Ewa Marzena Kalemba

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.

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.

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.

Invasive oaks escape pre-dispersal insect seed predation and trap enemies in their seeds

Species introduced to habitats outside their native range often escape control by their natural enemies. Besides competing with native species, an alien species might also affect the native herbivores by introducing a new source of different quality food. Here, we describe the case of northern red oak (Quercus rubra) invasion in Europe. We collected data on insect (moth Cydia spp. and weevil Curculio spp.) seed predation of northern red oak in its native (USA, North America) and invasive (Poland, Europe) range, as well as for sessile oaks (Quercus petrea) in Europe. We also evaluated the quality of acorns as hosts for weevil larvae by collecting infested acorns and measuring weevil developmental success, and quantifying acorn traits such as seed mass, tannins, lipids and protein concentration. We used DNA barcoding to identify insects to the species level. The predation by moths was similar and very low in both species and in both ranges. However, red oaks escape pre-dispersal seed predation by weevils in Europe. Weevil infestation rates of northern red oak acorns in their invasive range were 10 times lower than that of sessile oaks, and also 10 times lower than that of red oaks in North America. Furthermore, even when weevils oviposited into northern red oaks, the larvae failed to develop, suggesting that the exotic host created a trap for the insect. This phenomenon might gradually decrease the local abundance of the seed predator, and further aid the invasion.

The involvement of the mitochondrial peroxiredoxin PRXIIF in defining physiological differences between orthodox and recalcitrant seeds of two Acer species

Norway maple (Acer platanoides L., orthodox) and sycamore (Acer pseudoplatanus L., recalcitrant) belong to the same genus and grow under similar climatic conditions, but their seeds differ in their tolerance to desiccation. The initial water content (WC) of the seeds used in this study was 50%, and they were dried to 40, 20 and 7%. The mitochondrial peroxiredoxin IIF (PRXIIF) was identified in seeds of both species by immunoblotting. Semiquantitative RT-PCR analyses indicated that the transcript level of PRXIIF in both types of seeds increased during different stages of desiccation and was higher in seeds of Norway maple than in sycamore. General proteome analyses showed important differences between orthodox and recalcitrant seeds. In sycamore seeds that had been desiccated to a 7% WC, the number of protein spots and the levels of those spots were lower than in desiccation-tolerant Norway maple seeds. Post-translational modifications of PRXIIF in seeds at a 50% WC were detected via 2D electrophoresis and subsequent western blot analysis. The detected shift in the pI values (± 0.3) in A. pseudoplatanus was possibly caused by phosphorylation because several potential phosphorylation sites were predicted in silico for that protein. The gene and amino acid sequences were obtained and aligned with known sequences of other plant PRXIIF genes and proteins. High values of sequence identity were noted between the PRXIIF protein sequences of Acer species, Populus trichocarpa Torr. & A. Gray and Arabidopsis thaliana (L.) Heynh. The involvement of PRXIIF in defining the physiological differences between desiccation-tolerant and desiccation-sensitive Acer seeds is discussed in the context of its role in mitochondrial redox homeostasis.

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.

Nitrate simultaneously enhances lipid and protein accumulation in developing yellow lupin cotyledons cultured in vitro, but not under field conditions

The research was conducted on yellow lupin (Lupinus luteus L.) mature seeds, developing cotyledons, developing pods, and seedlings. The main storage compound in yellow lupin seeds is protein, whose content may reach up to 45%. Oil content in seeds of yellow lupin is about 6%. In such protein-storing seeds there is a strong negative relationship between accumulation of storage lipid and protein. An increase in protein content causes a decrease in lipid level, and vice versa. However, simultaneous increase in lipid and protein content is possible in developing lupin cotyledons (the main storage organs of lupin seeds) cultured in vitro. Such an effect was obtained by feeding the cotyledons with nitrate (35mM). The same positive relationship in storage lipid and protein accumulation was also obtained in developing lupin pods fed with nitrate (35mM), detached from the mother plant, and maintained under quasi in vitro conditions. Fertilization of lupin plants with nitrate under field conditions (40 or 80kgNha-1 applied before sowing, at the nodulation stage or at the flowering and pod formation stage) did not cause significant changes in lipid and protein contents in mature seeds. Experiments performed on lupin seedlings cultivated hydroponically showed that nitrate added to the medium was accumulated mainly in roots, and at a remarkably lower level in shoots. We hypothesize that the lack of stimulatory effect of nitrate on storage lipid and protein accumulation in seeds under field conditions is due to inefficient transport of nitrate from the root to developing pods in lupin plants. This causes that the level of nitrate inside the developing lupin seeds is not elevated under field conditions.

Age-related changes in protein metabolism of beech (Fagus sylvatica L.) seeds during alleviation of dormancy and in the early stage of germination

The long-term storage of seeds generally reduces their viability and vigour. The aim of this work was to evaluate the effect of long-term storage on beech (Fagus sylvatica L.) seeds at optimal conditions, over 9 years, on the total and soluble protein levels and activity of proteolytic enzymes, including endopeptidases, carboxypeptidases and aminopeptidases, as well as free amino acid levels and protein synthesis, in dry seeds, after imbibition and during cold stratification leading to dormancy release and germination. The same analyses were conducted in parallel on seeds gathered from the same tree in the running growing season and stored under the same conditions for only 3 months. The results showed that germination capacity decreased from 100% in freshly harvested seeds to 75% in seeds stored for 9 years. The levels of total and soluble proteins were highest in freshly harvested seeds and decreased significantly during storage, these proportions were retained during cold stratification and germination of seeds. Significant differences between freshly harvested and stored seeds were observed in the activities of proteolytic enzymes, including endopeptidases, aminopeptidases and carboxypeptidases, and in the levels of free amino acids. The neosynthesis of proteins during dormancy release and in the early stage of seed germination was significantly weaker in stored seeds. These results confirm the importance of protein metabolism for seed viability and the consequences of its reduction during seed ageing.