Identification of DNA Methylation Changes in European Beech Seeds during Desiccation and Storage.

Ageing and deterioration of seeds is a major problem for the maintenance of seed quality and viability during long-term storage. Prediction of early stages of seed deterioration in order to point out the plantlets' regeneration time is a major challenge of successful storage. In preserved seeds, damages accumulate within cells at the rate mainly related to their moisture content and temperature of storage. Current research reveals global alterations in DNA methylation in lipid-rich intermediate seeds during desiccation and storage at various regimes covering nonoptimal and optimal conditions. We show for the first time that monitoring of 5-methylcytosine (m5C) level in seeds can be used as a truly universal viability marker regardless of postharvest category of seeds and their composition. For seeds stored up to three years, in varied conditions, moisture content, temperature, and time of storage had significant influence on seedling emergence and DNA methylation (p < 0.05). Similarities among lipid-rich intermediate and orthodox seeds regarding different reactions of embryonic axes and cotyledons to desiccation are newly revealed. Along with previous studies on seeds dramatically different in desiccation tolerance (recalcitrant vs. orthodox), results regarding lipid-rich seeds positioned in-between (intermediate) prove that maintaining global DNA methylation status is crucial for maintaining seed viability.

Oxidative damage and DNA repair in desiccated recalcitrant embryonic axes of Acer pseudoplatanus L.

BACKGROUND: Most plants encounter water stress at one or more different stages of their life cycle. The maintenance of genetic stability is the integral component of desiccation tolerance that defines the storage ability and long-term survival of seeds. Embryonic axes of desiccation-sensitive recalcitrant seeds of Acer pseudoplatnus L. were used to investigate the genotoxic effect of desiccation. Alkaline single-cell gel electrophoresis (comet assay) methodology was optimized and used to provide unique insights into the onset and repair of DNA strand breaks and 8-oxo-7,8-dihydroguanine (8-oxoG) formation during progressive steps of desiccation and rehydration. RESULTS: The loss of DNA integrity and impairment of damage repair were significant predictors of the viability of embryonic axes. In contrast to the comet assay, automated electrophoresis failed to detect changes in DNA integrity resulting from desiccation. Notably, no significant correlation was observed between hydroxyl radical (Ù OH) production and 8-oxoG formation, although the former is regarded to play a major role in guanine oxidation. CONCLUSIONS: The high-throughput comet assay represents a sensitive tool for monitoring discrete changes in DNA integrity and assessing the viability status in plant germplasm processed for long-term storage.

Volatile signature indicates viability of dormant orthodox seeds.

All seeds eventually die even under optimal storage conditions. The moment of viability loss is difficult to predict and detect. In order to differentiate between dead and viable dormant orthodox seeds, GC-MS analysis was used to non-invasively evaluate the volatile signature of seeds of Pyrus communis L. and Sorbus aucuparia L. Dormant seeds are capable of extended metabolic depression. However, their low metabolic rate remains largely unquantified, and there are no measurements of metabolites, i.e. volatile organic compounds (VOC) for physiologically dormant seeds during storage. Therefore, to address this issue, seeds were stored at a broad range of moisture content (MC) ranging from 2 to 30% under cryogenic (-196°C), cool (5°C) and elevated (40°C) temperatures. Volatile emission was highly dependent on seed MC and storage temperature and was higher under conditions associated with seed viability loss. However, changes in the emission of volatiles entrapped in seeds and released during 24 h after storage were detected for all conditions, providing insight into the processes occurring in dry dormant seeds. Among the 36 volatiles identified, three (acetaldehyde, ethanol, ethyl acetate) were highly correlated with seed germinability and show potential for the non-invasive screening of viability. Significantly, all three VOC are derived mostly from glycolysis and peroxidation and were detected even under very low moisture and temperature storage conditions. This is the first study to report on VOC accumulation and emission from physiologically dormant seeds and provide a broader view into their viability.

Global 5-methylcytosine alterations in DNA during ageing of Quercus robur seeds.

BACKGROUND AND AIMS: Epigenetic regulation plays an important role in the management of plant growth, development and response to stress factors, and several reports have indicated that DNA methylation plays a critical role in seed development and viability. This study examines changes in 5-methylcytosine (m(5)C) levels in the DNA of seeds during ageing, a process that has important implications for plant conservation and agriculture. METHODS: Changes in the global level of m(5)C were measured in mature seeds of oak, Quercus robur. The extent of DNA methylation was measured using a protocol based on two-dimensional thin-layer chromatography. Viability of seeds was determined by germination and seedling emergence tests. KEY RESULTS: An ageing-related decrease in total m(5)C during storage of recalcitrant seeds was highly and significantly correlated with a decrease in seed viability, as reflected by a reduction in germination (r = 0·8880) and seedling emergence (r = 0·8269). CONCLUSIONS: The decrease in viability during ageing of Q. robur seeds is highly correlated with a global decline in the amount of m(5)C in genomic DNA, and it is possible that this may represent a typical response to ageing and senescence in recalcitrant seeds. Potential mechanisms that drive changes in genomic DNA methylation during ageing are discussed, together with their implications for seed viability.

The chances in the redox priming of nondormant recalcitrant seeds by spermidine.

The problems posed by seed sensitivity to desiccation and aging have motivated the development of various techniques for mitigating their detrimental effects. The redox priming of seeds in antioxidant solution to improve their postharvest performance is one of the approaches. Spermidine (Spd) was tested as an invigorating solution on nondormant recalcitrant (desiccation-sensitive) seeds of the silver maple (Acer saccharinum L.). The treatment resulted in an 8-10% increase in germination capacity in seeds subjected to mild and severe desiccation, while in aged seeds stored for 6 months, no significant change was observed. The cellular redox milieu, genetic stability, mitochondrial structure and function were investigated to provide information about the cellular targets of Spd activity. Spermidine improved the antioxidative capacity, especially the activity of catalase, and cellular membrane stability, protected genome integrity from oxidative damage and increased the efficiency of mitochondria. However, it also elicited a hydrogen peroxide burst. Therefore, it seems that redox priming in nondormant seeds that are highly sensitive to desiccation, although it positively affected desiccated seed performance, may not be a simple solution to reinvigorate stored seeds with a low-efficiency antioxidant system.

Epigenetic Marks, DNA Damage Markers, or Both? The Impact of Desiccation and Accelerated Aging on Nucleobase Modifications in Plant Genomic DNA.

Modifications of DNA nucleobases are present in all forms of life. The purpose of these modifications in eukaryotic cells, however, is not always clear. Although the role of 5-methylcytosine (m5C) in epigenetic regulation and the maintenance of stability in plant genomes is becoming better understood, knowledge pertaining to the origin and function of oxidized nucleobases is still scarce. The formation of 5-hydroxymetylcytosine (hm5C) in plant genomes is especially debatable. DNA modifications, functioning as regulatory factors or serving as DNA injury markers, may have an effect on DNA structure and the interaction of genomic DNA with proteins. Thus, these modifications can influence plant development and adaptation to environmental stress. Here, for the first time, the changes in DNA global levels of m5C, hm5C, and 8-oxo-7,8-dihydroguanine (8-oxoG) measured by ELISA have been documented in recalcitrant embryonic axes subjected to desiccation and accelerated aging. We demonstrated that tissue desiccation induces a similar trend in changes in the global level of hm5C and 8-oxoG, which may suggest that they both originate from the activity of reactive oxygen species (ROS). Our study supports the premise that m5C can serve as a marker of plant tissue viability whereas oxidized nucleobases, although indicating a cellular redox state, cannot.

Choosing the Right Path for the Successful Storage of Seeds.

Seeds are the most commonly used source of storage material to preserve the genetic diversity of plants. However, prior to the deposition of seeds in gene banks, several questions need to be addressed. Here, we illustrate the scheme that can be used to ensure that the most optimal conditions are identified to enable the long-term storage of seeds. The main questions that need to be answered pertain to the production of viable seeds by plants, the availability of proper protocols for dormancy alleviation and germination, seed tolerance to desiccation and cold storage at -20 °C. Finally, it is very important to fully understand the capability or lack thereof for seeds or their explants to tolerate cryogenic conditions. The proper storage regimes for orthodox, intermediate and recalcitrant seeds are discussed.

DNA Methylation as an Early Indicator of Aging in Stored Seeds of "Exceptional" Species Populus nigra L.

Ex situ preservation of genetic resources is an essential strategy for the conservation of plant biodiversity. In this regard, seed storage is the most convenient and efficient way of preserving germplasm for future plant breeding efforts. A better understanding of the molecular changes that occur during seed desiccation and aging is necessary to improve conservation protocols, as well as real-time methods for monitoring seed quality. In the present study, we assessed changes in the level of genomic 5-methylcytosine (5mC) in seeds of Populus nigra L. by 2D-TLC. Epigenetic changes were characterized in response to several seed storage regimes. Our results demonstrate that P. nigra seeds represent an intermediate type of post-harvest behavior, falling between recalcitrant and orthodox seeds. This was also true for the epigenetic response of P. nigra seeds to external factors. A crucial question is whether aging in seeds is initiated by a decline in the level of 5mC, or if epigenetic changes induce a process that leads to deterioration. In our study, we demonstrate for the first time that 5mC levels decrease during storage and that the decline can be detected before any changes in seed germination are evident. Once P. nigra seeds reached an 8-10% reduction in the level of 5mC, a substantial decrease in germination occurred. The decline in the level of 5mC appears to be a critical parameter underlying the rapid deterioration of intermediate seeds. Thus, the measurement of 5mC can be a fast, real-time method for assessing asymptomatic aging in stored seeds.

Characterisation of the novel spontaneously immortalized and invasively growing human skin keratinocyte line HaSKpw.

We here present the spontaneously immortalised cell line, HaSKpw, as a novel model for the multistep process of skin carcinogenesis. HaSKpw cells were established from the epidermis of normal human adult skin that, without crisis, are now growing unrestricted and feeder-independent. At passage 22, clonal populations were established and clone7 (HaSKpwC7) was further compared to the also spontaneously immortalized HaCaT cells. As important differences, the HaSKpw cells express wild-type p53, remain pseudodiploid, and show a unique chromosomal profile with numerous complex aberrations involving chromosome 20. In addition, HaSKpw cells overexpress a pattern of genes and miRNAs such as KRT34, LOX, S100A9, miR21, and miR155; all pointing to a tumorigenic status. In concordance, HaSKpw cells exhibit reduced desmosomal contacts that provide them with increased motility and a highly migratory/invasive phenotype as demonstrated in scratch- and Boyden chamber assays. In 3D organotypic cultures, both HaCaT and HaSKpw cells form disorganized epithelia but only the HaSKpw cells show tumorcell-like invasive growth. Together, HaSKpwC7 and HaCaT cells represent two spontaneous (non-genetically engineered) "premalignant" keratinocyte lines from adult human skin that display different stages of the multistep process of skin carcinogenesis and thus represent unique models for analysing skin cancer development and progression.

Reactive Oxygen Species as Potential Drivers of the Seed Aging Process.

Seeds are an important life cycle stage because they guarantee plant survival in unfavorable environmental conditions and the transfer of genetic information from parents to offspring. However, similar to every organ, seeds undergo aging processes that limit their viability and ultimately cause the loss of their basic property, i.e., the ability to germinate. Seed aging is a vital economic and scientific issue that is related to seed resistance to an array of factors, both internal (genetic, structural, and physiological) and external (mainly storage conditions: temperature and humidity). Reactive oxygen species (ROS) are believed to initiate seed aging via the degradation of cell membrane phospholipids and the structural and functional deterioration of proteins and genetic material. Researchers investigating seed aging claim that the effective protection of genetic resources requires an understanding of the reasons for senescence of seeds with variable sensitivity to drying and long-term storage. Genomic integrity considerably affects seed viability and vigor. The deterioration of nucleic acids inhibits transcription and translation and exacerbates reductions in the activity of antioxidant system enzymes. All of these factors significantly limit seed viability.

Changes in genomic 5-methylcytosine level mirror the response of orthodox (Acer platanoides L.) and recalcitrant (Acer pseudoplatanus L.) seeds to severe desiccation.

Poor storability of recalcitrant seeds is due to their inability to tolerate low moisture content. Understanding the processes underlying their recalcitrance is a prerequisite to developing a maintenance strategy and prolonging their lifespan. Multiple studies have investigated the differences between orthodox (desiccation-tolerant) and recalcitrant (desiccation-sensitive) seeds. Information on epigenetic regulation, however, is lacking and thus limits our understanding of the processes defining the physiology of seeds. In the present comparative study, changes in the global levels of 5-methylcytosine (m5C) in orthodox and recalcitrant seeds of Acer platanoides L. and Acer pseudoplatanus L. were characterized during progressive stages of severe drying. Concomitant with their differential sensitivity to desiccation stress, we demonstrate variation in the response of embryonic axes and cotyledons to water deficit at the level of DNA methylation. Results indicate that desiccation-induced changes in m5C are both tissue- and seed category-specific and are highly correlated with recalcitrant seed viability. Moreover, we demonstrate that m5C global changes in response to desiccation are not retained in DNA isolated from seedlings, except in seedlings that are derived from strongly desiccated orthodox seeds (moisture content of 3.5%). Finally, the potential utilization of m5C status as a universal seed viability marker is discussed.