Comparative study of storage compound breakdown in germinating seeds of three lupine species

Storage lipid and protein breakdown in germinating seeds of yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupine (L. mutabilis Sweet) and regulatory function of sucrose were investigated. Less oil bodies were detected in organs of yellow lupine seeds, whereas the highest content of oil bodies was noticed in the Andean lupine seeds. Mature, air-dried yellow, white and Andean lupine seeds do not contain starch. Starch grains appear the earliest in white lupine seeds during imbibition. Sucrose deficiency in tissues enhances breakdown of storage lipid, protein and temporary starch in cotyledons. In sucrose starved embryo axes of all investigated lupine species, an increased level of vacuolization was noted. Interconnections between catabolism of storage protein and storage lipid in germinating lupine seeds were identified by applying ¹⁴C-acetate. To assess the importance of key processes in storage lipid breakdown NaF (inhibitor of glycolysis and gluconeogenesis), KCN, NaN₃ and SHAM (inhibitors of mitochondrial electron transport chain) and MSO (inhibitor of glutamine synthetase) were used. Radioactivity coming from ¹⁴C-acetate was released as ¹⁴CO₂ but mostly was incorporated into ethanol-soluble fraction of embryo axes and cotyledons. Respiratory inhibitors caused a significant decrease in ¹⁴CO₂ and ethanol fractions in all three lupine species studied. MSO stimulated release of ¹⁴CO₂ and radioactivity of ethanol fractions in yellow lupine organs fed with sucrose, but in Andean lupine MSO enhanced the production of ¹⁴CO₂ and radioactivity of ethanol fractions both in organs fed and not fed with sucrose. Different strategies of storage compound breakdown are proposed, depending on relative proportion in storage protein and lipid content in lupine seeds.     

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 (10 mg/L) or water for 6 h. 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.     

Factors influencing the storability of Fagus sylvatica L. seeds after release from dormancy

Seeds of beech (Fagus sylvatica L.) that have been subjected to dormancy breaking consisting of 10 weeks of prechilling at 3 °C and 34 % water content (WC) and then desiccation to 10 % WC, are non-dormant (ND). ND seeds are characterised by greater sensitivity to storage conditions, than no prechilled, dormant (D) seeds. The aim of the present work was to investigate factors affecting the loss of seed viability during storage of D and ND beech seeds at different temperatures (4 and 20 °C) and humidity levels (45 and 75 % RH) for 3 weeks. In general, both D and ND seeds maintained a high germination capacity after storage at 4 °C. At 20 °C and 45 and 75 % RH the germination capacity of D seeds diminished to 80 and 28 %, respectively. Under the same conditions, ND seeds lost germination capacity to a greater degree, with only 62 and 7 % germinated seeds, respectively. At 20 °C, an increase in production of reactive oxygen species was observed, and the increase was significantly higher in ND seeds. The loss of germination capacity was coincident with an increase in electrolyte leakage and accumulation of free fatty acids, which suggests that membrane deterioration was the cause of the decline in germinability. ND seeds stored at 20 °C and 45 and 75 % RH showed a greater decrease than D seeds in contents of the primary phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as well as in polyunsaturated fatty acids (18:2 and 18:3). ND seeds possessed more unsaturated fatty acids, especially 18:3, than D seeds in the phospholipid fraction before storage. D seeds were characterised by a significantly higher level of α-tocopherol and UV-absorbing phenols. The level of ascorbate was similar in D and ND seeds. D seeds contained glutathione in both reduced (GSH) and oxidised (GSSG) forms, and GSSG dominated GSH. ND seeds contained more GSSG form than D seeds. We concluded that the membranes of ND seeds are exposed to greater oxidative stress during storage due to higher levels of unsaturation and lower levels of α-tocopherol, the main antioxidant that protects membranes against free radical attack.     

Selective Reconstitution of the Tonoplast H+-ATPase of the Crassulacean-Acid Metabolism Plant Kalanchoë daigremontiana

IA detergent removal technique was used to reconstitute solubilized tonoplast proteins of mesophyll cells of the CAM plant Kalanchoë daigremontiana into phosphatidylcholine liposomes. The proteoliposomes were able to hydrolyse ATP and to pump protons across the vesicle membrane. Both activities were inhibited by nitrate, an inhibitor of V-type ATPases. Freeze-fracture micrographs confirmed the incorporation of membrane proteins into liposomes. Increase of specific ATP-hydrolysis activity compared to solubilized tonoplast proteins and SDS-PAGE analysis of reconstituted proteins in comparison with the polypeptide pattern of the purified tonoplast H⁺-ATPase from the same plant source indicated a highly selective reconstitution of the tonoplast H⁺-ATPase.     

Interaction of bovine estrogen receptor with immobilized zinc

The metal-binding properties of partially purified untransformed or salt-dissociated bovine estrogen receptors were studied using zinc-chelated iminodiacetic acid gels. Only the salt-dissociated 5S receptor is retained by the metal-chelated resin, and this interaction is dependent on the presence of dithiothreitol. The untransformed 9S receptor is not retained, indicating that the zinc-interacting amino acid residues may be masked by receptor-associated proteins such as 90K heat-shock protein or because of an unfavorable receptor conformation.     

Metabolic transformations of some ent-kaurenes in Gibberella fujikuroi

The conversion of ent-kaur-16-enes to gibberellic acid in Gibberella fujikuroi is blocked by A-ring modifications. Thus ent-3β-hydroxykaur-16-en-19-yl succinate gives good conversion (46%) to the 7β-hydroxy derivative.* Under the same conditions the 3β-epimer gives 7β- or 6α-hydroxylation and the former occurs for the 3-oxo analogue. The succinoyloxy function acts as a less efficient block and ent-kaur-16-en-19-yl succinate is converted to 7β-hydroxy and 6β,7β-dihydroxy derivatives along with gibberellic acid. Hydrolysis of the succinate block of the metabolities provides the 7β, 19-diol and 6β,7β, 19-triol. Of this pair only the former was effectively metabolized to gibberellic acid in G. fujikuroi.