrate the use of the Brust-Schiffrin method based on coordination compound theory. We confirmed that the formation of stable complexes is the driving force for a series chemical reaction in the organic phase. We found that the stable Cu(I)-thiolate complex decreased the half-cell reduction potential of Cu(I)/Cu(0). Thus, when thiol ligands were in excess, thiolate-protected Cu(I) clusters formed rather than Cu(0)-cored nanoparticles. The thiolate-protected metal-hydride nanoclusters were the intermediate between the metal complexes and nanoparticles. The "metallophilic" interactions of the d10 closed-shell electronic configuration of the metal coordination centers were proposed as the driving force for nanocluster and nanoparticle formation. To confirm this mechanism, we synthesized Au, Ag, and Cu monometallic nanoparticles and bi- and trimetallic nanoparticles. We found that although thiolate-protected Cu(I) nanoclusters are not easily reduced, they can combine with Au and/or Ag nanoclusters to form nanoparticles. The proposed mechanism is expected to provide deeper insight into the Brust-Schiffrin method and further extend its application to metals other than Au, Ag and Cu.

Eight-Electron Superatomic Cu31 Nanocluster with Chiral Kernel and NIR-II Emission

Owing to the inherent instability caused by the low Cu(I)/Cu(0) half-cell reduction potential, Cu(0)-containing copper nanoclusters are quite uncommon in comparison to their Ag and Au congeners. Here, a novel eight-electron superatomic copper nanocluster [Cu31(4-MeO-PhC≡C)21(dppe)3](ClO4)2 (Cu31, dppe = 1,2-bis(diphenylphosphino)ethane) is presented with total structural characterization. The structural determination reveals that Cu31 features an inherent chiral metal core arising from the helical arrangement of two sets of three Cu2 units encircling the icosahedral Cu13 core, which is further shielded by 4-MeO-PhC≡C- and dppe ligands. Cu31 is the first copper nanocluster carrying eight free electrons, which is further corroborated by electrospray ionization mass spectrometry, X-ray photoelectron spectroscopy and density functional theory calculations. Interestingly, Cu31 demonstrates the first near-infrared (750-950 nm, NIR-I) window absorption and the second near-infrared (1000-1700 nm, NIR-II) window emission, which is exceptional in the copper nanocluster family and endows it with great potential in biological applications. Of note, the 4-methoxy groups providing close contacts with neighboring clusters are crucial for the cluster formation and crystallization, while 2-methoxyphenylacetylene leads only to copper hydride clusters, Cu6H or Cu32H14. This research not only showcases a new member of copper superatoms but also exemplifies that copper nanoclusters, which are nonluminous in the visible range may emit luminescence in the deep NIR region.

Distinct redox state regulation in the seedling performance of Norway maple and sycamore

Norway maple and sycamore, two Acer genus species, have an important ecological value and different sensitivity to stressing factors being currently aggravated by climate change. Seedling growth is postulated to be the main barrier for successful plant establishment under the climate change scenarios. Therefore, the differences in redox regulation during the seedling performance of Norway maple and sycamore were investigated. Seeds of the two Acer species exhibited an identical high germination capacity, whereas seedling emergence was higher in sycamores. PCA analyses revealed that there is more diversification in the leaf characteristics than roots. Norway maple displayed a higher chlorophyll content index (CCI) with a similar leaf mass whereas sycamore seedlings exhibited a higher normalized difference vegetation index (NDVI), higher water content, higher root biomass and higher shoot height. Based on NDVI, sycamore seedlings appeared as very healthy plants, whereas Norway maple seedlings displayed a moderate healthy phenotype. Therefore, redox basis of seedling performance was investigated. The total pool of glutathione was four times higher in sycamore leaves than in Norway maple leaves and was reflected in highly reduced half-cell reduction potential of glutathione. Sycamore leaves contained more ascorbate because the content of its reduced form (AsA) was twice as high as in Norway maple. Therefore, the AsA/DHA ratio was balanced in sycamore leaves, reaching 1, and was halved in Norway maple leaves. Nicotinamide adenine dinucleotide phosphate content was twice as high in sycamore leaves than in Norway maples; however, its reduced form (NADPH) was predominant in Norway maple seedlings. Norway maple leaves exhibited the highest anabolic and catabolic redox charge. The higher reduction capacity and the activity of NADPH-dependent reductases in Norway maple leaves possibly resulted in higher CCI, whereas the larger root system contributed to higher NDVI in sycamore. The different methods of controlling redox parameters in Acer seedlings grown at controlled conditions provided here can be useful in understanding how tree species can cope with a changing environment in the future.

Antioxidant depletion during seed storage under ambient conditions

AbstractCumulative oxidative damage from the unavoidable formation of reactive oxygen species (ROS) contributes to seed ageing. Low-molecular-weight (LMW) antioxidants, such as water-soluble glutathione (GSH) and lipid-soluble tocochromanols, can prevent ROS from causing damage, especially when antioxidant enzymes are inactive due to desiccation. However, loss of tocochromanols does not always accompany seed ageing, such as during accelerated ageing or controlled deterioration, despite the presence of oxygen and prevalent loss of GSH. To assess relationships between total germination (TG) and antioxidant changes under storage conditions with practical relevance, commercial seeds of Cucumis sativus, Daucus carota, Helianthus cucumerifolius, Latuca sativa, Lepidium sativum, Phaseolus vulgaris and Raphinus sativus of the same cultivar were obtained over multiple years and stored under ambient conditions (21.9 ± 2.1°C; 36.8 ± 6.6% relative humidity). Sigmoidal fitting of TG revealed time to when 50% of seeds had lost viability, which ranged from <5 years (D. carota) to >15 years (C. sativus). Cellular redox states were quantified via the half-cell reduction potential of LMW thiol/disulphide couples. These negatively correlated with TG (i.e. cell redox states were more oxidized in lots with lower TG), with an average R2 value of 0.62 for the most abundant thiol (GSH, or γ-glutamyl-cysteine in P. vulgaris). Concentrations of tocochromanols positively correlated with TG, with an average R2 value of 0.50 for the most abundant tocochromanol (γ or α in L. sativa and H. cucumerifolius). Therefore, during viability loss under ambient ageing conditions leading to the cytoplasm having a glassy state, the lipid domain in all species experienced oxidative damage.

Cold stratification-induced dormancy removal in apple (Malus domestica Borkh.) seeds is accompanied by an increased glutathione pool in embryonic axes

A reduced form of glutathione (GSH) is an essential metabolite that participates in the control of reactive oxygen species (ROS) levels in cells. GSH plays a pivotal role in seed biology as a modulator of seed viability and germination. The GSH:GSSG ratio and half-cell reduction potential (EGSSG/2GSH) serve as indicators of the oxidative status in seeds. Apple (Malus domestica Borkh.) seeds are deeply dormant, and this state is removed by long-term cold stratification. The aim of our work was to examine the modification of GSH and GSSG content, GSH:GSSG ratio and EGSSG/2GSH in the embryonic axes isolated from apple seeds subjected to cold stratification for 7, 14, 21 and 40 d. Our data indicated that cold stratification increased the generation of free radicals in the embryonic axes, which correlated with an alteration in the expression of genes encoding Rboh, particularly RbohC. GSH and GSSG levels increased during prolonged cold stratification of apple seeds. This was accompanied by the modification of glutathione reductase and glutathione peroxidase-like activities, which did not match their transcript levels. The steady-state GSH:GSSG ratio and EGSSG/2GSH in the axes of embryos subjected to cold stratification indicated no impact of the dormancy removal treatment on apple seed viability. We suggest that the glutathione system is an important component of the redox network and is involved in the management of the seed transition from dormant to nondormant states.

Nicotinamide adenine dinucleotides are associated with distinct redox control of germination in Acer seeds with contrasting physiology.

Seed germination is a complex process enabling plant reproduction. Germination was found to be regulated at the proteome, metabolome and hormonal levels as well as via discrete post-translational modification of proteins including phosphorylation and carbonylation. Redox balance is also involved but less studied. Acer seeds displaying orthodox and recalcitrant characteristics were investigated to determine the levels of redox couples of nicotinamide adenine dinucleotide (NAD) phosphate (NADP) and integrated with the levels of ascorbate and glutathione. NAD and NADP concentrations were higher in Norway maple seeds and exceptionally high at the germinated stage, being the most contrasting parameter between germinating Acer seeds. In contrast, NAD(P)H/NAD(P)+ ratios were higher in sycamore seeds, thus exhibiting higher reducing power. Despite distinct concentrations of ascorbate and glutathione, both seed types attained in embryonic axes and cotyledons had similar ratios of reduced/oxidized forms of ascorbate and half-cell reduction potential of glutathione at the germinated stage. Both species accomplished germination displaying different strategies to modulate redox status. Sycamore produced higher amounts of ascorbate and maintained pyridine nucleotides in reduced forms. Interestingly, lower NAD(P) concentrations limited the regeneration of ascorbate and glutathione but dynamically drove metabolic reactions, particularly in this species, and contributed to faster germination. We suggest that NAD(P) is an important player in regulating redox status during germination in a distinct manner in Norway maple and sycamore seeds.

Cu61(StBu)26S6Cl6H14]+: A Core–Shell Superatom Nanocluster with a Quasi-J36 Cu19 Core and an “18-Crown-6” Metal-Sulfide-like Stabilizing Belt

Although core–shell copper metal nanoclusters are important emerging materials for practical applications and fundamental scientific research, their synthesis lags behind that of gold and silver nanoclusters–challenged by copper’s much lower half-cell reduction potential, M(I)/M(0). To overcome this synthetic hurdle, we introduce a simple reaction strategy, involving the mild reducing agent borane tert-butylamine complex, to produce a core–shell superatom copper nanocluster, [Cu61(StBu)26S6Cl6H14]+ (−StBu; tert-butyl thiolate), which is the largest Cu(0)-containing structurally-solved core–shell copper cluster to-date. The nanocluster exhibits a quasi-elongated triangular gyrobicupola (quasi-J36, J36 = Johnson solid) Cu19 core and a shell held together by a novel “18-crown-6” metal-sulfide-like belt. Because of its stability, this cluster displays a single molecular ion peak in mass spectrometry measurements without any cluster fragmentation signals—a first observation of its kind for copper nanoclusters that paves the way for researchers to study nanocluster composition, charge, stability, and reaction mechanisms with atomic precision that only mass spectrometry could afford.

Wheat seed ageing viewed through the cellular redox environment and changes in pH

To elucidate biochemical mechanisms leading to seed deterioration, we studied 23 wheat genotypes after exposure to seed bank storage for 6–16 years compared to controlled deterioration (CD) at 45 °C and 14 (CD14) and 18% (CD18) moisture content (MC) for up to 32 days. Under two seed bank storage conditions, seed viability was maintained in cold storage (CS) at 0 °C and 9% seed MC, but significantly decreased in ambient storage (AS) at 20 °C and 9% MC. Under AS and CS, organic free radicals, most likely semiquinones, accumulated, detected by electron paramagnetic resonance, while the antioxidant glutathione (GSH) was partly lost and partly converted to glutathione disulphide (GSSG), detected by HPLC. Under AS the glutathione half-cell reduction potential (EGSSG/2GSH) shifted towards more oxidising conditions, from −186 to −141 mV. In seeds exposed to CD14 or CD18, no accumulation of organic free radicals was observed, GSH and seed viability declined within 32 and 7 days, respectively, GSSG hardly changed (CD14) or decreased (CD18) and EGSSG/2GSH shifted to −116 mV. The pH of extracts prepared from seeds subjected to CS, AS and CD14 decreased with viability, and remained high under CD18. Across all treatments, EGSSG/2GSH correlated significantly with seed viability (r = 0.8, p<.001). Data are discussed with a view that the cytoplasm is in a glassy state in CS and AS, but during the CD treatments, underwent transition to a liquid state. We suggest that enzymes can be active during CD but not under the seed bank conditions tested. However, upon CD, enzyme-based repair processes were apparently outweighed by deteriorative reactions. We conclude that seed ageing by CD and under seed bank conditions are accompanied by different biochemical reactions.

Occurrence of Alkaloids in Grass Seeds Symbiotic With Vertically-Transmitted Epichloë Fungal Endophytes and Its Relationship With Antioxidants

Host organisms can acquire new functional traits through symbiosis. Seed-transmitted Epichloe fungal endophytes are known to protect host plants against herbivores and increase tolerance to abiotic stresses by alkaloids and antioxidants, respectively (currencies of mutualism). Whereas alkaloids are fungal products with demonstrated effects at plant vegetative stage, few studies have focused on alkaloids in seeds. We assessed the occurrence of fungal alkaloids and determined their concentration in seeds of two host grasses, Festuca rubra and Lolium multiflorum. Then, we sought for a relationship with the antioxidants tocochromanols and glutathione, which are involved in the control of oxidative stress. Different alkaloids were detected depending on the species and plant genotype. Most notably, loline alkaloids were not detected in F. rubra seeds, whereas ergovaline and peramine were absent in L. multiflorum. In F. rubra, ergovaline concentration was dependent on the maternal line in interaction with the production year, diminishing in seeds after one year of storage. The exposure of L. multiflorum plants to ozone had no effect on the seed concentration of lolines. There was a positive relationship between concentrations of ergovaline and tocochromanol in both species, and between ergovaline concentration and EGSSG/2GSH (glutathione half-cell reduction potential) in RAB maternal line of F. rubra. These results suggest that alkaloid and antioxidants have a close association in seeds of host grasses, and that the alkaloid bioactivity could be related with the antioxidant capacity to control stress. This has important implications for the ecology of partner species, thus supporting its consideration for further research.

Identification of a redox-dependent regulatory network of miRNAs and their targets in wheat.

Reactive oxygen species and antioxidants have an important role in the regulation of plant growth and development under both optimal and stress conditions. In this study, we investigate a possible redox control of miRNAs in wheat (Triticum aestivum ssp. aestivum). Treatment of seedlings with 10 mM H2O2 via the roots for 24 h resulted in decreased glutathione content, increased half-cell reduction potential of the glutathione disulphide/glutathione redox pair, and greater ascorbate peroxidase activity compared to the control plants. These changes were accompanied by alterations in the miRNA transcript profile, with 70 miRNAs being identified with at least 1.5-fold difference in their expression between control and treated (0, 3, 6 h) seedlings. Degradome sequencing identified 86 target genes of these miRNAs, and 6722 possible additional target genes were identified using bioinformatics tools. The H2O2-responsiveness of 1647 target genes over 24 h of treatment was also confirmed by transcriptome analysis, and they were mainly found to be related to the control of redox processes, transcription, and protein phosphorylation and degradation. In a time-course experiment (0-24 h of treatment) a correlation was found between the levels of glutathione, other antioxidants, and the transcript levels of the H2O2-responsive miRNAs and their target mRNAs. This relationship together with bioinformatics modelling of the regulatory network indicated glutathione-related redox control of miRNAs and their targets, which allows the adjustment of the metabolism to changing environmental conditions.

The Occurrence of Peroxiredoxins and Changes in Redox State in Acer platanoides and Acer pseudoplatanus During Seed Development

Norway maple (Acer platanoides L.) and sycamore (A. pseudoplatanus L.) are genetically closely related species that produce desiccation-tolerant (orthodox) and desiccation-sensitive (recalcitrant) seeds, respectively. Norway maple and sycamore seeds were analyzed during their development from the 14th to 24th weeks after flowering (WAF) and 11th to 21st WAF, respectively, to explore redox-related biochemical properties related to their contrasting physiology. Selected similar stages of seed development were characterized during the course of gradual decreasing water content in both seed types. The levels of protein and non-protein thiols peaked at the 18th WAF in Norway maple embryonic axes, whereas these levels constantly increased in maturing sycamore seeds. The glutathione half-cell reduction potential revealed that the cell environment adopted a more oxidized state in sycamore seeds. Peroxiredoxins (Prxs), including cytosolic/nuclear 1-Cys-Prx, cytosolic PrxIIC, mitochondrial PrxIIE, and plastidic PrxIIF, 2-Cys-Prx, and PrxQ, were detected in both species, but Norway maple embryonic axes contained higher levels of PrxIIC and PrxIIE, two Prxs with the highest peroxide detoxification potential in Arabidopsis. Redox proteomics revealed that 2-Cys-Prx was present in reduced form in both species, whereas 1-Cys-Prx was reduced uniquely in Norway maple seeds. Several enzymes, including glucose and ribitol dehydrogenase as well as fructose-bisphosphate aldolase, were oxidation-sensitive at all developmental stages in sycamore embryonic axes. Redox signaling as manifested by reactive oxygen species signals, and the oxidation of protein thiols to disulfides are discussed with respect to their significance in determining orthodox or recalcitrant seed characteristics.

The effect of a doubled glutathione level on parameters affecting the germinability of recalcitrant Acer saccharinum seeds during drying

Approximately 20% of plant species, including silver maple (Acer saccharinum L.), produce seeds that are sensitive to desiccation, which is reflected in their poor storage potential and viability. In the search for a compound that can improve seed recalcitrance, freshly harvested seeds were soaked in either 2.5 mM reduced glutathione (GSH) or water and desiccated to comparable water levels of 55–20%. We examined the impact of a doubled endogenous level of glutathione on the seed germination capacity, the activity of enzymes involved in glutathione metabolism, the cell membrane components and integrity, reactive oxygen species, and ascorbate levels. GSH treatment resulted in slower dehydration and a higher germination capacity. The increased glutathione was mainly consumed by glutathione S-transferase, leading to more efficient detoxification, and by dehydroascorbate reductase (DHAR), accelerating the ascorbate regeneration. As a result, the cellular environment became more reduced, and protection of the membrane structures was enhanced. The ameliorated membrane integrity was manifested via a lower electrolyte leakage and a lower lipid peroxide level despite the higher level of hydrogen peroxide (H2O2) detected in the GSH-treated seeds. The degradation of phospholipids (PLs) was less intense and related to the phosphatidylinositol (PI) level, which is the precursor of the phospholipase D cofactor, whereas in water-soaked seeds, PL degradation was promoted by H2O2. The germination capacity of the dehydrated seeds depended primarily on the level of H2O2, lipid hydroxyperoxides, electrolyte leakage, GSH, the half-cell reduction potential of glutathione, PI, and the activity of DHAR and γ-glutamylcysteine synthetase. Interestingly, H2O2 affected all of the parameters. The germination of GSH-boosted seeds was strongly impacted by the pool of ascorbate, the half-cell reduction potential of ascorbate, and the glutathione peroxidase activity. In general, germination was DHAR activity-dependent. A strong negative correlation was detected in the water-soaked seeds, whereas a strong positive correlation was detected in the GSH-treated seeds. The enhanced level of glutathione likely improved the efficiency of the ascorbate–glutathione cycle, confirming its effect on seed germinability after dehydration.

A DFT investigation of the blue bottle experiment: E∘half-cell analysis of autoxidation catalysed by redox indicators.

The blue bottle experiment is a collective term for autoxidation reactions catalysed by redox indicators. The reactions are characterized by their repeatable cycle of colour changes when shaken/left to stand and intricate chemical pattern formation. The blue bottle experiment is studied based on calculated solution-phase half-cell reduction potential of related reactions. Our investigation confirms that the reaction in various versions of the blue bottle experiment published to date is mainly the oxidation of an acyloin to a 1,2-dicarbonyl structure. In the light of the calculations, we also propose new non-acyloin reducing agents for the experiment. These results can help guide future experimental studies on the blue bottle experiment.

Monitoring of oxidative status in three native Australian species during cold acclimation and cryopreservation.

Three wild species exhibited a significant reduction in antioxidants throughout the cryopreservation protocol, whilst the half-cell reduction potential became more oxidised. Antioxidant content recuperated in recovering shoot tips. Cryopreservation is the most efficient and cost-effective long-term storage solution for the conservation of a wide range of plant species and material. Changes in the levels of antioxidants during the process of cryopreservation are known to reduce post-cryogenic survival due to oxidative stress. Low-molecular-weight thiols (cysteine, γ-glutamylcysteine, and glutathione) and ascorbic acid, which represent the two major water-soluble antioxidants in plants, were analysed at specific stages during cryopreservation of shoot tip material of three native Australian plant species [Anigozanthos viridis (Haemodoraceae), Lomandra sonderi (Asparagaceae), and Loxocarya cinerea (Restionaceae)] to quantify the oxidative stress experienced during cryopreservation. Post-cryogenic regeneration of shoot tips was greatest in A. viridis (78%) followed by L. sonderi (50%), whilst L. cinerea did not show any post-cryogenic regeneration. The application of a 3-week cold (5°C) preconditioning regime, commonly used to increase post-cryogenic survival, resulted in significantly lower post-cryogenic regeneration for A. viridis (33%), but had little effect on the other two species. Total antioxidant concentration in shoot material decreased significantly with each step throughout the cryopreservation process, particularly in the cryoprotection and washing stages. Antioxidant levels in shoot tips then increased during the subsequent 7-day post-cryopreservation recovery period, with the greatest increase measured in A. viridis. Concentrations of thiols and their corresponding disulphides were used to calculate the corresponding half-cell reduction potentials, whereby the ability of these plant species to maintain a strong reducing environment in shoot tissues throughout the cryopreservation protocol was found to correlate with post-cryogenic survival.

Analyses of several seed viability markers in individual recalcitrant seeds of Eugenia stipitata McVaugh with totipotent germination.

The use of biochemical seed viability markers is often compromised by the unknown partitioning of analytes in bulk seed lots consisting of inseparable populations of viable and nonviable seeds. We took advantage of an unusual morphological syndrome found in the recalcitrant, undifferentiated seeds of Eugenia stipitata: one seed can be cut into several parts, each of which can germinate and develop into seedlings. We used four seed parts from one individual seed to analyse seed moisture content (MC), seed viability and the antioxidant glutathione (γ-glutamyl-cysteinyl-glycine; GSH), glutathione disulphide (GSSG) and intermediates of glutathione synthesis and breakdown. Seeds were exposed to different environmental MC to induce various levels of desiccation stress. Upon storage at high seed MC, seed viability was maintained, while GSH concentration increased and the glutathione half-cell reduction potential (EGSSG/2GSH ) was less negative than -215mV, indicating GSH production and highly reducing conditions. Storage at low seed MC led to loss of GSH, resulting in a shift in EGSSG/2GSH , and seed death. In contrast, the cyst(e)ine half-cell reduction potential (ECySS/2CYS ) could not distinguish between the viability categories. Previous studies on seed populations revealed that the probability for a seed being alive is 50% at EGSSG/2GSH values between -180 and -160mV. The single seed approach revealed that the window in which seed viability was lost could be slightly shifted towards more negative values. We discuss the contribution of cellular pH to EGSSG/2GSH and recommend E.stipitata as a recalcitrant seed model to study stress response on a single seed basis.

Impact of ozone on the viability and antioxidant content of grass seeds is affected by a vertically transmitted symbiotic fungus

Ozone gas is a rising pollutant in the troposphere and is a consequence of human-driven global change. As a novel environmental stressor, interest in the impact of ozone on symbiotic systems is increasing. Focusing on the symbiosis between grasses and Epichloe species, we evaluated the effect of ozone exposure on the relative fitness of symbiotic and endophyte free plants and its transgenerational effects including seed performance and endophyte persistence. Endophyte symbiotic and endophyte free Lolium multiflorum plants were exposed to high ozone concentration at pre-anthesis. Seed production of symbiotic plants was 23% higher than that of endophyte free plants, being positively correlated with number of spikes and independent of ozone. Seed viability was negatively affected by the endophyte and improved by ozone. A dramatic negative effect of ozone on endophyte viability was manifested only after 25-day seed storage under accelerating ageing conditions. On average, seeds from plants exposed to ozone had higher levels of reduced glutathione (GSH), whilst seeds from symbiotic plants were associated with higher content of glutathione disulfide (GSSG). Consistent with the pattern of seed viability dynamics, the glutathione half-cell reduction potential (EGSSG/2GSH) was higher (i.e. less negative) in E+ seeds and slightly lower (i.e. more negative) in seeds from plants exposed to high ozone. The relationship between endophyte symbiosis and ozone stress with the levels of tocochromanol antioxidants was unclear, and irrespective of seed or endophyte viability. The concentration of some tocochromanols were not affected, whereas others were positively (β-tocopherol) or negatively (α-tocotrienol and γ-tocopherol) affected by the endophyte, or positively affected (γ-tocopherol) by ozone alone. The fungal symbiont modified the effect of ozone exposure in the maternal environment and thus, grass seed viability and antioxidant content. Although the grass-endophyte symbiosis may promote plant yield under rising ozone levels associated with global change, it may be at the expense of seed viability and endophyte persistence.

Antioxidants in Festuca rubra L. seeds affected by the fungal symbiont Epichloë festucae

Vertically transmitted fungal endophytes can be beneficial for host grasses. While the alkaloid-mediated mechanism for herbivore resistance has been widely studied, underlying physiological mechanisms for increased tolerance to abiotic stress remain scarcely explored. In this study we used three maternal lines of perennial grass Festuca rubra to examine the role of antioxidants in endophyte-mediated effects on seed viability over long-term storage. Uncolonized plants (E−) were generated by removing the endophyte from ramets of naturally endophyte-colonized (E+) plants. The E + and E− ramets were planted in a common garden in Salamanca, Spain. Seeds produced in 2009, 2010 and 2011 were harvested at maturity, dried and stored at 10 °C until 2011 when we tested seed and endophyte viability, and measured antioxidants. Seed viability and α-tocopherol antioxidant were negatively affected by the endophyte in two maternal lines. In these same lines, the endophyte viability was lowest at the longest storage time. In the maternal line that showed the highest negative effect of endophyte on seed viability, the pattern of glutathione was opposite to that observed for tocopherols since it was higher for E + than for E− seeds. In all maternal lines, the glutathione half-cell reduction potential (EGSSG/2GSH) and % glutathione disulphide (GSSG) increased with storage time but there was no clear pattern associated with endophyte symbiosis. Whether these parameters are good predictors of seed and endophyte longevity in storage and natural conditions should be further explored.

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

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 (EGSSG/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 (ECySS/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’ (Ethiol–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 ECySS/2 Cys to Ethiol–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.