Ewelina Ratajczak

Age-related biochemical changes during storage of beech (Fagus sylvatica L.) seeds

Substances that could play a role in maintaining seed viability (phenolic compounds, a-tocopherol, sterols, ascorbic acid, glutathione and soluble proteins) were estimated in beech (Fagus sylvatica L.) seed lots that had been stored for 2, 5, 7 and 10 years at 2108C. Germination capacity was strongly and positively correlated with amounts of total phenolic compounds, ultraviolet (UV)-absorbing phenols and soluble proteins. Moderately strong relationships appeared between germination capacity and a-tocopherol and ascorbic acid contents. Total sterol and glutathione contents were not correlated with germination capacity. A strong, negative correlation was found between germination capacity and reactive oxygen species (ROS), such as superoxide radical (O ·2 2 ) and hydrogen peroxide (H2O2), as well as with lipid hydroxyperoxides (LHPOs). The putative role of these compounds in the maintenance of beech seed viability during long-term storage is discussed.

The protective role of selenium in recalcitrant Acer saccharium L. seeds subjected to desiccation

Freshly harvested silver maple (Acer saccharinum L.) seeds were soaked in either sodium selenite (10mg/L) or water for 6h. After washing and air drying, seeds were desiccated at 22°C at a RH of 45-50% to comparable water levels from 50 to 12%. Germination capacity was significantly higher in seeds treated with selenium and desiccated [from 50 to 40, 35 and 30% of water content (WC)] than in water-soaked seeds. At 20% WC, the seeds from both treatments had low viability (approximately 20%). The electrolyte leakage and the MDA content were significantly lower in the embryonic axes of seeds soaked in selenite than in seeds soaked in water. We also found that the activity of glutathione peroxidase (GPX) of embryonic axes from selenium-treated seeds that were not desiccated, or from seeds that were desiccated to 40 and 35% WC, was significantly higher than that of non-treated axes. No difference in GPX activity was detected in cotyledons. This was confirmed by activity staining of GPX after native PAGE of proteins extracted from embryonic axes and cotyledons. An increase in glutathione reductase (GR) activity was also observed in embryonic axes of seeds treated with selenium and dried to 35 and 30% WC compared to non-treated samples. Selenium appeared to have no such effect on cotyledons.

Ascorbate and glutathione metabolism during development and desiccation of orthodox and recalcitrant seeds of the genus Acer

The ascorbate-glutathione system was studied during development and desiccation of seeds of two Acer species differing in desiccation tolerance: Norway maple (Acer platanoides L., orthodox) and sycamore (Acer pseudoplatanus L., recalcitrant). The results showed remarkable differences in the concentration and redox balance of ascorbate and glutathione between these two kinds of seeds during development, and a significant dependence between glutathione content and acquisition of desiccation tolerance in Norway maple seeds. There were relatively small differences between the species in the activities of enzymes of the ascorbate-glutathione cycle: ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.6.4.2). At the end of seed maturation, ascorbic acid content and the activities of the above enzymes was about the same in both species The electrophoretic pattern of APX isoenzymes was also similar for both species, and the intensity of the bands decreased at the end of seed maturation in both species. When sycamore seeds were desiccated to a moisture content of less than 26%, there was a marked decrease in seed viability and an increase in the production of reactive oxygen species. During desiccation, Norway maple seeds had a more active defence system, which was reflected in a higher glutathione content, a higher glutathione redox status, a higher ascorbate redox status, and higher activities of APX, MR, DHAR, GR and GPX (glutathione peroxidase). During desiccation, sulfhydryl-to-disulfide transition into proteins was more intense in Norway maple seeds than sycamore seeds. All of these results suggest that, in orthodox seeds, the ascorbate-glutathione cycle plays an important role in the acquisition of tolerance to desiccation, in protein maturation, and in protection from reactive oxygen species.

Decrease in beech [Fagus sylvatica] seed viability caused by temperature and humidity conditions as related to membrane damage and lipid composition

The aim of this study was to determine if the loss of germinability and viability of beech (Fagus sylvatica L.) seeds stored at different variants of temperature (4, 20, and 30 °C) and relative humidity (RH: 45 and 75 %) is associated with a loss of membrane integrity and changes in lipid composition. Beech seeds stored for 9 weeks gradually lost viability at a rate dependent on temperature and humidity. The harmful effect of temperature increased with growing humidity. The loss of seed viability was strongly correlated with an increase in membrane permeability and with production of lipid hydroxyperoxides (LHPO), which was regarded as an indicator of peroxidation of unsaturated fatty acids. The condition of membranes was assessed on the basis of their permeability and the state of lipid components: phospholipids and fatty acids. During seed storage we observed a decline in concentration of individual phospholipids and fatty acids, proportional to the loss of seeds viability. We also detected a decrease in concentrations of α-tocopherol and sterols, which play an important role in protection of membranes against the harmful influence of the environment. Our results show that the germinability of beech seeds declines rapidly at temperature above 0 °C and growing humidity. This is due mainly to the loss of membrane integrity, caused by peroxidation of unsaturated fatty acids.

Non-reducing sugar levels in beech (Fagus sylvatica) seeds as related to withstanding desiccation and storage.

Levels of sucrose and raffinose family oligosaccharides (RFOs) (raffinose and stachyose) were determined in beech (Fagus sylvatica L.) seeds during development, maturation, desiccation and storage. An increase in RFOs and a marked decrease in the S:(R+St) ratio (i.e. mass ratio of sucrose to the sum of RFOs) were observed at the time of desiccation tolerance (DT) acquisition by seeds. In seeds stored at -10 degrees C through 1, 4, 7, and 12 years, changes in sucrose, raffinose and stachyose levels and in alpha-galactosidase activity were noted. The S/R+St ratio and alpha-galactosidase activity significantly increased in seeds after 7 and 12 years of storage, when a marked decrease in viability, measured as germination capacity, was recorded. Germination capacity was found to be strongly correlated with sucrose content, the S:(R+St) ratio, and alpha-galactosidase activity. A strong positive correlation was found between germination capacity and stachyose content. The results clearly indicated that the composition of RFOs in beech seeds is closely related to DT acquisition and seed viability during storage.

The production, localization and spreading of reactive oxygen species contributes to the low vitality of long-term stored common beech (Fagus sylvatica L.) seeds.

The common beech (Fagus sylvatica L.) is propagated by seeds, but the seed set is irregular with five to ten years in between crops. It is therefore necessary to store the seeds. However, beech seeds lose germinability during long-term storage. In this study, beech seeds were stored at -10°C under controlled conditions for 2, 5, 8, 11 and 13 years. Our results show that beech seeds lose germinability during storage in proportion to the duration of storage. The decrease in germinability correlated with increased electrolyte leakage and accumulation of superoxide anion radicals, hydrogen peroxide and hydroxyl radicals. Furthermore, a strong positive correlation was observed among the releases of superoxide anion radicals, hydrogen peroxide and hydroxyl radicals. In situ localization showed that superoxide anion radicals and hydrogen peroxide were first detectable in root cap cells. When the seed storage time was extended, the reactive oxygen species fluorescence expanded to more areas of the radicle, reaching the root apical meristem. A storage time-dependent decrease in catalase activity, observed in both embryonic axes and cotyledons, was also positively correlated with germinability. DNA fragmentation was observed in beech seeds during storage and occurred predominantly in embryonic axes stored for 5 years and more. Altogether, these results suggest that the loss of germinability in beech seeds during long-term storage depends on several factors, including strong of reactive oxygen species accumulation accompanied by reduced catalase activity as well as membrane injury and DNA alternations, which may be aging-related and ROS-derived. We suggest that the accumulating reactive oxygen species that spread to the root apical meristem are key factors that affect seed germinability after long-term storage.

ROS production and antioxidative system activity in embryonic axes of Quercus robur seeds under different desiccation rate conditions

The seeds of pedunculate oak (Quercus robur L.) were subjected to slow (S) and rapid (R) desiccation at desiccation rates of 0.16 and 0.39% H2O per hour, respectively. Till ca. 40% water content (WC) the germination capacity of seeds in the S and R variants was high (ca. 100%). Between 40 and 28% WC, germination capacity declined to 20 and 50% in S and R variants, respectively. The decrease in seed viability was accompanied by a significant increase of electrolyte leakage from embryonic axes (28% for S and 15% for R variants). In the embryonic axes of seeds subjected to slow desiccation, malondialdehyde (MDA) and free fatty acid (FFA) contents were significantly higher than those in R variants, indicating greater membrane damage due to lipid peroxidation. The production of ROS (H2O2 and O2·−) was significantly higher in S than in R variants. The low molecular weight antioxidants α-tocopherol, ascorbic acid (ASA), and phenolic compounds indicated different reactions in response to desiccation stress. ASA levels decreased during desiccation to a similar degree in both the S and R variants. A significant decrease of total phenols was observed in R variant, which coincided with a significant increase of guaiacol peroxidase (POX) activity. α-Tocopherol content was significantly higher in the embryonic axes of seeds subjected to rapid drying. The activities of the enzymatic scavengers APX and GR had similar runs and were slightly higher in R variant. The activities of POX and SOD were significantly higher in the embryonic axes of seeds subjected to rapid drying. These results show that rapid dehydration of Q. robur seeds leads to the greater mobilization of antioxidant system in embryonic axes, particularly increased levels of α-tocopherol and POX and SOD activities, in the first stages of water loss. This mobilization has a greater impact on maintenance of higher viability of seeds after drying to lower level of WC.

The role of oxidative stress in determining the level of viability of black poplar (Populus nigra) seeds stored at different temperatures

Black poplar (Populus nigra L.) is one of the most threatened tree species in Europe since up to 99% of its natural habitat has disappeared. Black poplar seeds are characterised by short longevity. It was recently demonstrated that black poplar seeds can be successfully stored at -10°C, -20°C and -196°C for at least 2 years but not at higher temperatures. In the present study, the role of oxidative stress in determining the level of viability of black poplar seeds stored at -196°C, -20°C, -10°C, -3°C and 3°C for 3 months, 1 year and 2 years was monitored. The superoxide anion radicals (O2-•) and hydrogen peroxide (H2O2) increased during storage and had an impact on membrane integrity as determined by changes in the content of fatty acids and phospholipids and increases in electrolyte leakage. The level of non-enzymatic and enzymatic components of the ascorbate-glutathione (AsA-GSH) cycle was also investigated. The level of O2-• was strongly correlated with the level of seed germination after 1 and 2 years of storage. This was accompanied by changes in the redox potential, as well as changes in the content of linoleic acid and phosphatydiloglycerol over the same period of time. In particular, the deleterious effect of H2O2 was observed after 2 years of storage when its accumulation was highly correlated with changes in the composition of fatty acids and phospholipids. Despite increased activity of AsA-GSH cycle enzymes, the level of reducing agents was insufficient and seeds exhibited large increases in the redox potential when stored at -3°C and still higher when stored at 3°C. Overall, the results of the study demonstrate that oxidative stress increases during seed storage, especially at the warmer temperatures and injures seed tissues; resulting in a loss of viability.

Ascorbate and glutathione metabolism during development and desiccation of beech (Fagus sylvatica L.) seeds

The ascorbate-glutathione system was studied during development and maturation of beech (Fagus sylvatica L.) seeds, the classification of which in the orthodox category is controversial. This study revealed an increase in glutathione content after acquisition of desiccation tolerance, which was more intensive in embryonic axes than in cotyledons. During seed maturation, the redox status of glutathione markedly changed toward the more reducing state, especially in cotyledons. Ascorbic acid content decreased during maturation, mostly in cotyledons. Activities of the enzymes of the ascorbate-glutathione cycle—ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2)—were markedly higher in embryonic axes than in cotyledons throughout the study period. In the course of seed maturation, the activities of these enzymes decreased. Importance of the ascorbate-glutathione cycle in desiccation tolerance of beech seeds was discussed in relation to results for typical orthodox and recalcitrant seeds of other broadleaved species.

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.