Glutathione Half-cell Reduction Potential Research Articles

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.

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.

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.

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.

Glutathione half-cell reduction potential as a seed viability marker of the potential oilseed crop Vernonia galamensis

Seeds of Vernonia galamensis (Cass.) Less. contain a high proportion of vernolic acid that is highly desirable for industrial use and emphasises the potential of this species for commercialisation. For successful commercialisation, seed quality during storage must be maintained and regularly assessed, which can be rapidly achieved by using biochemical markers of seed viability. In V. galamensis, three potential viability markers electrolyte leakage (as a measure of membrane integrity) and the antioxidants tocopherol and glutathione were investigated in highly viable, aged and dead seeds at 6% and 12% moisture content (MC). Both electrolyte leakage and tocopherol were not correlated with seed viability whereas the glutathione half-cell reduction potential ( E GSSG/2GSH) correlated strongly with viability loss during seed ageing at both MCs. However, an increase in E GSSG/2GSH associated with viability loss was delayed in the drier seeds (6% MC), highlighting that the performance of this seed viability marker could be decreased in seeds which lose viability in, or close to, the glassy state.

Introduction of the interdependence between the glutathione half-cell reduction potential and thermodynamic parameters during accelerated aging of maize seeds

Two maize hybrids with a different ability to maintain seed germination were examined during the course of accelerated aging (AA). Initially, the similar seed reduction potential of the GSSG/2GSH half-cell increased in H1 (dent hybrid) without influencing the seed germination ability up to the 6th day of AA, while in H2 (sweet corn hybrid), it was not changed up to the 6th day of AA but with a significant later loss of seed germination ability. During the AA course, the amount of free thiol decreased in H1 and increased in H2. Irrespective of the continual increase of the differential Gibbs energy during AA, the characteristics of the examined hybrids are possibly connected to the different metabolic pathways of the seeds: H1 is characterized by higher entropy and positive enthalpy values, while H2 had negative entropy values and a decreasing trend of enthalpy, indicating a shift of the system from a relatively ordered to a disordered state. The different types of nanomolecular switches, resulting in a faster decrease of GSH in the H2 than in the H1 hybrids, indicate that a combination of the GSSG/2GSH half-cell potential and thermodynamics could be a useful tool to quantify plant stress.

Introduction of the interdependence between the glutathione half-cell reduction potential and thermodynamic parameters during accelerated aging of maize seeds

Introduction of the interdependence between the glutathione half-cell reduction potential and thermodynamic parameters during accelerated aging of maize seeds

Glutathione half-cell reduction potential and α-tocopherol as viability markers during the prolonged storage of Suaeda maritima seeds

AbstractAntioxidants protect seeds from oxidative damage during storage, supporting the maintenance of seed viability and the ability to germinate post-storage. No data on antioxidants during long-term storage are available for the seeds of the halophyte Suaeda maritima. Therefore, changes in lipid-soluble antioxidants in the tocopherol family (α-, γ-, δ-tocopherol), were investigated in seeds stored for up to 16 years at 4°C at a seed moisture content of 10–13%, as well as changes in the water-soluble antioxidant glutathione (GSH) and electrolyte leakage. Seed oil content was also measured and determined to be 22%. During the first 3 years of storage, seed viability remained high and the concentration of total tocopherol was stable. Thereafter, both seed viability and α-tocopherol concentration rapidly decreased and electrolyte leakage increased, while γ-tocopherol and δ-tocopherol concentrations did not correlate with seed viability. Although the concentrations of neither GSH nor glutathione disulphide (GSSG) alone were correlated with seed viability, the glutathione half-cell reduction potential (EGSSG/2GSH) was strongly correlated with viability throughout storage and increased before the onset of viability loss. Hence, in this species EGSSG/2GSH appeared to be an ‘early warning’ system preceding viability loss while α-tocopherol concentration changed concomitantly with viability.

Extracellular superoxide production, viability and redox poise in response to desiccation in recalcitrantCastanea sativaseeds

Reactive oxygen species (ROS) are implicated in seed death following dehydration in desiccation-intolerant 'recalcitrant' seeds. However, it is unknown if and how ROS are produced in the apoplast and if they play a role in stress signalling during desiccation. We studied intracellular damage and extracellular superoxide (O(2)(.-)) production upon desiccation in Castanea sativa seeds, mechanisms of O(2)(.-) production and the effect of exogenously supplied ROS. A transient increase in extracellular O(2)(.-) production by the embryonic axes preceded significant desiccation-induced viability loss. Thereafter, progressively more oxidizing intracellular conditions, as indicated by a significant shift in glutathione half-cell reduction potential, accompanied cell and axis death, coinciding with the disruption of nuclear membranes. Most hydrogen peroxide (H(2)O(2))-dependent O(2)(.-) production was found in a cell wall fraction that contained extracellular peroxidases (ECPOX) with molecular masses of approximately 50 kDa. Cinnamic acid was identified as a potential reductant required for ECPOX-mediated O(2)(.-) production. H(2)O(2), applied exogenously to mimic the transient ROS burst at the onset of desiccation, counteracted viability loss of sub-lethally desiccation-stressed seeds and of excised embryonic axes grown in tissue culture. Hence, extracellular ROS produced by embryonic axes appear to be important signalling components involved in wound response, regeneration and growth.

Glutathione half-cell reduction potential: A universal stress marker and modulator of programmed cell death?

The elucidation of factors that contribute to cell viability loss is presently compromised by the lack of a universal measure that quantifies “stress.” We have investigated mechanisms of viability loss in plant seeds to find a reliable marker of stress response. Oxidative damage has previously been correlated with degenerative processes and death, but how exactly this contributes to viability loss is unknown. We show in four species subjected to ageing or desiccation that seed viability decreased by 50% when the half-cell reduction potential of glutathione ( E GSSG/2GSH), a major cellular antioxidant and redox buffer, increased to −180 to −160 mV. We then conducted a metaanalysis of data representative of 13 plant and fungal orders to show that plant stress generally becomes lethal when E GSSG/2GSH exceeds −160 mV. We put forward that this change in E GSSG/2GSH is part of the signaling cascade that initiates programmed cell death (PCD), finally causing internucleosomal DNA fragmentation in the final, or execution phase, of PCD. E GSSG/2GSH is therefore a universal marker of plant cell viability and allows us to predict whether a seed will live, germinate, and produce a new plant, or if it will die.