Stanisława Pukacka

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.

Water and lipid relations in beech (Fagus sylvatica L.) seeds and its effect on storage behaviour

Beech (Fagus sylvatica L.) seeds indicate intermediate storage behaviour. Properties of water in seed tissues were studied to understand their requirements during storage conditions. Water sorption isotherms showed that at the same relative humidity (RH) the water content is significantly higher in embryo axes than cotyledons. This tendency maintains also after recalculating the water content for zero amount of lipids in tissues. Differential thermal analysis (DTA) indicated water crystallization exotherms in the embryo axes at moisture content (MC) higher than 29% and 16% in the cotyledons. In order to examine the occurrence of glassy state in the cytoplasm of beech embryos as a function of water content, isolated embryo axes were examined using electron spin resonance (ESR) of nitroxide TEMPO probe located inside axes cells. TEMPO molecules undergo fast reorientations with correlation time varied from 2 x 10(-9) s at 180 K to 2 x 10(-11) s at 315 K. Although the TEMPO molecules label mainly the lipid bilayers of cell membranes, they are sensitive to the dynamics and phase transformation of the cytoplasmic cell interior. The label motion is clearly affected by a transition between liquid and glassy state of the cytoplasm. The glass transition temperature (T(g)) raises from 253 to 293 K when water content decreases from 18% to 8%. Far from T(g) the motion is described by Arrhenius equation with very small activation energy E(a) in the liquid state and is relatively small in the glassy state where E(a)=1.5 kJ/mol for 28% H(2)O and E(a)=4.7 kJ/mol for 8% H(2)O or less. The optimal storage conditions of beech seeds are proposed in the range from 255 K for 15% H(2)O to 280 K for 9% H(2)O.

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.

Lipid and protein accumulation in developing seeds of three lupine species: Lupinus luteus L., Lupinus albus L., and Lupinus mutabilis Sweet

A comparative study was carried out on the dynamics of lipid accumulation in developing seeds of three lupine species. Lupine seeds differ in lipid content; yellow lupine (Lupinus luteus L.) seeds contain about 6%, white lupine (Lupinus albus L.) 7–14%, and Andean lupine (Lupinus mutabilis Sweet) about 20% of lipids by dry mass. Cotyledons from developing seeds were isolated and cultured in vitro for 96 h on Heller medium with 60 mM sucrose (+S) or without sucrose (–S). Each medium was additionally enriched with 35 mM asparagine or 35 mM NaNO3. Asparagine caused an increase in protein accumulation and simultaneously decreased the lipid content, but nitrate increased accumulation of both protein and lipid. Experiments with [1-14C]acetate and [2-14C]acetate showed that the decrease in lipid accumulation in developing lupine seeds resulted from exhaustion of lipid precursors rather than from degradation or modification of the enzymatic apparatus. The carbon atom from the C-1 position of acetate was liberated mainly as CO2, whereas the carbon atom from the C-2 position was preferentially used in anabolic pathways. The dominant phospholipid in the investigated lupine seed storage organs was phosphatidylcholine. The main fatty acid in yellow lupine cotyledons was linoleic acid, in white lupine it was oleic acid, and in Andean lupine it was both linoleic and oleic acids. The relationship between stimulation of lipid and protein accumulation by nitrate in developing lupine cotyledons and enhanced carbon flux through glycolysis caused by the inorganic nitrogen form is discussed.

Carbohydrate metabolism in Norway maple and sycamore seeds in relation to desiccation tolerance

Summary The work reports on sucrose and oligosaccharide metabolism in Norway maple (Acer platanoides L.) and sycamore (Acer pseudoplatanus L.) embryo axes and cotyledons during seed maturation. Enzyme activities of sucrose synthase (SuS), sucrose phosphate synthase (SPS), acid and alkaline invertases, α-galactosidase and galactinol synthase (GS) were studied. These species are closely related, but differ with regard to seed tolerance to desiccation. Norway maple possesses strictly orthodox seeds, while sycamore seeds are recalcitrant. In contrast to sycamore, maturation of Norway maple seeds was associated with an increase in raffinose and stachyose concentrations and a decrease in sucrose to oligosaccharides ratios. Activities of SuS and SPS as well as α-galactosidase were higher in seeds of sycamore compared to Norway maple. The activity of GS in Norway maple seeds was higher than in sycamore and was coincident with increased level of raffinose and stachyose in mature seeds. The results confirmed a finding of increased sucrose metabolism during maturation in recalcitrant sycamore seeds compared to orthodox Norway maple seeds.

Seasonal changes in antioxidant level of Scots pine (Pinus sylvestris L.) needles exposed to industrial pollution. I. Ascorbate and thiol content.

Current and previous year needles from three 16 years-old populations of Scots pine (Pinus sylvestris L.) trees were seasonally collected at the three experimental areas: Luboń- close to the phosphate fertiliser factory, Głogów — close to the copper foundry and Kórnik — control site. Głogów is the most polluted site, where at 1998 monthly mean daily concentrations of different pollutants were: SO2 - 17 µg·m−3, NOx - 12 µg·m−3 and dust containing heavy metals as Cu, Pb, Cd - 29 µg·m−3. Trees growing in Luboń were influenced for many years by high concentration of SO2 and fluor compounds. A few years ago emissions were markedly reduced, but low pH of soil and high concentration of aluminium ions still influence the growth of trees. Seasonal changes of ascorbate and thiol content were observed in each needle class and population, with the maximum in the winter and minimum in the summer. In needles from trees growing on polluted sites higher level of ascorbic acid and thiols comparing to control site was observed. Significant differences appeared in each population of Scots pine growing under higher pollution stress in the Głogów site. In needles from trees growing in Luboń significant differences in ascorbic acid and thiols content were evidently less numerous. Needles from polluted sites in some seasons contained significantly more malondialdehyde (MDA) and those was more frequent in Głogów than in Luboń. The results indicated that in the Głogów site trees are more influenced by pollution stress than in Luboń and the defense reaction measured as an increase of the antioxidant level is more evident.

Seasonal changes in antioxidant level of Scots pine (Pinus sylvestris L.) needles exposed to industrial pollution. II. Enzymatic scavengers activities.

The involvement of enzymic antioxidant system, superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase in defense reaction to environmental stress evoked by air and soil pollution, was seasonally studied on three populations of Scots pine (Pinus sylvestris L.) growing on experimental areas close two industrial objects in Poland. The first of them (Luboón) is localised near a phosphate fertiliser factory, the second (Głogów) near a copper foundry, and control stand is placed in Kórnik. Głogów is the most polluted site, where in 1998 monthly mean daily concentrations was: SO2 - 17 µg·m−3, NOx- 12 µg·m−3 and dust containing heavy metals (Cu, Pb, Cd) - 29 µg·m−3. Trees in Luboń were influenced for many years by high concentration of SO2 and fluor compounds. Few years ago emissions were markedly reduced, but changes in the soil (low pH and high concentration of aluminium ions) still influence the growth of trees. In needles of two populations: 3 (Russia) and 8 (Poland), from the polluted sites Głogów and Luboń, activities of superoxide dismutase (SOD) and guaiacol peroxidase (PO) were significantly higher compared to Kórnik. However, in one population (16 - Slovakia), such dependance was not evident. Activity of ascorbate peroxidase (AP) measured in winter was also higher in needles from polluted sites. The results indicated that the sensitivity of free radical scavenging system in Scots pine needles differs among populations.

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.