ABSTRACT
BACKGROUND: European beech (Fagus sylvatica L.) trees produce seeds irregularly; therefore, it is necessary to store beech seeds for forestation. Despite the acquisition of desiccation tolerance during development, beech seeds are classified as intermediate because they lose viability during long-term storage faster than typical orthodox seeds. In this study, beech seeds stored for short (3 years) or long (20 years) periods under optimal conditions and displaying 92 and 30% germination capacity, respectively, were compared. RESULTS: Aged seeds displayed increased membrane damage, manifested as electrolyte leakage and lipid peroxidation levels. Analyses have been based on embryonic axes, which contained higher levels of reactive oxygen species (ROS) and higher levels of protein-bound methionine sulfoxide (MetO) in aged seeds. Using label-free quantitative proteomics, 3,949 proteins were identified, of which 2,442 were reliably quantified pointing to 24 more abundant proteins and 35 less abundant proteins in beech seeds under long-term storage conditions. Functional analyses based on gene ontology annotations revealed that nucleic acid binding activity (molecular function), ribosome organization or biogenesis and transmembrane transport (cellular processes), translational proteins (protein class) and membranous anatomical entities (cellular compartment) were affected in aged seeds. To verify whether MetO, the oxidative posttranslational modification of proteins that can be reversed via the action of methionine sulfoxide reductase (Msr) enzymes, is involved in the aging of beech seeds, we identified and quantified 226 MetO-containing proteins, among which 9 and 19 exhibited significantly up- and downregulated MetO levels, respectively, in beech seeds under long-term storage conditions. Several Msr isoforms were identified and recognized as MsrA1-like, MsrA4, MsrB5 and MsrB5-like in beech seeds. Only MsrA1-like displayed decreased abundance in aged seeds. CONCLUSIONS: We demonstrated that the loss of membrane integrity reflected in the elevated abundance of membrane proteins had a higher impact on seed aging progress than the MetO/Msr system. Proteome analyses enabled us to propose protein Sec61 and glyceraldehyde-3-phosphate dehydrogenase as potential longevity modulators in beech seeds.
Subject(s)
Fagus , Methionine , Plant Proteins , Proteomics , Seeds , Fagus/metabolism , Methionine/metabolism , Methionine/analogs & derivatives , Seeds/metabolism , Plant Proteins/metabolism , Plant Proteins/genetics , Germination , Reactive Oxygen Species/metabolism , Gene Expression Regulation, PlantABSTRACT
The conservation of the genetic resources of old trees is crucial to their ecological role but is extremely difficult, especially for oak species (Quercus spp.) displaying recalcitrance in seed and vegetative propagation methods. Our study aimed to assess the regenerative potential of Quercus robur trees of different ages (up to 800 years) during micropropagation. We also aimed to determine how in vitro conditions can influence in vitro regeneration responses. Lignified branches collected from 67 selected trees were cultivated ex vitro in culture pots at 25 °C to obtain epicormic shoots (explant sources). The explants were cultivated on an agar medium supplemented with 0.8 mg L-1 6-benzylaminopurine (BAP) for at least 21 months. In a second experiment, two different shoot multiplication conditions (temporary immersion-RITA® bioreactor and agar medium) and two culture medium formulations (Woody Plant Medium and modified Quoirin and Lepoivre medium) were tested. The results showed that the mean length of the epicormic shoots obtained in a pot culture was a function of donor age and was similar among the group of younger trees (ca. 20-200 years), and varied between older trees (ca. 300-800 years). The efficiency of in vitro shoot multiplication strictly depended on the genotype. A sustainable in vitro culture (defined as survival after 6 months) was only possible for half of the tested old donor trees, even when they survived the first month of in vitro growth. A continuous monthly increase in the number of in vitro cultured shoots was reported in younger oaks and in some old oaks. We found a significant effect of the culture system and the macro- and micronutrient composition on in vitro shoot growth. This is the first report demonstrating that the in vitro culture can be successfully applied to the propagation of even 800-year-old pedunculate oak trees.
ABSTRACT
Norway maple and sycamore, two Acer genus species, have an important ecological value and different sensitivity to stressing factors being currently aggravated by climate change. Seedling growth is postulated to be the main barrier for successful plant establishment under the climate change scenarios. Therefore, the differences in redox regulation during the seedling performance of Norway maple and sycamore were investigated. Seeds of the two Acer species exhibited an identical high germination capacity, whereas seedling emergence was higher in sycamores. PCA analyses revealed that there is more diversification in the leaf characteristics than roots. Norway maple displayed a higher chlorophyll content index (CCI) with a similar leaf mass whereas sycamore seedlings exhibited a higher normalized difference vegetation index (NDVI), higher water content, higher root biomass and higher shoot height. Based on NDVI, sycamore seedlings appeared as very healthy plants, whereas Norway maple seedlings displayed a moderate healthy phenotype. Therefore, redox basis of seedling performance was investigated. The total pool of glutathione was four times higher in sycamore leaves than in Norway maple leaves and was reflected in highly reduced half-cell reduction potential of glutathione. Sycamore leaves contained more ascorbate because the content of its reduced form (AsA) was twice as high as in Norway maple. Therefore, the AsA/DHA ratio was balanced in sycamore leaves, reaching 1, and was halved in Norway maple leaves. Nicotinamide adenine dinucleotide phosphate content was twice as high in sycamore leaves than in Norway maples; however, its reduced form (NADPH) was predominant in Norway maple seedlings. Norway maple leaves exhibited the highest anabolic and catabolic redox charge. The higher reduction capacity and the activity of NADPH-dependent reductases in Norway maple leaves possibly resulted in higher CCI, whereas the larger root system contributed to higher NDVI in sycamore. The different methods of controlling redox parameters in Acer seedlings grown at controlled conditions provided here can be useful in understanding how tree species can cope with a changing environment in the future.
Subject(s)
Acer , Seedlings , Acer/chemistry , Acer/physiology , NADP/analysis , NADP/metabolism , Oxidation-Reduction , Glutathione/metabolism , Plant Leaves/metabolismABSTRACT
Pedunculate oak (Quercus robur L.) is an economically important forest-forming species in Poland that produces seeds that are sensitive to desiccation; therefore, short-lived seeds are classified as recalcitrant. Such seeds display active metabolism throughout storage. Acorns stored under controlled conditions (moisture content of 40%, temperature -3 °C) maintain viability for up to 1.5-2 years. Meanwhile, oaks only produce large numbers of seeds every few years during so-called mast years. This results in a scarcity of good-quality seeds for continuous nursery production and restoration. The recalcitrant storage behavior and the requirements of foresters make it necessary to develop a new protocol for longer acorn storage at lower temperatures. Two storage temperatures were tested: -3 °C (currently used in forest practice) and -7 °C. Our results showed that acorns stored for six months exhibited deterioration and reduced germination capacity, as well as reduced seedling performance, particularly when acorns were stored at -7 °C. To elucidate the decrease in quality during storage, an untargeted metabolomics study was performed for the first time and supported with the analysis of carbohydrates and percentages of carbon (C) and nitrogen (N). Embryonic axes were characterized by a lower C:N ratio and higher hydration. A total of 1985 metabolites were detected, and 303 were successfully identified and quantified, revealing 44 known metabolites that displayed significantly up- or downregulated abundance. We demonstrated for the first time that the significant deterioration of seed germination potential, particularly in seeds stored at -7 °C, was accompanied by an increased abundance of phenolic compounds and carbohydrates but also amino acids and phosphorylated monosaccharides, particularly in the embryonic axes. The increased abundance of defense-related metabolites (1,2,4-Benzenetriol; BTO), products of ascorbic acid degradation (threonic and isothreonic acid), as well as antifreezing compounds (sugar alcohols, predominantly threitol), was reported in seed stored at -7 °C. We hypothesize that seed deterioration was caused by freezing stress experienced during six months of storage at -7 °C, a decline in antioxidative potential and the unsuccessful rerouting of the energy-production pathways. Additionally, our data are a good example of the application of high-throughput metabolomic tools in forest management.
ABSTRACT
In recent years, several reports pointed out the role of protein oxidation in seed longevity, notably regarding the oxidation of methionine (Met) residues to methionine sulfoxide (MetO) in proteins. To further consider this question, we present a handy proteomic method based on the use of two-dimensional diagonal electrophoresis (2Dd) and cyanogen bromide (CNBr) cleavage, which we refer to as 2Dd-CNBr. CNBr treatment of proteins causes the non-enzymatic hydrolysis of peptide bonds on the carboxyl side of reduced Met residues. However, Met oxidation causes a lack of cleavage, thus modifying the electrophoretic mobility of CNBr-induced peptides. This approach was first validated using bovine serum albumin as a model protein, which confirmed the possibility of distinguishing between oxidized and non-oxidized forms of Met-containing peptides in gels. Then, the 2Dd-CNBr method was applied to the Arabidopsis thaliana seed protein extract in a control (non-oxidized) condition and in an oxidized one (as obtained following hypochlorous acid treatment). Twenty-four oxidized Met residues in 19 proteins identified by mass spectrometry were found to be surface exposed in these proteins. In the three-dimensional environment of the oxidized Met, we detected amino acid residues that could be converted by oxidation (carbonylation) or by phosphorylation, suggesting a possible interplay between Met oxidation and the other protein modifications. The identification of the proteins oxidatively modified in Met residues revealed the finding that MetO-containing proteins are related to seed longevity. Based on these results, we suggest that the method presently described also has the potential for wider applications.
ABSTRACT
European beech is an important component of European lowland forests in terms of ecology, and produces irregular seeds categorized as intermediate due to their limited longevity. Removal of the excess of reactive oxygen species is crucial for redox homeostasis in growing plant tissues. Hydrogen peroxide (H2O2) is detoxified via the plant-specific ascorbate-glutathione cycle, and enzymatically, mainly by catalase (CAT). The reduced and oxidized (redox) forms of ascorbate (AsA, DHA) and glutathione (GSH, GSSG) decreased during maturation as the content of redox forms of nicotinamide adenine dinucleotide (NADH, NAD+) phosphate (NADPH, NADP+), cofactors of ascorbate-glutathione enzymes, declined and limited this cycle. The degree of oxidation of glutathione peaked at approximately 80%, at the exact time when the NADP content was the lowest and the NADPH/NADP+ ratio reached the highest values. The glutathione pool was reflected in changes in the NADP pool, both in embryonic axes (R2 = 0.61) and in cotyledons (R2 = 0.98). A large excess of NADPH was reported in embryonic axes, whereas cotyledons displayed more unified levels of NADP redox forms. As a result, anabolic redox charge and reducing power were higher in embryonic axes. CAT was recognized as two proteins, and the abundance of the 55 kDa protein was correlated with all redox forms of ascorbate, glutathione, NAD, and NADP, whereas the 37 kDa protein was oppositely regulated in embryonic axes and cotyledons. Here, we discuss the role of NAD(P) in the regulation of the ascorbate-glutathione cycle, catalase, and seed longevity concerning a putative role of NAD(P)H as a redox biomarker involved in predefining seed quality, because NAD(P)H-derived redox homeostasis was found to be better controlled in embryonic axes than cotyledons.
ABSTRACT
Seed germination is a complex process enabling plant reproduction. Germination was found to be regulated at the proteome, metabolome and hormonal levels as well as via discrete post-translational modification of proteins including phosphorylation and carbonylation. Redox balance is also involved but less studied. Acer seeds displaying orthodox and recalcitrant characteristics were investigated to determine the levels of redox couples of nicotinamide adenine dinucleotide (NAD) phosphate (NADP) and integrated with the levels of ascorbate and glutathione. NAD and NADP concentrations were higher in Norway maple seeds and exceptionally high at the germinated stage, being the most contrasting parameter between germinating Acer seeds. In contrast, NAD(P)H/NAD(P)+ ratios were higher in sycamore seeds, thus exhibiting higher reducing power. Despite distinct concentrations of ascorbate and glutathione, both seed types attained in embryonic axes and cotyledons had similar ratios of reduced/oxidized forms of ascorbate and half-cell reduction potential of glutathione at the germinated stage. Both species accomplished germination displaying different strategies to modulate redox status. Sycamore produced higher amounts of ascorbate and maintained pyridine nucleotides in reduced forms. Interestingly, lower NAD(P) concentrations limited the regeneration of ascorbate and glutathione but dynamically drove metabolic reactions, particularly in this species, and contributed to faster germination. We suggest that NAD(P) is an important player in regulating redox status during germination in a distinct manner in Norway maple and sycamore seeds.
Subject(s)
Acer/metabolism , Germination/physiology , NADP/metabolism , NAD/metabolism , Seeds/metabolism , Oxidation-ReductionABSTRACT
Seeds stored in controlled conditions in gene banks, faster or slower lose their viability. The effects of seed moisture content levels (ca. 5, 8, 11%) combined with storage temperatures (-3°, -18°, -196°C) were investigated in terms of the description of seeds defined as orthodox under oxidative stress after seed storage, during germination, and initial seedling growth. Hydrogen peroxide (H2O2), thiobarbituric acid reactive substances (TBARS) and ascorbate (Asc) were analyzed in relation to seed germinability and seedlings emergence in three species: Malus sylvestris L., Prunus avium L. and Prunus padus L. The effect of seed storage conditions on H2O2 levels appeared in germinated seeds after the third year of storage in each species. The H2O2 levels were negatively correlated with the germination and seedling emergence of P. avium seeds after three years of storage under all examined combinations. The emergence of P. padus seedlings was not linked to any of the stress markers tested. The P. padus seed biochemical traits were least altered by storage conditions, and the seeds produced tolerant seedlings of relatively high levels of H2O2 and TBARS. To cope with different H2O2 levels, TBARS levels, and Asc levels in seeds of three species varying storage conditions different molecular responses, i.e. repairing mechanisms, were applied during stratification to compensate for the storage conditions and, as a result, seeds remained viable and seedlings were successfully established.
Subject(s)
Malus/metabolism , Prunus avium/metabolism , Seedlings/genetics , Germination/drug effects , Hydrogen Peroxide/metabolism , Malus/growth & development , Oxidation-Reduction/drug effects , Prunus avium/growth & development , Seedlings/growth & development , Seeds/genetics , Seeds/growth & development , Seeds/metabolism , TemperatureABSTRACT
Norway maple and sycamore produce desiccation-tolerant (orthodox) and desiccation-sensitive (recalcitrant) seeds, respectively. Drying affects reduction and oxidation (redox) status in seeds. Oxidation of methionine to methionine sulfoxide (MetO) and reduction via methionine sulfoxide reductases (Msrs) have never been investigated in relation to seed desiccation tolerance. MetO levels and the abundance of Msrs were investigated in relation to levels of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion radical and hydroxyl radical (â¢OH), and the levels of ascorbate and glutathione redox couples in gradually dried seeds. Peptide-bound MetO levels were positively correlated with ROS concentrations in the orthodox seeds. In particular, â¢OH affected MetO levels as well as the abundance of MsrB2 solely in the embryonic axes of Norway maple seeds. In this species, MsrB2 was present in oxidized and reduced forms, and the latter was favored by reduced glutathione and ascorbic acid. In contrast, sycamore seeds accumulated higher ROS levels. Additionally, MsrB2 was oxidized in sycamore throughout dehydration. In this context, the three elements â¢OH level, MetO content and MsrB2 abundance, linked together uniquely to Norway maple seeds, might be considered important players of the redox network associated with desiccation tolerance.
ABSTRACT
Desiccation tolerance is a developmental program enabling seed survival in a dry state and is common in seeds categorized as orthodox. We focused on NAD and its phosphorylated form (NADP) because their continual switching between reduced (NAD(P)H) and oxidized (NAD(P)+) forms is involved in the modulation of redox signaling and the determination of the reducing power and further antioxidant responses. Norway maple and sycamore seeds representing the orthodox and recalcitrant categories, respectively, were used as models in a comparison of responses to water loss. The process of desiccation up to 10% water content (WC) was monitored in Norway maple seeds, while dehydration up to 30% WC was monitored in desiccation-sensitive sycamore seeds. Norway maple and sycamore seeds, particularly their embryonic axes, exhibited a distinct redox status during dehydration and desiccation. High NADPH levels, NAD+ accumulation, low and stable NAD(P)H/NAD(P)+ ratios expressed as reducing power and high NADPH-dependent enzyme activity were reported in Norway maple seeds and were considered attributes of orthodox-type seeds. The contrasting results of sycamore seeds contributed to their low antioxidant capacity and high sensitivity to desiccation. NADPH deficiency, low NADPH-dependent enzyme activity and lack of NAD+ accumulation were primary features of sycamore seeds, with implications for their NAD(P)H/NAD(P)+ ratios and reducing power and with effects on many seed traits. Thus, we propose that the distinct levels of pyridine nucleotides and their redox status contribute to orthodox and recalcitrant phenotype differentiation in seeds by affecting cellular redox signaling, metabolism and the antioxidant system.
Subject(s)
Acer/metabolism , NADP/metabolism , Oxidation-Reduction , Seeds/metabolism , Acer/physiology , Dehydration , NADP/physiology , Seeds/physiologyABSTRACT
Reactive oxygen species (ROS) are constantly produced by metabolically active plant cells. The concentration of ROS may determine their role, e.g., they may participate in signal transduction or cause oxidative damage to various cellular components. To ensure cellular homeostasis and minimize the negative effects of excess ROS, plant cells have evolved a complex antioxidant system, which includes ascorbic acid (AsA). AsA is a multifunctional metabolite with strong reducing properties that allows the neutralization of ROS and the reduction of molecules oxidized by ROS in cooperation with glutathione in the Foyer-Halliwell-Asada cycle. Antioxidant enzymes involved in AsA oxidation and reduction switches evolved uniquely in plants. Most experiments concerning the role of AsA have been performed on herbaceous plants. In addition to extending our understanding of this role in additional taxa, fundamental knowledge of the complex life cycle stages of woody plants, including their development and response to environmental factors, will enhance their breeding and amend their protection. Thus, the role of AsA in woody plants compared to that in nonwoody plants is the focus of this paper. The role of AsA in woody plants has been studied for nearly 20 years. Studies have demonstrated that AsA is important for the growth and development of woody plants. Substantial changes in AsA levels, as well as reduction and oxidation switches, have been reported in various physiological processes and transitions described mainly in leaves, fruits, buds, and seeds. Evidently, AsA exhibits a dual role in the photoprotection of the photosynthetic apparatus in woody plants, which are the most important scavengers of ozone. AsA is associated with proper seed production and, thus, woody plant reproduction. Similarly, an important function of AsA is described under drought, salinity, temperature, light stress, and biotic stress. This report emphasizes the involvement of AsA in the ecological advantages, such as nutrition recycling due to leaf senescence, of trees and shrubs compared to nonwoody plants.
ABSTRACT
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.
Subject(s)
Cotyledon/metabolism , Lipid Metabolism , Lupinus/metabolism , Nitrates/metabolism , Plant Proteins/metabolism , Asparagine/metabolism , Biomass , Fatty Acids/metabolism , Phospholipids/metabolism , Plant Roots/metabolism , Plant Shoots/metabolism , SolubilityABSTRACT
A strong increase in the level of dehydrin/response ABA transcripts expression reported from the 14th week after flowering coincident with the accumulation of 26 and 44 kDa dehydrins in the embryonic axes of developing beech (Fagus sylvatica L.) seeds. Both transcript and protein levels were strongly correlated with maturation drying. These results suggest that the 44-kDa dehydrin protein is a putative dimer of dehydrin/response ABA protein migrating as a 26-kDa protein. Dehydrins and dehydrin-like proteins form large oligomeric complexes under native conditions and are shown as several spots differing in pI through isoelectrofocusing analyses. Detailed prediction of specific sites accessible for various post-translational modifications (PTMs) in the dehydrin/response ABA protein sequence revealed sites specific to acetylation, amidation, glycosylation, methylation, myristoylation, nitrosylation, O-linked ß-N-acetylglucosamination and Yin-O-Yang modification, palmitoylation, phosphorylation, sumoylation, sulfation, and ubiquitination. Thus, these results suggest that specific PTMs might play a role in switching dehydrin function or activity, water binding ability, protein-membrane interactions, transport and subcellular localization, interactions with targeted molecules, and protein stability. Despite the ability of two Cys residues to form a disulfide bond, -SH groups are likely not involved in dimer arrangement. His-rich regions and/or polyQ-tracts are potential candidates as spatial organization modulators. Dehydrin/response ABA protein is an intrinsically disordered protein containing low complexity regions. The lack of a fixed structure and exposition of amino acids on the surface of the protein structure enhances the accessibility to 40 predicted PTM sites, thereby facilitating dehydrin multifunctionality, which is discussed in the present study.
Subject(s)
Computer Simulation , Fagus/metabolism , Plant Proteins/metabolism , Seeds/metabolism , Amino Acid Sequence , Electrophoresis, Gel, Two-Dimensional , Fagus/embryology , Fagus/genetics , Gene Expression Regulation, Plant , Oxidation-Reduction , Plant Proteins/chemistry , Plant Proteins/genetics , Protein Processing, Post-Translational , RNA, Messenger/genetics , RNA, Messenger/metabolism , Seeds/genetics , Water/metabolismABSTRACT
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-â¢) 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.
ABSTRACT
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.