Function of the ascorbate-glutathione cycle in aged sunflower seeds

The function of the ascorbate-glutathione (AsA/GSH) cycle was analyzed in seeds of sunflower ( Helianthus annuus L. cv. Peredovik) subjected to accelerated ageing at 43°C and 75% relative humidity for 1 to 11 days. The study was performed using dry seeds and seeds hydrated by imbibition in distilled water for 4 h at 25 °C. Lipid peroxidation was also determined by measuring the malondialdehyde (MDA) level. As the ageing period increased, a progressive loss of seed viability became increasingly evident. Even though high levels of MDA were delected, the MDA level did not change during accelerated ageing, suggesting that lipid peroxidation might occur to some extent. The study of the ascorbate/glutathione (AsA/GSH) cycle revealed that the GSH system is the major detoxifying mechanism in both dry and imbibed sunflower seeds. The GSH system is mainly located in the embryo, and its protective role is mediated by reactions that consume the GSH pool and, thereby, minimize the increase of the oxidized form (GSSG). Seed imbibition activates cellular metabolism and allows some antioxidant enzymes like glutathione reductase (EC 1,6,4,2) to act upon toxic agents. These reactions provide a reducing status, so that repair of damage becomes possible. However, prolonged ageing conditions (11 days) result in an irreversible damage, as evidenced by the appearance of dead seeds when the germination period ended. Multiple regression analysis revealed the effectiveness of the GSH system in aged seeds, especially upon imbibition and until the AsA/GSH cycle became completely functional.     

Accelerated ageing and subsequent imbibition affect seed viability and the efficiency of antioxidant system in macaw palm seeds

Accelerated ageing is an accurate test indicator of seed vigor and storability that helps to understand the mechanisms of cellular and biochemical deterioration that occur during seed ageing. This study was carried out to elucidate the mechanisms of ageing in macaw palm embryos. Seeds were artificially aged during 4, 8 and 12 days at 45 °C and 100% relative humidity. After ageing, seeds were tested for viability (tetrazolium), electrical conductivity, lipid peroxidation (MDA) and hydrogen peroxide (H₂O₂) content. Part of the aged seeds was imbibed for 8 days and then determined the hydrogen peroxide content and the activity of antioxidant system enzymes (superoxide dismutase, catalase and glutathione reductase). Ageing reduced the embryo viability from 8 days of treatment and increased malondialdehyde content (MDA) and solute leakage. Hence, membrane permeability correlated with both loss of viability and lipid peroxidation. Imbibition after ageing significantly increased H₂O₂ content along with superoxide dismutase activity. Catalase activity was significantly higher than control in embryos aged from 8 days and imbibed, and glutathione reductase activity did not change. Our results suggest that macaw palm seed deterioration during accelerated ageing is closely related to lipid peroxidation, and that enzymatic antioxidant system is not completely efficient in reducing reactive oxygen species after imbibition, a critical phase to germination. Moreover, accelerated ageing test can be used as a reliable model to understand the mechanisms involved in palm seeds deterioration.     

Free radical scavenging as affected by accelerated ageing and subsequent priming in sunflower seeds

Lipid peroxidation resulting from loss of free radical scavenging is thought to be involved in deterioration of sunflower (Helianthus annuus L.) seeds during accelerated ageing. In other respects, presoaking of seeds in a solution of low water potential (osmopriming) has been demonstrated to reinvigorate aged seeds. The aim of the present work was to study the effect of osmopriming on the germination of aged sunflower seeds and to investigate whether this effect was associated with the restoration of antioxidant defence systems. Seeds were aged for 5 days at 45°C and 100% relative humidity and then primed for various durations up to 7 days at 15°C in a solution of polyethylene glycol 6000 at ?2 MPa. Lipid peroxidation was estimated by measuring malondialdehyde (MDA) and conjugated diene contents, and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) were measured throughout the treatments. Accelerated ageing resulted in a marked decrease in the germination rate, and was associated with an increase in the levels of MDA and conjugated dienes, thus indicating lipid peroxidation. Ageing was also characterized by a decrease in the activities of CAT and GR. The activities of SOD and DHAR were much less altered. No APX activity was detected whatever the seed treatment. Priming of aged seeds progressively restored the initial germinative ability and resulted in a marked decrease in the levels of MDA and conjugated dienes, indicating a fall in lipid peroxidation processes. These effects of priming were also well correlated to the recovery of SOD, CAT and GR activities. Priming treatment for 7 days led to full restoration of the cell detoxifying mechanisms which were strongly altered during ageing. Glutathione content showed the same changes as GR activity. There existed a clear-cut relationship between seed germinative energy, expressed as the germination rate, and the efficiency of free radical scavenging systems, in particular CAT and GR activities and glutathione content. The results suggest that the antioxidant defence systems might play a key role in seed vigour.     

Ageing-induced changes in glutathione system of sunflower seeds

The glutathione system is thought to be involved in defence mechanisms present in plant tissues. The efficacy of this system was evaluated in large seeds of sunflower ( Helianthus annuus L. cv. Peredovik) in response to accelerated ageing (43°C/75% relative humidity from 1 to 11 days). Differences between the embryo axis and cotyledons in relation to the glutathione system were also investigated. Additionally, lipid peroxidation was determined by measuring the malondialdehyde (MDA) content. All assays were performed using dry seeds and seeds subsequently hydrated by imbibition in distilled water for 12 h at 25°C. Accelerated ageing caused a marked decrease in seed viability, accompanied by an increase in mean germination time. There were no changes in total glutathione in dry seeds. However, the distribution in its reduced (GSH) and oxidized (GSSG) forms revealed that ageing produced a slow conversion from GSH to GSSG. As the ageing period increased, this effect was accompanied by a decrease in glutathione reductase (GR, EC 1.6.4.2) activity. The results also indicated that the GSH system exerts a different response in the embryo axis as compared with the cotyledon: (1) the GSH levels decreased less in the cotyledons than in axes of aged seeds, and (2) the GSSG level in cotyledons was independent of ageing, while its amount increased in aged embryo axes. These different responses, in conjunction with the lower MDA levels in large as compared with small seeds, indicate a possible protective role of the reserve lipids. The efficacy of the GSH system in aged seeds was associated with seed viability, as revealed by multiple regression analysis. Upon imbibition, aged seeds were able to restore their GSH levels, reaching values approximating those of unaged seeds.     

Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated aging

Sunflower ( Helianthus annuus L.) seeds progressively lost their ability to germinate at 25°C, the optimal temperature for germination, after accelerated aging was carried out at 45°C (a temperature too high to permit germination) in water or at 76 or 100% relative humidity (RH). The deleterious effects of the high-temperature treatment increased with increasing seed moisture content. Incubation of seeds at 45°C in water resulted in electrolyte leakage, which indicated a loss of membrane integrity. A relationship between leakage and loss of seed viability could not be assumed, since no increase in electrolyte efflux occurred after aging al 100% RH. Accelerated aging induced accumulation of malondialdehyde, suggesting that seed deterioration was associated with lipid peroxidation. However, there was no direct relationship between lipid peroxidation and deterioration in membrane integrity. Loss of seed viability was also associated with a decrease in superoxide dismutase, catalase and glutathione reductase activities. Finally, the results obtained suggest that sunflower seed deterioration during accelerated aging is closely related to a decrease in the activities of detoxifying enzymes and to lipid peroxidation.     

Kinetin-Mediated Prolongation of Viability in Recalcitrant Sal (Shorea robusta Gaertn. f.) Seeds at Low Temperature: Role of Kinetin in Delaying Membrane Deterioration during Desiccation-Induced Injury

The effects of kinetin (6-furfurylaminopurine) on viability during storage of recalcitrant sal (Shorea robusta Gaertn. f.) seeds at low temperature (15°C) were investigated. The freshly mature sal seeds showed an absolute loss of viability within 6–7 dah (days after harvest) when stored at ambient or at 15°C (control). Storage of these seeds at 15°C after kinetin (10 ppm) treatment prolonged the viability period up to 35 days with 20% germination. The kinetin-treated seeds exhibited 100% germination up to 10 days compared with 3 days in controls. Measurements of leachate conductivity, ·O⁻ ₂ and lipid peroxidation registered gradual increases from 0 dah onward to 35 dah with significantly low levels compared with controls. On the other hand, an enormous increase in superoxide dismutase activity was discernible for a longer duration (0–35 dah) in kinetin-treated seeds than in control seeds where it remained for 3 dah. The role of kinetin in prolonging seed viability by reducing the loss of leachates, lipid peroxidation, ·O⁻ ₂, and enhancing of superoxide dismutase is discussed. Received October 7, 1997; accepted January 27, 1998     

电场处理油葵种子后对其萌发期抗旱性的影响

用不同电场条件处理油葵种子,以PEG(聚乙二醇)模拟水分胁迫,通过高渗溶液萌发法、种子吸水率和膜脂过氧化检测法测定电场处理对油葵种子萌发期抗旱性的影响。结果表明,不同处理条件对油葵种子在萌发期对水分胁迫的适应性影响不同。通过电场处理能够提高油葵种子吸水速率及在水分胁迫条件下的发芽势、发芽率;降低水分胁迫对种子细胞膜的伤害,提高种子萌发期体内超氧化物歧化酶、过氧化物醇的活性,减少了膜脂过氧化产物丙二醛的积累。所有这些变化都有利于缓解水分胁迫对油葵种子的伤害,提高其对水分胁迫的适应性。     

Storage elicits a fast antioxidant enzyme activity in <Emphasis Type="Italic">Araucaria angustifolia</Emphasis> embryos

Storage of recalcitrant seeds leads to the initiation of subcellular damage or to the initiation of germination process, and both may result in viability loss. This study aimed to elucidate the biochemical basis of embryos survival of Araucaria angustifolia recalcitrant seeds during storage. After harvesting, seeds were stored at ambient conditions (without temperature and humidity control) and in a cold chamber (temperature of 10 ± 3 °C, and relative humidity of 45 ± 5 %). Moisture content, viability, H₂O₂ content, lipid peroxidation, protein content, and activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), at 0, 15, 45 and 90 days of storage, were evaluated. Seed viability reduced about 40 % during the storage period accompanied by a reduction in soluble protein (about 64 % of reduction) in both storage conditions, and increased lipid peroxidation (about 115 % and 66 % for ambient and cold chamber conditions, respectively). H₂O₂ content used as a marker of oxidative stress was reduced during the period, possibly controlled by the action of CAT and APX, for which increased activities were observed. The results allowed the identification of seven SOD isoenzymes (one Mn-SOD, five Fe-SOD and one Cu/Zn-SOD), whose activities also increased in response to storage. Some biochemical damage resulting from storage was observed, but viability reduction was not due to failure of enzymatic protection mechanisms.     

Lipid peroxidation and peroxide-scavenging in soybean seeds during aging

The possible role of lipid peroxidation in seed deterioration was investigated during natural aging and accelerated aging of seeds of edible soybean ( Glycine max [L], Merr. cv. Kaohsiung Selection No. 1). Natural aging was achieved by sealing the seeds in aluminum foil bags coated with polyethylene and storing the seeds at room temperature for 3 to 12 months. Accelerated aging was obtained by incubating the seeds at 45°C and close to 100% relative humidity for 3 to 12 days, after which the seeds were air dried to their original moisture level (8%). The results indicate that both natural and accelerated aging enhanced lipid peroxidation, as germination was depressed. Aging also inhibited the activity of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase. The changes in germination and physiological activities, expressed as a function of aging duration, were somewhat similar in the two aging treatments.     

Lipid peroxidation and peroxide-scavenging enzymes associated with accelerated aging and hydration of watermelon seeds differing in ploidy

Hydration offers an effective means for raising seed performance in many crop species. The objective of this study was to evaluate the effect of vermiculite hydration on germinability and several physiological activities related to vigor in artificially aged watermelon seeds differing in ploidy. Aging was achieved by incubating the seeds at 45°C and 79% relative humidity for 6 days, then the seeds were air-dried to their original moisture level (4.7%). Hydration was achieved by mixing the untreated and aged seeds with moist vermiculite No. 3 at 25°C for 24 h. The partially hydrated seeds were air-dried at 25°C for 36 h to 4.7% moisture level. Significant differences existed between unaged and aged seeds, with lower germination percentage and slower germination speed in the latter. Aging also increased lipid peroxidation and reduced the activity of peroxide-scavenging enzymes. The germinability of aged watermelon seed was restored partially by vermiculite hydration. The activities of protein synthesis and peroxide-scavenging enzymes in axis and cotyledon portions of the seeds were also increased by hydration treatment. The changes in germination and related physiological responses in relation to aging and hydration are similar in seeds differing in ploidy, despite differences in their germination performance, seed leakage, extent of lipid peroxidation and activities of peroxide-scavenging enzymes.     

The effects of desiccation on seeds of Acer saccharinum and Aesculus pavia: recalcitrance in temperate tree seeds

This study was undertaken to determine how the results from lipid, moisture, and differential scanning calorimetry analyses conducted on silver maple (Aceraceae: Acer saccharinum L.) and red buckeye (Hippocastanaceae: Aesculus pavia L.) compared with those obtained from previous studies on white and water oaks (Fagaceae: Quercus alba and Q. nigra), and the tropical zone species American muskwood (Meliaceae: Guarea guidonia) and carapa (Meliaceae: Carapa guianensis). Seeds were air-dried at room temperature for 9-11 days. At intervals, germination was tested, moisture determined, and lipids extracted. It was found that, like the other recalcitrant seeds, (1) viability was greatly reduced or lost after 11 days of drying, (2) percentage changes in individual fatty acids were not related to seed viability, and (3) results from the differential scanning calorimetry studies revealed a strong relationship between enthalpy/onset data from the embryo and cotyledon tissues and loss of viability. Also, silver maple seeds experienced a 50% reduction in viability by day 5 of drying and retained an axis moisture content over 25% throughout the experiment. However, unlike the other recalcitrant seeds surveyed, both silver maple and red buckeye had a significant reduction in the total amount (mg/g) of cotyledon lipids as the experiment progressed. However, no decrease in the unsaturated/saturated fatty acid ratio was found, so we conclude that in these species lipid peroxidation is not a marker of declining seed viability. Also, red buckeye seeds did not lose 50% viability until after day 8 of the experiment, and axis moisture content fell well below 20% as the seeds dried.     

Desiccation-induced changes in lipid peroxidation, superoxide level and antioxidant enzymes activity in neem (Azadirachta indica A. Juss) seeds

The freshly harvested mature neem seeds (42.2 % seed moisture content) with 100 % viability deteriorate when naturally desiccated to below 10.9 %. The desiccation-induced loss of viability was closely associated with over accumulation of superoxide anion and lipid peroxidation products both in the embryonic axes and cotyledons. The levels of superoxide anion and lipid peroxidation products were higher in axes compared to cotyledons. Superoxide dismutase activity was not much affected, both in the axes and cotyledons of 100 % viable seeds during desiccation from 42.2 % to 10.9 % seed moisture content. Steep rise in its activity was observed during drying below lowest safe moisture content (LSMC). Activities of catalase and peroxidase exhibited substantially higher levels in the 100 % viable seeds dehydrated up to LSMC. Their activities declined sharply in seeds with water content below LSMC. Impairment of catalase and peroxidase activities possibly lead to enhanced accumulation of reactive oxygen species. The accumulation of superoxide anion, lipid peroxidation and differential expression of superoxide dismutase and catalse/peroxidase activities in response to desiccation (below LSMC) is discussed to explain the intermediate storage physiology of neem seeds.     

Lipid peroxidation and peroxide-scavenging enzymes associated with accelerated aging of peanut seed

Accelerated aging is known to reduce seed viability and vigor in many crop species. The phenomenon is due in part to aging-induced lipid peroxidation, which has the potential to damage membranes of the seed tissues. This study was undertaken to evaluate the effect of accelerated aging on germinability and several physiological characteristics related to peroxidation in the seed of two peanut cultivars. Accelerated aging was achieved by incubating seed at 45°C and 79% relative humidity in a closed chamber for 3, 6, or 9 days. The results indicate that accelerated aging inhibited seed germination and seedling growth. Enhanced lipid peroxidation and increased peroxide accumulation were observed in the axis and cotyledons of aged seed. Accelerated aging also inhibited the activity of superoxide dismutase, peroxidase, ascorbate peroxidase, and lipoxygenase. Seed axes appeared to be more susceptible to aging than cotyledons. The changes in germination and physiological activities, expressed as a function of aging duration, were similar in the two cultivars, despite differences in their seed weight.     

Lipid peroxidation associated with accelerated aging of soybean axes

Soybean seeds age rapidly during storage at high temperature and high relative humidity. The axes of such aged seeds contain high levels of malondialdehyde, a product of the peroxidation of unsaturated fatty acids. The levels of linoleic (18:2) and linolenic (18:3) acids in a polar lipid (phospholipid) fraction decrease during aging and more dramatically during postaging deterioration. None of these changes occurred in seeds that have been stored at high temperature but low relative humidity. No superoxide dismutase activity was detected in any nonimbibed seed. In viable seeds, activity was detectable 1.5 hours after the onset of imbibition, but none was found in aged seeds up to 5 hours. It is suggested that aging leads to peroxidative changes to lipids and that these could contribute to loss of viability.     

Amelioration of the effects of ageing in onion seeds by osmotic priming and associated changes in oxidative metabolism

Osmotic priming of aged onion seeds with 25% polyethylene glycol-8000 for 5 d resulted in a marked increase in the rate of germination and early seedling growth. Priming reduced electrolyte leakage as well as lipid peroxidation in seeds implying the activation of membrane repair processes. Priming was also associated with increased levels of antioxidants,i.e. ascorbic acid and tocopherols particularly the latter and the activities of catalase and peroxidase involved in the mitigation of oxidative damage. In comparison with the priming of unaged seeds, the aged seeds experienced a diminution of response in terms of changes in the levels of antioxidants and scavenging enzymes.     

Decrease in beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis>) 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.     

Decrease in sunflower (Helianthus annuus) seed viability caused by high temperature as related to energy metabolism, membrane damage and lipid composition

The aim of the present work was to investigate whether loss of germination ability and viability of sunflower (Helianthus annuus L.) seeds during incubation at a high temperature (45°C) was related to changes in energy metabolism, loss of membrane integrity, and/or changes in lipid composition. Pre‐treatment of seeds at 45°C progressively reduced subsequent germination at the optimal temperature (25°C). Seeds did not germinate at 45°C and almost all of them were dead after 72 h of soaking at this high temperature. This loss of seed viability was associated with a large increase in leakage of K⁺ and total electrolytes into the incubation medium, and with production of malondialdehyde in the embryonic axis and cotyledons, suggesting a loss of membrane integrity probably due to lipid peroxidation. ATP and ADP levels increased sharply during the first hours of imbibition at 45°C, remained high for about 24 h and then decreased. As a consequence, the energy charge followed a similar pattern. If the treatment at 45°C did not exceed 48 h, seeds recovered an apparently normal energy metabolism after transfer to 25°C, even though they lost their ability to germinate at this temperature. Therefore, energy metabolism at the whole embryo level cannot be considered as an indicator of germination ability. Incubation of seeds at 45°C resulted in an increase in triacylglycerols and diacylglycerols without a significant change in their fatty acid composition. It also induced a slight increase in phospholipid content with an increase in C_16:0, C_18:0 and C_18:1, but with no change in C_18:2. In phospholipids, the C_18:2/C_18:1 and (C_18:1 + C_18:2)/ (C_16:0 + C_18:0) ratios thus declined during treatment at 45°C. The results obtained suggest that deterioration of sunflower seeds during incubation at a high temperature is mainly related to membrane damage and alteration of energy metabolism, and that accumulation of malondialdehyde, which is an index of lipid peroxidation, does not correspond to a decrease in total lipids and phospholipids nor to a significant change in fatty acid composition, except in PL in which the C_18:2/C_18:1 and (C_18:1 + C_18:2)/ (C_16:0 + C_18:0) ratios slightly declined.     

Age-related Changes in the Ultrastructure and Membrane Properties of Brassica napus L. Seeds

The ultrastructure was studied of imbibed non-aged winter rape(Brassica napus L.) seeds in comparison with that of artificiallyaged seeds in which viability was partially or completely impaired.In parallel, measurements were made of lipid-phosphorus content,the leakage of phosphate from the seeds and their vigour andgerminability. Decreases in lipid-phosphorus which accompaniedthe loss of viability corresponded to an increase in phosphateleakage, suggesting damage to cellular membranes. Three ultrastructuralsymptoms possibly related to age-induced membrane deteriorationwere observed: (i) the lowering of electron contrast in allcellular membranes excluding plasmalemma; (ii) coalescence ofsmall storage lipid bodies to larger units presumably as a resultof the degradation of enclosing half-unit membranes; and (iii)the appearance of protoplasmic inclusions inside the storageprotein bodies, possibly resulting from rupture of the enclosingunit membranes. It is suggested that the presence of enlarged fibrillar centresin nucleoli of low viability seeds observed here for the firsttime in aged seed material may be the morphological manifestationof age-induced damage to nucleic acids. Brassica napus L, seeds, accelerated ageing, ultrastructure, leakage     

RNases and nucleases in embryos and endosperms from naturally aged wheat seeds stored in different conditions

Temperature and moisture content are particularly important factors influencing the longevity of seeds, and therefore the ageing of seeds is closely tied to storage conditions. The ageing process is characterised by many physiological and biochemical changes: membranes tend to leak, enzymes lose catalytic activity, and chromosomes accumulate mutations. Since viability loss is also associated with the breakdown of nucleic acids, the aim of the study was to determine whether the damage induced by ageing could be associated with changes in the activity of RNases and nucleases in embryos and endosperms of differently stored wheat seeds. In order to better characterise seed conditions, the damage to membranes during seed ageing was evaluated by measuring the conductivity of the soaking solution during imbibition, and by using the Evans Blue colorant; lipid peroxidation was also recorded. RNases and nucleases were studied by SDS-PAGE and activity staining. Ageing of seeds stored in a dry state involved a progressive loss of membrane integrity, which increased with the degree of ageing, while lipid peroxidation remained unchanged. Changes in nucleolytic enzyme activity were recorded in embryos: a decrease in RNases and an increase in nucleases. In the endosperm compartment there were no significant differences in ribonuclease and nuclease patterns during seed ageing. Moreover, neutral RNases were absent in endosperms of dry seeds and were activated following imbibition. Present studies reveal that embryos and endosperms have different enzymatic patterns, thus highlighting that the two seed compartments age independently. A different nucleolytic pattern was present in seeds of comparable viability and membrane damage, which were stored differently, and nuclease metabolism was subject to regulation according to both ageing and the length of the storage period.