TY - JOUR
AU - Michalak, Marcin
AB - Abstract 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. Introduction The ability to survive severe levels of desiccation without mortality is only found in a limited number of microbes, invertebrates, several species of ‘resurrection plants’ and plant seeds. Even though this ability is rather an unique feature in some organisms, the survival of completely dry seeds is an expected feature (Barbedo et al. 2013, Sershen et al. 2016), especially for seeds of grain crops, which have played such an essential role in the evolution of human cultures. Roberts (1973) established a distinct classification for seeds based on their desiccation tolerance, categorizing desiccation-tolerant seeds as orthodox, and desiccation-sensitive seeds as recalcitrant. This classification nomenclature has been widely used for over 40 years in defining seed types and in establishing seed storage management protocols. Many studies on seed desiccation tolerance and storage have been published since Roberts proposed this classification system. Some of these studies revealed that a precise division between orthodox and recalcitrant seeds is difficult to establish and thus another seed category, intermediate seeds, was recommended by Ellis et al. (1990). It should be kept in mind, however, that seeds exhibit a wide spectrum of responses to drying rather than the rigid ones defined in the different categories (Walters 2015a). Due to the economic importance and conservational efforts related to species preservation, the desiccation tolerance of seeds has received a significant amount of research focus. Recalcitrance of seeds is a problem since this property precludes their storage for long periods of time using the conventional methods developed for orthodox seeds. Further studies of desiccation tolerance are also required due to the need to partially dry seeds when preparing them for long-term storage in liquid nitrogen (−196 °C), which currently represents the most promising option for the preservation of recalcitrant seeds (Walters et al. 2013, Walters 2015a, 2015b, Sershen et al. 2016). Thus, the sensitivity of seeds to desiccation is the fundamental physiological feature dictating the success or failure of germplasm conservation (Sershen et al. 2016). The structural and metabolic features of seeds in relation to their storage properties have been recently reviewed (Obroucheva et al. 2016). Although numerous processes have been implicated in the acquisition and maintenance of desiccation tolerance, our understanding of this feature is still limited and consequently an integrated, comprehensive view cannot be presented (Berjak and Pammenter 2008, Obroucheva et al. 2016). This is mainly due to the lack of a universal biomarker that connects desiccation-related changes with seed viability, irrespective of seed category, the rate of drying or storage conditions (temperature and humidity). Importantly, some areas of seed biology have been insufficiently investigated. For example, the number of reports characterizing the influence of desiccation on the epigenome of tree seeds are limited. This is surprising given the fact that forests are the most important global repositories of biodiversity (Liang et al. 2016). Therefore, conducting studies that improve our knowledge of woody plant seed biology, especially in relation to seed preservation, appears to be well justified. The premise that genome-wide changes in gene expression modulate the physiology and development of plants in response to environmental conditions (Zhou et al. 2007) is the underlying basis for conducting epigenetic studies. The role of epigenetic changes in regulating the biology of plants throughout their life cycle has been thoroughly reviewed (Valledor et al. 2007, Braeutigam et al. 2013, Baulcombe and Dean 2014). The most extensively characterized epigenetic modification is the methylation of cytosine (m5C) in DNA, which is catalyzed by DNA methyltransferases. The level of m5C in plants varies from 6 to 30% (Chen and Li 2004). In seeds of woody plants, such as Acer platanoides L., Pyrus communis L. and Quercus robur L., global methylation levels have been reported to range from ∼13–22% (Michalak et al. 2013, 2015, Plitta et al. 2014a, 2014b). Information on the epigenetic changes occurring in seeds of woody plants subjected to abiotic stress (e.g., desiccation) or during seed aging is still limited (Kim et al. 2015). We previously reported that changes in m5C amount can be observed in seeds of P. communis and A. platanoides, both of which are classified as orthodox seeds, when they are subjected to a reduction in water content (WC) (Michalak et al. 2013, Plitta et al. 2014a) and may represent an indicator of deterioration in Q. robur recalcitrant seeds (Michalak et al. 2015). To the best of our knowledge, a detailed comparison of the epigenetic processes in orthodox and recalcitrant seeds in response to severe desiccation, and the relationship of these epigenetic changes to seed viability and metabolic competence, has not yet been conducted. Therefore, embryonic axes and cotyledons excised from the seeds of two Acer species, A. planatoindes (orthodox) and Acer pseudoplatanus L. (recalcitrant), that differ in their response to desiccation and storage, were used in the present study. Epigenetic mechanisms, such as DNA methylation, can be inherited by subsequent generations (Moliner et al. 2006, Verhoeven et al. 2010, Avramova 2015). While the majority of stress-induced modifications are reset to a pre-stress status upon relief from the stress, some of the epigenetic modifications may be carried forward as a ‘stress memory’ that is inherited across mitotic or even meiotic cell divisions; helping plants to effectively cope with subsequent stress (Avramova 2015). Determining if desiccation experienced by the embryo can have an effect on the epigenetic status of later life stages, is also an interesting question. Therefore, the potential to transfer the variation in the level of genomic DNA methylation from seeds (separately for embryonic axes and cotyledons) to seedlings was also analyzed. Materials and methods Plant material, assessment of WC and desiccation of seeds Samaras of A. platanoides L. (Norway maple) and A. pseudoplatanus L. (sycamore) were collected from individual trees in Mosina (N52° 14′ 43.9″, E16° 50′ 38.2) and Drużyna (N52° 12′ 22.9″, E16° 49′ 35.1″), Poland, respectively. These provenances are situated in the middle-west of Poland. Both regions are characterized by similar agroclimatic conditions. The WC of seeds was calculated on a dry weight basis and expressed as g H2O g−1 dry mass (g g−1), as well as on a fresh weight basis as percent of moisture content (MC) using a previously described formula (Michalak et al. 2013). In order to obtain a MC lower than 46% for A. platanoides and 44.7% for A. pseudoplatanus, samaras were placed in a drying box on blotting paper and desiccated over silica gel to the required MC. The duration of the desiccation protocol ranged from several days to 2–6 weeks in the case of the lowest MC in seeds of both species. Moisture content of samaras, seeds, embryonic axes and cotyledons were individually assessed by drying each entity at 103 °C ± 2 °C for 24 h. The MC of samaras, embryonic axes and cotyledons are reported in Tables 1 and 2. Table 1. Moisture and corresponding WCs of samaras, seeds and embryonic tissues of Acer platanoides after collection and desiccation.   Freshly collected seeds  First level of desiccation  Second level of desiccation  Third level of desiccation    MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  Samaras  43.6 ± 1.49  9.6 ± 0.02  6.0 ± 0.04  3.0 ± 0.06  0.78 ± 0.05  0.11 ± 0.001  0,06 ± 0.001  0.03 ± 0.001  Seeds  46.0 ± 1.26  10.3 ± 0.13  6.7 ± 0.06  3.5 ± 0.08  0.86 ± 0.05  0.12 ± 0.002  0.07 ± 0.001  0.03 ± 0.004  Embryonic axes  50.8 ± 2.30  8.6 ± 0.31  5.5 ± 0.13  3.9 ± 0.1  1.04 ± 0.09  0.10 ± 0.004  0.06 ± 0.001  0.04 ± 0.002  Cotyledons  43.7 ± 1.66  8.2 ± 0.33  5.8 ± 0.03  3.3 ± 0.07  0.78 ± 0.05  0.09 ± 0.004  0.06 ± 0.001  0.03 ± 0.001    Freshly collected seeds  First level of desiccation  Second level of desiccation  Third level of desiccation    MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  Samaras  43.6 ± 1.49  9.6 ± 0.02  6.0 ± 0.04  3.0 ± 0.06  0.78 ± 0.05  0.11 ± 0.001  0,06 ± 0.001  0.03 ± 0.001  Seeds  46.0 ± 1.26  10.3 ± 0.13  6.7 ± 0.06  3.5 ± 0.08  0.86 ± 0.05  0.12 ± 0.002  0.07 ± 0.001  0.03 ± 0.004  Embryonic axes  50.8 ± 2.30  8.6 ± 0.31  5.5 ± 0.13  3.9 ± 0.1  1.04 ± 0.09  0.10 ± 0.004  0.06 ± 0.001  0.04 ± 0.002  Cotyledons  43.7 ± 1.66  8.2 ± 0.33  5.8 ± 0.03  3.3 ± 0.07  0.78 ± 0.05  0.09 ± 0.004  0.06 ± 0.001  0.03 ± 0.001  Table 1. Moisture and corresponding WCs of samaras, seeds and embryonic tissues of Acer platanoides after collection and desiccation.   Freshly collected seeds  First level of desiccation  Second level of desiccation  Third level of desiccation    MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  Samaras  43.6 ± 1.49  9.6 ± 0.02  6.0 ± 0.04  3.0 ± 0.06  0.78 ± 0.05  0.11 ± 0.001  0,06 ± 0.001  0.03 ± 0.001  Seeds  46.0 ± 1.26  10.3 ± 0.13  6.7 ± 0.06  3.5 ± 0.08  0.86 ± 0.05  0.12 ± 0.002  0.07 ± 0.001  0.03 ± 0.004  Embryonic axes  50.8 ± 2.30  8.6 ± 0.31  5.5 ± 0.13  3.9 ± 0.1  1.04 ± 0.09  0.10 ± 0.004  0.06 ± 0.001  0.04 ± 0.002  Cotyledons  43.7 ± 1.66  8.2 ± 0.33  5.8 ± 0.03  3.3 ± 0.07  0.78 ± 0.05  0.09 ± 0.004  0.06 ± 0.001  0.03 ± 0.001    Freshly collected seeds  First level of desiccation  Second level of desiccation  Third level of desiccation    MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  Samaras  43.6 ± 1.49  9.6 ± 0.02  6.0 ± 0.04  3.0 ± 0.06  0.78 ± 0.05  0.11 ± 0.001  0,06 ± 0.001  0.03 ± 0.001  Seeds  46.0 ± 1.26  10.3 ± 0.13  6.7 ± 0.06  3.5 ± 0.08  0.86 ± 0.05  0.12 ± 0.002  0.07 ± 0.001  0.03 ± 0.004  Embryonic axes  50.8 ± 2.30  8.6 ± 0.31  5.5 ± 0.13  3.9 ± 0.1  1.04 ± 0.09  0.10 ± 0.004  0.06 ± 0.001  0.04 ± 0.002  Cotyledons  43.7 ± 1.66  8.2 ± 0.33  5.8 ± 0.03  3.3 ± 0.07  0.78 ± 0.05  0.09 ± 0.004  0.06 ± 0.001  0.03 ± 0.001  Table 2. Moisture and corresponding WCs of samaras, seeds and embryonic tissues of Acer pseudoplatanus after collection and desiccation.   Freshly collected seeds  First level of desiccation  Second level of desiccation  Third level of desiccation  Freshly collected seeds    MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  Samaras  29.5 ± 0.55  23.9 ± 0.34  19.4 ± 0.48  15.5 ± 0.4  10.6 ± 0.18  0.71 ± 0.01  0.42 ± 0.01  0.24 ± 0.01  0.18 ± 0.01  0.12 ± 0.002  Seeds  44.7 ± 0.66  35.0 ± 0.58  28.7 ± 0.48  21.7 ± 1.04  13.9 ± 0.3  1.35 ± 0.04  1.06 ± 0.11  0.41 ± 0.01  0.28 ± 0.02  0.16 ± 0.004  Embryonic axes  1.8 ± 1.16  35.0 ± 1.89  25.4 ± 1.39  17.8 ± 2.12  14.2 ± 0.84  1.33 ± 0.01  0.72 ± 0.03  0.34 ± 0.02  0.22 ± 0.03  0.17 ± 0.008  Cotyledons  44.9 ± 0.97  39.8 ± 1.84  27.8 ± 1.22  19.3 ± 2.04  12.7 ± 0.58  1.51 ± 0.04  0.82 ± 0.04  0.39 ± 0.03  0.24 ± 0.03  0.15 ± 0.01    Freshly collected seeds  First level of desiccation  Second level of desiccation  Third level of desiccation  Freshly collected seeds    MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  Samaras  29.5 ± 0.55  23.9 ± 0.34  19.4 ± 0.48  15.5 ± 0.4  10.6 ± 0.18  0.71 ± 0.01  0.42 ± 0.01  0.24 ± 0.01  0.18 ± 0.01  0.12 ± 0.002  Seeds  44.7 ± 0.66  35.0 ± 0.58  28.7 ± 0.48  21.7 ± 1.04  13.9 ± 0.3  1.35 ± 0.04  1.06 ± 0.11  0.41 ± 0.01  0.28 ± 0.02  0.16 ± 0.004  Embryonic axes  1.8 ± 1.16  35.0 ± 1.89  25.4 ± 1.39  17.8 ± 2.12  14.2 ± 0.84  1.33 ± 0.01  0.72 ± 0.03  0.34 ± 0.02  0.22 ± 0.03  0.17 ± 0.008  Cotyledons  44.9 ± 0.97  39.8 ± 1.84  27.8 ± 1.22  19.3 ± 2.04  12.7 ± 0.58  1.51 ± 0.04  0.82 ± 0.04  0.39 ± 0.03  0.24 ± 0.03  0.15 ± 0.01  Table 2. Moisture and corresponding WCs of samaras, seeds and embryonic tissues of Acer pseudoplatanus after collection and desiccation.   Freshly collected seeds  First level of desiccation  Second level of desiccation  Third level of desiccation  Freshly collected seeds    MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  Samaras  29.5 ± 0.55  23.9 ± 0.34  19.4 ± 0.48  15.5 ± 0.4  10.6 ± 0.18  0.71 ± 0.01  0.42 ± 0.01  0.24 ± 0.01  0.18 ± 0.01  0.12 ± 0.002  Seeds  44.7 ± 0.66  35.0 ± 0.58  28.7 ± 0.48  21.7 ± 1.04  13.9 ± 0.3  1.35 ± 0.04  1.06 ± 0.11  0.41 ± 0.01  0.28 ± 0.02  0.16 ± 0.004  Embryonic axes  1.8 ± 1.16  35.0 ± 1.89  25.4 ± 1.39  17.8 ± 2.12  14.2 ± 0.84  1.33 ± 0.01  0.72 ± 0.03  0.34 ± 0.02  0.22 ± 0.03  0.17 ± 0.008  Cotyledons  44.9 ± 0.97  39.8 ± 1.84  27.8 ± 1.22  19.3 ± 2.04  12.7 ± 0.58  1.51 ± 0.04  0.82 ± 0.04  0.39 ± 0.03  0.24 ± 0.03  0.15 ± 0.01    Freshly collected seeds  First level of desiccation  Second level of desiccation  Third level of desiccation  Freshly collected seeds    MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  MC (%) WC (g g−1)  Samaras  29.5 ± 0.55  23.9 ± 0.34  19.4 ± 0.48  15.5 ± 0.4  10.6 ± 0.18  0.71 ± 0.01  0.42 ± 0.01  0.24 ± 0.01  0.18 ± 0.01  0.12 ± 0.002  Seeds  44.7 ± 0.66  35.0 ± 0.58  28.7 ± 0.48  21.7 ± 1.04  13.9 ± 0.3  1.35 ± 0.04  1.06 ± 0.11  0.41 ± 0.01  0.28 ± 0.02  0.16 ± 0.004  Embryonic axes  1.8 ± 1.16  35.0 ± 1.89  25.4 ± 1.39  17.8 ± 2.12  14.2 ± 0.84  1.33 ± 0.01  0.72 ± 0.03  0.34 ± 0.02  0.22 ± 0.03  0.17 ± 0.008  Cotyledons  44.9 ± 0.97  39.8 ± 1.84  27.8 ± 1.22  19.3 ± 2.04  12.7 ± 0.58  1.51 ± 0.04  0.82 ± 0.04  0.39 ± 0.03  0.24 ± 0.03  0.15 ± 0.01  Viability assessment Prior to germination tests, stratification of dormant A. platanoides and A. pseudoplatanus seeds within their samaras was required after desiccation. Samaras with seed coats that had been cut to leave two-thirds of each seed, were placed in a substrate consisting of a moist mixture (1:1, v/v) of quartz sand (<1 mm fraction) and sieved peat (pH 3.5–4.5). Seeds, mixed with the substrate (1:3, v/v), were placed in 0.25 l plastic bottles, until the first germinated seeds (<5%), defined as seeds with a 2–3 mm long radical, were observed. This was used as a visible indicator that the seeds were released from dormancy. Water was added to the substrate as needed to keep it moist and the seeds were monitored for fungal infections throughout the stratification treatment. In order to complete stratification, A. platanoides and A. pseudoplatanus required 10–13 and 12–13 weeks at 3 °C, respectively. Seeds were subjected to a germination assay (Plitta et al. 2014a) after the completion of stratification by transferring them to a cyclically alternating temperature (20 °C/3 °C for 8 h/16 h, light/dark photoperiod). Seeds were germinated in plastic boxes containing the same substrate used for stratification. They were sown at a depth of 1 cm and covered with a layer of substrate. The germination test was conducted on five biological replicates consisting of 30 seeds in each replicate. The boxes were covered with a transparent lid to allow light penetration. The germination assay conditions were kept until seedlings were approximately 20–30 mm high, after which seedlings were grown in a chamber set at a constant 20 °C temperature with a 16 h/8 h light/dark photoperiod at light intensity of 60 μmol m−2 s−1. Seed germination assessments were conducted using the International Rules for Seed Testing (2016) where only seeds with both a developed radicle and a shoot were counted as ‘germinated’. After a 3-month period of growth, all viable seedlings were collected for the determination of biomass. For this purpose, after a careful clean-up of roots, whole seedlings were dried in a forced-air oven at 80 °C for 72 h and dry weight was assessed separately for five biological replicates and expressed as an average. A tetrazolium chloride (TTC) assay was conducted according to the International Rules for Seed Testing (2016). Embryos were soaked in a solution of 1% 2,3,5-triphenyltetrazolium chloride. The test was carried out in 50 ml covered vessels containing 25 ml aqueous sterile solutions of 1% TTC. The TTC test was conducted on five biological replicates, each with 30 seeds. Seeds were incubated in the TTC solution in the dark at 30 °C ± 1 for 24 h. Embryos that remained green (unstained) or had unstained areas close to embryonic axes that exhibited evidence of staining were considered as dead seeds, while embryos that were stained pink to red were classified as living. An in vitro regrowth assay was conducted on five biological replicates, each with 30 embryonic axes that were isolated from non-stratified seeds of both A. pseudoplatanus and A. platanoides. Excised embryonic axes were surface sterilized in 10% commercial bleach (≤5% NaClO and ≤1% NaOH) for 5 min and then rinsed four times in sterile water. The embryonic axes were then cultured on MS medium (Murashige and Skoog 1962) containing sucrose (30 g l−1), 0.8 mg l−1 6-benzylaminopurine (BAP) and agar (7.0 g l−1) for solidification. The pH of the medium was adjusted to 5.7 prior to autoclaving. Embryonic axes were cultured at 25 °C with a 16 h/8 h, light/dark photoperiod and a light intensity of 77 μmol m−2 s−1. Regrowth of the embryonic axes was assessed after 8 weeks of in vitro culture, and counted as regenerated only if the embryonic axis had developed either a shoot or a shoot with a root. DNA isolation and assessment of global m5C levels Total genomic DNA was separately extracted from embryonic axes, cotyledons and leaves of 3-month-old seedlings with a Qiagen DNAeasy Plant Mini KitTM (Qiagen, Hilden, Germany). Each assay consisted of five biological replicates that were comprised of either five embryonic axes, three cotyledons or three leaves. A TLC-based method was used for the chromatographic separation of m5C from other DNA bases, as well as RNA contamination, since it provides very precise separation (Figure 1). All nucleobases were labeled with radioactive phosphate to enable a highly sensitive determination of the level of genome-wide methylation in DNA samples derived from plant tissues of limited size, such as the embryonic axes used in this study (Figure 2). Analysis of the global level of m5C in DNA of seeds was carried out and calculated as previously described (Michalak et al. 2013, 2015, Plitta et al. 2014a, 2014b). Dried DNA (1 μg) was digested to completion (6 h) with 0.001 U of spleen phosphodiesterase II and 0.02 U of microccocal nuclease in 20 mM succinate buffer containing 10 mM CaCl2 at 37 °C. The resulting hydrolysate (0.3 μg) was then labeled with 1 μCi [γ-32P] ATP (6000 Ci mmol−1 Hartmann Analytic, Braunschweig, Germany) and 1.5 U of T4 polynucleotide kinase in 10 mM bicine-NaOH buffer (pH 9.7) containing 10 mM MgCl2, 10 mM DTT and 1 mM spermidine. After incubation for 30 min at 37 °C, 0.03 U of apyrase in 10 mM bicine-NaOH buffer was added and the mixture was incubated for 30 min. Subsequently, 0.2 μg of RNase P1 in 500 mM ammonium acetate buffer pH 4.5 was used for 3′ phosphate cleavage. Analysis of [γ-32P] m5C was performed with 2D TLC on cellulose plates (Merck, Darmstadt, Germany) in isobutyric acid/NH4OH/H2O (66/1/17) (first direction) and 0.1 M sodium phosphate pH 6.8/ammonium sulfate/n-propanol (100 ml/60 g/1.5 ml) (second direction). Radioactivity was measured with a FLA-5100 Fluoro Image Analyzer and Multi Gauge 3.0 Software. Figure 1. View largeDownload slide Assessment of global DNA methylation by 2D TLC. [γ-32P] 2′deoxynucleotides derived from DNA hydrolysis (labeled spots) and RNA contaminations (unlabeled spots). S, start of separation. The first and second dimensions of separation are labeled on chromatograms. A, adenine; C, cytosine; m5C, 5-methylcytosine; T, thymine; G, guanine. Figure 1. View largeDownload slide Assessment of global DNA methylation by 2D TLC. [γ-32P] 2′deoxynucleotides derived from DNA hydrolysis (labeled spots) and RNA contaminations (unlabeled spots). S, start of separation. The first and second dimensions of separation are labeled on chromatograms. A, adenine; C, cytosine; m5C, 5-methylcytosine; T, thymine; G, guanine. Figure 2. View largeDownload slide Seeds and explants of Acer platanoides and Acer pseudoplatanus Figure 2. View largeDownload slide Seeds and explants of Acer platanoides and Acer pseudoplatanus The image analysis procedure used to quantify the level of m5C accounts for the level of both cytosine (C) and m5C (Figure 1). The level of m5C in each biological replicate was measured five times. The R ratio was calculated using the following formula:   R(%)=m5C/m5C + C × 100 Statistical analysis STATISTICA version 11.0 (StatSoft, Tulsa, OK, USA) software was used for the statistical analyses. All data were logit transformed prior to analysis. In all figures, however, non-transformed data are presented to simplify the interpretation of biological relevance. To test for differences within species, germination, in vitro regrowth, viability of the embryonic axes (TTC assay), seedling dry weight and the levels of global DNA methylation data were subjected to analysis of variance (ANOVA) across desiccation levels. A Fisher test was used to determine significant differences between sample means at P ≤ 0.05. For each species, separate ANOVA and post hoc tests were performed on data for germination, in vitro regrowth, the TTC assay, seedling dry weight and global DNA methylation levels. ANOVA and post hoc tests were only used to determine the effect of desiccation on each separate variable and were not used to investigate relationship between variables. The correlations were tested using the Pearson correlation coefficient analysis. Error bars indicate standard errors (SE) of the mean within an individual treatment. Two multivariate analyses (principal component analysis (PCA) and linear discriminant analysis (LDA)) were performed using JMP 12 (SAS Institute Inc., Cary, USA). A PCA on correlations was applied to investigate the relationship of seed germination, TTC, m5C level in embryonic axes, cotyledons and seedlings in both species after logit transformation. An LDA was conducted to differentiate two analyzed species according to m5C level in embryonic axes, cotyledons and seedlings. Results Viability assessments The rate of germination in stratified, non-desiccated control seeds of A. platanoides (MC of 46%) and A. pseudoplatanus (MC of 44.7%) was 68.6 and 87.3%. Desiccation of samaras to a lower seed MC resulted in a statistically significant decline in germination of seeds from both species (Figure 3A and B). After a period of 8 weeks, the regrowth of embryonic axes from control seeds of A. platanoides or A. pseudoplatanus was high and only a slight, but statistically significant, decrease was observed when seeds of A. platanoides were dried to an MC of 10.3 and 6.7%. A substantial decline in regrowth to 43.1% was observed, however, for seeds desiccated to 3.5% (Figure 3C). The regrowth of A. pseudoplatanus embryonic axes declined after seed desiccation to 35–21.7%, but the reductions were not statistically significant until seeds were dried to the lowest MC (Figure 3D). According to the TTC staining assay, ~90% of the embryos extracted from control seeds of A. platanoides or A. pseudoplatanus were viable. In both cases, the level of viability remained comparable high except at the lowest MC (Figure 3E and F). Figure 3. View largeDownload slide Effect of seed MC on the germination (A, B), in vitro regrowth of embryonic axes (C, D), and on the respiratory competence (E, F) of Acer platanoides and Acer pseudoplatanus seeds. Values labeled with different letters are significantly different at P ≤ 0.05, according to Fisher test. Data represent the mean ± SE (n = 5). Figure 3. View largeDownload slide Effect of seed MC on the germination (A, B), in vitro regrowth of embryonic axes (C, D), and on the respiratory competence (E, F) of Acer platanoides and Acer pseudoplatanus seeds. Values labeled with different letters are significantly different at P ≤ 0.05, according to Fisher test. Data represent the mean ± SE (n = 5). DNA methylation in embryonic axes and cotyledons Embryonic axes of control A. platanoides seeds at an MC of 46% (Table 1) exhibited a 21.7% of m5C, while the m5C level in cotyledons was 21.9% of methylated cytosines (Figure 4A and C). A decrease in the MC of seeds to 10.3% induced a statistically significant decrease in global DNA methylation level in embryonic axes to 19%. Further drying of seeds decreased the m5C content to 18.7 and 17.8%, respectively. Only the decrease to 17.8%, however, was statistically significant. The level of DNA methylation in cotyledons ranged from an m5C level of 21.7–19.4%, but these changes were not statistically significant. Figure 4. View largeDownload slide Effect of seed MC on the global DNA methylation of embryonic axes (A, B) and cotyledons (C, D) of Acer platanoides and Acer pseudoplatanus. Values labeled with different letters are significantly different at P ≤ 0.05, according to Fisher test. Data represent the mean ± SE (n = 5). Figure 4. View largeDownload slide Effect of seed MC on the global DNA methylation of embryonic axes (A, B) and cotyledons (C, D) of Acer platanoides and Acer pseudoplatanus. Values labeled with different letters are significantly different at P ≤ 0.05, according to Fisher test. Data represent the mean ± SE (n = 5). The embryonic axes extracted from control seeds of A. pseudoplatanus exhibited an m5C level of 28.5%, while the m5C level in cotyledons was 32.5% (Figure 4B and D). Desiccation of seeds to lower levels of MC resulted in a concomitant decline in the m5C levels in embryonic axes to a level as low as 20.9%. Similar results were obtained for cotyledons, where a decline in MC also resulted in a decline in m5C levels to as low as 20.5%. Dry weight of 3-month-old seedlings Three-month-old A. platanoides seedlings obtained from control seeds had an average dry weight of 0.072 g. Severe desiccation of seeds did not significantly affect the average dry weight of seedlings (Figure 5A). Whereas, 3-month-old seedlings derived from control seeds of A. pseudoplatanus had an average dry weight (0.1 g) that was higher than all other seedlings that were obtained from desiccated seeds (Figure 5B). However, in both species, the seeds that were desiccated to the lowest MC produced seedlings with the lowest dry mass. Figure 5. View largeDownload slide Effect of seed MC on the average dry weight of 3-month-old seedlings of Acer platanoides (A) and Acer pseudoplatanus (B). Values labeled with different letters are significantly different at P ≤ 0.05, according to Fisher test. Data represent the mean ± SE (n = 5). Figure 5. View largeDownload slide Effect of seed MC on the average dry weight of 3-month-old seedlings of Acer platanoides (A) and Acer pseudoplatanus (B). Values labeled with different letters are significantly different at P ≤ 0.05, according to Fisher test. Data represent the mean ± SE (n = 5). DNA methylation in 3-month-old seedlings The level of DNA methylation (m5C) in 3-month-old seedlings of A. platanoides derived from untreated seeds was 16.8%. The m5C levels in seedlings derived from desiccated seeds ranged from 16.8 to 14.4%. The change in m5C levels between control and desiccated seeds was statistically significant only at the lowest MC (Figure 6A). In the case of A. pseudoplatanus seedlings, no statistically significant changes in m5C levels were observed between 3-month-old seedlings obtained from untreated and desiccated seeds (Figure 6B). Figure 6. View largeDownload slide Effect of seed MC on the global DNA methylation of Acer platanoides (A) and Acer pseudoplatanus (B) seedlings. Values labeled with different letters are significantly different at P ≤ 0.05, according to Fisher test. Data represent the mean ± SE (n = 5). Figure 6. View largeDownload slide Effect of seed MC on the global DNA methylation of Acer platanoides (A) and Acer pseudoplatanus (B) seedlings. Values labeled with different letters are significantly different at P ≤ 0.05, according to Fisher test. Data represent the mean ± SE (n = 5). Multivariate statistical analyses The results of PCA indicated strong relationships between seed germination, results of TTC staining and m5C level in embryonic axes and cotyledons. It was expressed in a high loading of these variables in principal component 1 (Prin1), while m5C level in seedlings had a major impact on Prin2. The first two principal components accounted for 56.2% and 19.5% of variance, respectively (in total 75.6%) (see Supplementary Data available at Tree Physiology Online). Acer platanoides differed significantly from A. pseudoplatanus in terms of Prin1 (Mann–Whitney U-test, P = 0.0005), while no significant distinction was detected in Prin2 values. Subsequently, an LDA was conducted to differentiate two analyzed species according to m5C levels in embryonic axes, cotyledons and seedlings. Since the m5C level in seedlings did not have a significant effect on the discrimination between the species, we discarded it from the analysis. Because two groups were analyzed, only one discriminant function was computed (Canonical 1, Figure 7). The function was able to effectively differentiate the two groups (the following tests: Wilks’ Lambda, Pillai’s Trace, Hotelling-Lawley and Roy’s Max Root gave results with P < 0.0001). After application of the analysis, 82% of observations were correctly classified. The m5C level in embryonic axes had more impact on the discriminant function than the m5C level in cotyledons (standardized scoring coefficients were 0.75 and 0.42, respectively). Figure 7. View largeDownload slide The LDA of m5C level in embryonic axes and cotyledons for Acer platanoides and Acer pseudoplatanus (P < .0001). The point corresponding to each multivariate mean is denoted by ‘+’ marker. The outer ellipses are plotted for 95% confidence level, while the inner ellipses enclose 50% of the observations. The rays represent loading of the two covariates onto Canonical 1. They emanate from the point (0), which represents the grand mean of the data. Figure 7. View largeDownload slide The LDA of m5C level in embryonic axes and cotyledons for Acer platanoides and Acer pseudoplatanus (P < .0001). The point corresponding to each multivariate mean is denoted by ‘+’ marker. The outer ellipses are plotted for 95% confidence level, while the inner ellipses enclose 50% of the observations. The rays represent loading of the two covariates onto Canonical 1. They emanate from the point (0), which represents the grand mean of the data. Discussion At the present time, the critical factors that trigger metabolic changes in desiccation-tolerant plants which prepare them to survive in a desiccated state are still unknown (Fernández-Marín et al. 2013). The current study was conducted in order to determine the epigenetic changes occurring in tree seeds in response to severe dehydration, and to examine the relationship of these changes to the viability and metabolic competence in the relation to orthodox and recalcitrant seeds. Global levels of m5C in genomic DNA isolated from embryonic axes and cotyledons were monitored at different degrees of drying. A global approach provides the ability to monitor the whole genome, rather than just a small, arbitrarily chosen fraction of it. This approach is advantageous since the justification of epigenetic studies has been based upon the premise that plants modulate their physiology and development through genome-wide changes in gene expression in response to environmental conditions (Zhou et al. 2007). Indeed, epigenetic regulation affects a large number of genes, and in one study, 630 genes were reported to be involved in the writing, erasing or reading of epigenetic marks (Yakovlev et al. 2016). This clearly demonstrates the scale of epigenetic regulation. Importantly, however, the analysis of a single epigenetic marker on a particular gene may not establish an obvious link to a distinctive phenotype due to the functional redundancy of epigenetic regulators and the bivalent state of many genes containing both repressive and permissive chromatin marks (van Zanten et al. 2013a, 2013b). Intriguingly, chromatin modifications often seem to be selective and the expression of some genes is strongly affected by the absence or presence of a chromatin modification. However, there are also many cases where this does not hold true (van Zanten et al. 2013a, 2013b, Seymour and Becker 2017). For example, moderately transcribed genes are most likely to be methylated, whereas genes at either extreme are least likely (Zilberman et al. 2007). Additionally, the methylation of transposable elements is typically associated with dampened gene expression, but the opposite relationship has also been reported (Seymour and Becker 2017). In regard to seeds, Michalak et al. (2015) and Ogneva et al. (2016) reported that genome-wide changes in m5C were highly correlated with the aging (gradual loss of viability) of recalcitrant and orthodox seeds. Based on the described premises and the previous reports on methylation in tree seeds, global levels of m5C changes were examined in the present study in embryos and seedlings derived from A. planatoindes (orthodox) and A. pseudoplatanus (recalcitrant) seeds subjected to a various levels of desiccation. The stages of seed drying selected for each species were different because a severe level of desiccation in the two species indicates different levels of MC. Samaras of A. platanoides were artificially dried to achieve a final MC of 3.9%. Since a 10% MC is recommended for their safe storage (Tylkowski 1989), we wanted to examine changes in the epigenome of seeds dried to this MC level and below, in order to determine their response to a severe desiccation stress. It was also our intention to complement previous research (Plitta et al. 2014a) and to use different and lower levels of MC, as compared with those that were used in the previous study where changes in m5C changes exhibited a sine-wave like response to moderate desiccation. In the current study, the A. platanoides seeds in samaras were more severely dried to a level below 5% of MC, since this value is considered safe for the storage of true orthodox seeds (Roberts 1973, Walters 2015a, 2015b). In contrast, in order to make category-related comparisons, here we also used samaras of A. pseudoplatanus that were collected when the embryonic axes had a 41.8% MC (Table 2), and were subsequently dried to a final MC of 14.2%. Drying the seeds to lower levels of MC served no purpose since the total germination was only 4% at this MC. The explants generated in the regrowth assay were embryonic axes in which all of the processes related to the initiation of germination occur. Furthermore, the cotyledons attached to the embryonic axis contain stored reserves (sucrose) that are utilized after radicle emergence for seedling development (Obroucheva et al. 2016). The functional and structural heterogeneity of the investigated tissues are reflected in different responses to desiccation. Free water, during seed desiccation, is first removed from the embryonic axis (Obroucheva et al. 2016), and several studies (e.g., Prichard 1991, Finch-Savage 1992, Pence 1992, Leprince et al. 1999) have demonstrated that the embryonic axes in recalcitrant seeds are more tolerant to drying than the cotyledons. This is based on the observation that the two tissues (embryonic axis vs cotyledons) differ in their ability to shut down respiration in response to desiccation. Respiration in the cotyledons within recalcitrant seed remains unabated, which allows oxygen-based metabolism to continue and thus promotes the generation of injurious levels of reactive oxygen species (ROS) (Leprince et al. 1999, Sershen et al. 2016). Based on these tissue-related differences, in contrast to our previous research (Plitta et al. 2014a, 2014b, Michalak et al. 2015), we investigated both tissues separately. A gradual decline in the DNA methylation level was observed in the DNA obtained from the embryonic axes of A. platanoides (orthodox) in response to the applied desiccation treatment (Figure 4A). Results indicated that m5C levels in the embryonic axes were stable when seed MC levels ranged from 10.3 down to 6.7%. These results are consistent with our previous data showing that changes in m5C levels were insignificant as seed MC levels decreased from 9 to 6%, and only became significant when MC levels dropped to 5% (Plitta et al. 2014a). When the impact of desiccation on m5C levels in cotyledons was investigated in the current study, however, no changes in m5C levels were observed (Figure 4C). Changes in seed viability, as measured in the germination assay, were also monitored in the current study (Figure 3A). Results confirmed that desiccation of A. platanoides seeds caused a significant gradual decline in germination. These data indicated that even though A. platanoides seeds are commonly classified as orthodox, severe desiccation can induce a significant decline in viability, particularly at the lowest seed MC. The negative effect of severe drying on the viability of A. platanoides seeds was also demonstrated in the in vitro regrowth assay utilizing embryonic axes (Figure 3C), as well as in a TTC assay (Figure 3E). Significantly, the desiccation levels were correlated with the viability of orthodox seeds of A. platanoides and m5C levels in the DNA of embryonic axes, whereas no such significant correlations were observed for cotyledons (Table 3). Table 3. Correlation coefficients (r) between MC of seeds, embryonic axes and cotyledons, germination, in vitro regrowth of embryonic axes, seed metabolic competence measured in TTC staining assay and global DNA methylation level in embryonic axes and cotyledons of Acer platanoides.   Germination  In vitro regrowth  TTC  m5C in embryonic axes  m5C in cotyledons  m5C in 3-month-old seedlings  MC of seeds  0.849***  0.826***  0.749***  0.820***  0.250  0.339  MC of embryonic axes  0.776***  0.756***  0.661**  0.784***  x  0.258  MC of cotyledons  0.823***  x  0.707***  x  0.247  0.306  Germination  x  0.967***  0.956***  0.773***  0.286  0.616**  In vitro regrowth  0.967***  x  0.900***  0.765***  0.306  0.558*  TTC  0.956***  0.900***  x  0.746***  0.298  0.630**  m5C level in embryonic axes  0.773***  0.765***  0.746***  x  0.182  0.415  m5C level in cotyledons  0.286  0.306  0.298  0.182  x  0.203  m5C level in 3-month-old seedlings  0.616**  0.558*  0.630**  0.415  0.203  x    Germination  In vitro regrowth  TTC  m5C in embryonic axes  m5C in cotyledons  m5C in 3-month-old seedlings  MC of seeds  0.849***  0.826***  0.749***  0.820***  0.250  0.339  MC of embryonic axes  0.776***  0.756***  0.661**  0.784***  x  0.258  MC of cotyledons  0.823***  x  0.707***  x  0.247  0.306  Germination  x  0.967***  0.956***  0.773***  0.286  0.616**  In vitro regrowth  0.967***  x  0.900***  0.765***  0.306  0.558*  TTC  0.956***  0.900***  x  0.746***  0.298  0.630**  m5C level in embryonic axes  0.773***  0.765***  0.746***  x  0.182  0.415  m5C level in cotyledons  0.286  0.306  0.298  0.182  x  0.203  m5C level in 3-month-old seedlings  0.616**  0.558*  0.630**  0.415  0.203  x  Bold numbers indicate significant correlations at the level of *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 were tested using the Pearson correlation coefficient analysis. MC, moisture content; m5C, 5-methylcytosine; TTC, 2,3,5-triphenyltetrazolium chloride. Table 3. Correlation coefficients (r) between MC of seeds, embryonic axes and cotyledons, germination, in vitro regrowth of embryonic axes, seed metabolic competence measured in TTC staining assay and global DNA methylation level in embryonic axes and cotyledons of Acer platanoides.   Germination  In vitro regrowth  TTC  m5C in embryonic axes  m5C in cotyledons  m5C in 3-month-old seedlings  MC of seeds  0.849***  0.826***  0.749***  0.820***  0.250  0.339  MC of embryonic axes  0.776***  0.756***  0.661**  0.784***  x  0.258  MC of cotyledons  0.823***  x  0.707***  x  0.247  0.306  Germination  x  0.967***  0.956***  0.773***  0.286  0.616**  In vitro regrowth  0.967***  x  0.900***  0.765***  0.306  0.558*  TTC  0.956***  0.900***  x  0.746***  0.298  0.630**  m5C level in embryonic axes  0.773***  0.765***  0.746***  x  0.182  0.415  m5C level in cotyledons  0.286  0.306  0.298  0.182  x  0.203  m5C level in 3-month-old seedlings  0.616**  0.558*  0.630**  0.415  0.203  x    Germination  In vitro regrowth  TTC  m5C in embryonic axes  m5C in cotyledons  m5C in 3-month-old seedlings  MC of seeds  0.849***  0.826***  0.749***  0.820***  0.250  0.339  MC of embryonic axes  0.776***  0.756***  0.661**  0.784***  x  0.258  MC of cotyledons  0.823***  x  0.707***  x  0.247  0.306  Germination  x  0.967***  0.956***  0.773***  0.286  0.616**  In vitro regrowth  0.967***  x  0.900***  0.765***  0.306  0.558*  TTC  0.956***  0.900***  x  0.746***  0.298  0.630**  m5C level in embryonic axes  0.773***  0.765***  0.746***  x  0.182  0.415  m5C level in cotyledons  0.286  0.306  0.298  0.182  x  0.203  m5C level in 3-month-old seedlings  0.616**  0.558*  0.630**  0.415  0.203  x  Bold numbers indicate significant correlations at the level of *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 were tested using the Pearson correlation coefficient analysis. MC, moisture content; m5C, 5-methylcytosine; TTC, 2,3,5-triphenyltetrazolium chloride. When m5C levels were measured in DNA isolated from the embryonic axes and cotyledons excised from A. pseudoplatanus (recalcitrant) seeds, changes in m5C levels were detectable in both tissues in response to progressive desiccation (Figure 4B and D). The results of the germination assay, the respiratory competence assay (TTC) and the in vitro regrowth assay of embryonic axes confirmed the recalcitrant nature of these seeds with the stringent evidence of a negative impact of desiccation reducing total germination down to 4% at an MC of 13.8% (Figure 3B). The decrease in germination, as an indicator of seed viability, was highly correlated with a decline in m5C levels in both embryonic axes and cotyledons (Table 4). Table 4. Correlation coefficients (r) between MC of seeds, embryonic axes and cotyledons, germination, in vitro regrowth of embryonic axes, seed metabolic competence measured in TTC staining assay and global DNA methylation level in embryonic axes and cotyledons of Acer pseudoplatanus.   Germination  In vitro regrowth  TTC  m5C in embryonic axes  m5C in cotyledons  m5C in 3- month-old seedlings  MC of seeds  0.964***  0.705***  0.750***  0.745***  0.760***  −0.197  MC of embryonic axes  0.909***  0.566**  0.640***  0.771***  x  −0.145  MC of cotyledons  0.942***  x  0.708***  x  0.725***  −0.198  Germination  x  0.675***  0.833***  0.705***  0.711***  −0.276  In vitro regrowth  0.675***  x  0.675***  0.434*  x  −0.091  TTC  0.830***  0.675***  x  0.440*  0.604***  −0.256  m5C in embryonic axes  0.705***  0.439*  0.440*  x  0.512**  0.144  m5C in cotyledons  0.711***  x  0.604***  0.512**  x  −0.229  m5C in month-old seedlings  −0.276  −0.091  −0.256  0.144  −0.229  x    Germination  In vitro regrowth  TTC  m5C in embryonic axes  m5C in cotyledons  m5C in 3- month-old seedlings  MC of seeds  0.964***  0.705***  0.750***  0.745***  0.760***  −0.197  MC of embryonic axes  0.909***  0.566**  0.640***  0.771***  x  −0.145  MC of cotyledons  0.942***  x  0.708***  x  0.725***  −0.198  Germination  x  0.675***  0.833***  0.705***  0.711***  −0.276  In vitro regrowth  0.675***  x  0.675***  0.434*  x  −0.091  TTC  0.830***  0.675***  x  0.440*  0.604***  −0.256  m5C in embryonic axes  0.705***  0.439*  0.440*  x  0.512**  0.144  m5C in cotyledons  0.711***  x  0.604***  0.512**  x  −0.229  m5C in month-old seedlings  −0.276  −0.091  −0.256  0.144  −0.229  x  Bold numbers indicate significant correlations at the level of *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 were tested using the Pearson correlation coefficient analysis. MC, moisture content, m5C, 5-methylcytosine, TTC, 2,3,5-triphenyltetrazolium chloride. Table 4. Correlation coefficients (r) between MC of seeds, embryonic axes and cotyledons, germination, in vitro regrowth of embryonic axes, seed metabolic competence measured in TTC staining assay and global DNA methylation level in embryonic axes and cotyledons of Acer pseudoplatanus.   Germination  In vitro regrowth  TTC  m5C in embryonic axes  m5C in cotyledons  m5C in 3- month-old seedlings  MC of seeds  0.964***  0.705***  0.750***  0.745***  0.760***  −0.197  MC of embryonic axes  0.909***  0.566**  0.640***  0.771***  x  −0.145  MC of cotyledons  0.942***  x  0.708***  x  0.725***  −0.198  Germination  x  0.675***  0.833***  0.705***  0.711***  −0.276  In vitro regrowth  0.675***  x  0.675***  0.434*  x  −0.091  TTC  0.830***  0.675***  x  0.440*  0.604***  −0.256  m5C in embryonic axes  0.705***  0.439*  0.440*  x  0.512**  0.144  m5C in cotyledons  0.711***  x  0.604***  0.512**  x  −0.229  m5C in month-old seedlings  −0.276  −0.091  −0.256  0.144  −0.229  x    Germination  In vitro regrowth  TTC  m5C in embryonic axes  m5C in cotyledons  m5C in 3- month-old seedlings  MC of seeds  0.964***  0.705***  0.750***  0.745***  0.760***  −0.197  MC of embryonic axes  0.909***  0.566**  0.640***  0.771***  x  −0.145  MC of cotyledons  0.942***  x  0.708***  x  0.725***  −0.198  Germination  x  0.675***  0.833***  0.705***  0.711***  −0.276  In vitro regrowth  0.675***  x  0.675***  0.434*  x  −0.091  TTC  0.830***  0.675***  x  0.440*  0.604***  −0.256  m5C in embryonic axes  0.705***  0.439*  0.440*  x  0.512**  0.144  m5C in cotyledons  0.711***  x  0.604***  0.512**  x  −0.229  m5C in month-old seedlings  −0.276  −0.091  −0.256  0.144  −0.229  x  Bold numbers indicate significant correlations at the level of *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 were tested using the Pearson correlation coefficient analysis. MC, moisture content, m5C, 5-methylcytosine, TTC, 2,3,5-triphenyltetrazolium chloride. The premise that seeds with greater resilience against the injury that occurs to biological systems subjected to dry conditions can survive longer periods of storage is widely accepted. Recognition of the factors underlying differences in seed longevity and desiccation tolerance are still the subject of many investigations (Walters 2015a). We perceived a link between the oxidative damage that occurs to cells during desiccation, which has been described in multiple studies (e.g., Pukacka and Ratajczak 2007, Berjak and Pammenter 2013, Walters 2015b), and ROS-related effects on DNA methylation (Cerda and Weitzman 1997). Injury to the cellular components of seeds is due to the fact that aqueous-based metabolism continues in an unbalanced manner, even as water is lost from cells (Ballesteros et al. 2014). Consequently, this leads to an increase in the generation and unregulated activity of ROS, resulting from the concurrent failure in antioxidant system. This process is considered as a major source of injury, which leads to a loss in seed viability (Umarani et al. 2015). Aberrant, metabolism-based injury has been demonstrated to occur at both high and intermittent moisture levels (Umarani et al. 2015); however, the initial lowering of WC did not have such a negative impact on metabolism as deep dehydration (Obroucheva et al. 2016). The changes that occur in m5C levels from the initial, small changes in MC are possibly due to either enzymatic (demethylases) or non-enzymatic ROS activity, and it may still be plausible that these changes are controlled by cells (Obroucheva et al. 2016). This was perhaps also true in the observations made in recalcitrant seeds, where the initial drying stages did not cause dramatic changes in seed respiration competence and seedling regeneration potential. Seed germination, however, decreased by 10 and 26% in seeds in which the MC of embryonic axes was 35 and 25.4%, respectively. No changes in m5C levels were observed in these tissues at those MC levels. When the MC of A. pseudoplatanus embryonic axes was lower than 25.4%, however, m5C levels started decreasing, which perhaps suggests that methylation/demethylation processes could no longer be actively controlled. This hypothesis is consistent with the results obtained in previous investigations on recalcitrant seeds which indicated that only bound water is present in cells once a MC of 26% is reached (Obroucheva et al. 2016). At this level of cellular dehydration, maintaining the proper conformation of enzymes, and the delivery of substrates to active centers becomes problematic; thus enzymatic activity becomes limited (Obroucheva et al. 2016). The mechanism underlying demethylation, whether the result of the bifunctional activity of demethylases or the oxidative damage imposed by ROS, still needs to be determined. We do not consider the observed decrease in global DNA methylation to be due to a failure in the post-replicative maintenance of DNA methylation, because no DNA replication has been observed in dormant Acer seeds (Finch-Savage et al. 1998). Similar conclusions have been reported in the investigations of desiccation response in orthodox seeds. Although orthodox seeds are generally characterized by a pattern of increased longevity with decreasing WC, they may deviate from the patterns at threshold WC that range between 0.03 and 0.07 g g−1, which represents an MC of 3–7% (Walters 2015b). At a WC lower than the threshold WC, longevity is either unaffected or decreases as WC approaches zero (Walters 2015b). This characterization is in accordance with the changes in seed viability (germination, TTC and in vitro regrowth of embryonic axes) and epigenetic changes (m5C levels) observed in A. platanoides seeds in the current study, where seed viability significantly decreased at MC levels below the threshold WC. Apparently, the existing seed protective mechanisms are ineffective and/or inhibited at these severe desiccation levels, which leads to a significant increase in cellular injury and thus, reduced seed viability. This occurs despite the fact that enzyme activity can occur in dry seeds at low water levels when the cytosol is in a rubbery state. Nonetheless, cellular components as well as enzyme activity are differentially affected in a rubbery state (Bewley and Black 1982, Fernández-Marín et al. 2013). The m5C level in cotyledons, however, remained unchanged throughout all of the stages of desiccation. It is tempting to speculate that the lack of significant changes in DNA methylation in cotyledons contributes to the general desiccation resistance of orthodox seeds. Even though a decline in viability is observed, a portion of the embryo tissue remains viable at an extremely low MC. There is growing evidence that epigenetic mechanisms, especially DNA methylation, play a significant role in stress response and stress memory in plants (Henderson and Jacobsen 2007, Mathieu et al. 2007, Zhang et al. 2010, Kinoshita and Seki 2014). Most of the conducted research, however, has focused on how the exposure of the parent plants is transmitted to the next generation, but little information exists on whether or not epigenetic stress signals can be transmitted from latent life (seeds) to seedlings. This question is the basis for our interest in determining if the epigenetic changes occurring in embryos while they are within seeds are stable throughout multiple mitoses and if they are transferred from seeds to seedlings once the seeds germinate. Therefore, in contrast to our previous research (Plitta et al. 2014a), we observed the influence of a stress factor (desiccation) on the development and biomass accumulation of seedlings. No statistically significant differences were observed in seedling dry weights from dried orthodox seeds in relation to the controls. Nevertheless, the dry mass of seedlings obtained from the seeds at a MC of 3.5% was the lowest. Whereas, a constant reduction of this parameter was observed for seedlings obtained from recalcitrant seeds at all stages of desiccation (Figure 5A and B). However, the level of m5C in DNA isolated from leaves of 3-month-old seedlings derived from desiccated recalcitrant seeds was unchanged relative to control seedlings, and only decreased in plants derived from orthodox seeds when the seeds were desiccated to the lowest MC (Figure 6A and B). This may due to the fact that after removal of the stress factor (desiccation), the global level of m5C was equalized by DNA methylation machinery in seedlings derived from seeds of both categories that survived desiccation. Indeed, it has been noted that induced epigenetic changes facilitating the rapid adaptation to short-term environmental fluctuations are reset afterwards, in the absence of stress, and any methylation-related signs of adaptation require repetitive exposure to stress over a few generations (Wibowo et al. 2016). Nevertheless, it is significant that severe desiccation of orthodox seeds (MC of 3.5%) impacted m5C level in seedlings, as the level of methylated cytosine was lower. In addition, the accumulation of biomass was also affected by the desiccation as well. This observation is in agreement with our previous research on 3-month-old seedlings of P. communis, which produces orthodox seeds (Michalak et al. 2013). Even though the overall content of m5C may not change significantly, it should be noted that the distribution of these marks within the genome is unknown. However, it should be kept in mind that within a plant genome, DNA methylation is highly concentrated in non-transcribed centromeric regions and repetitive sequences (He et al. 2011). As a result, it is plausible that any massive deviation in the m5C amount resulting from acute stress and causing highly deteriorating changes to plant physiology may have the greatest effects on these genome fragments. Indeed, it was shown that hyperosmotic stress primarily changed the methylation of non-CG sites located in heterochromatin and the silenced mobile elements (Meng et al. 2016, Wibowo et al. 2016). Results of the present study indicate that orthodox and recalcitrant seeds both respond to severe desiccation and exhibit significant changes in m5C levels in the DNA obtained from embryonic axes. Interestingly, changes in m5C levels in cotyledons appear to be specific to seed category, as they were only observed in recalcitrant seeds. The current study provides new information on desiccation sensitivity in orthodox and recalcitrant seeds of tree species, in relation to seed viability, metabolic activity and changes in m5C levels. Such knowledge is essential for developing appropriate storage regimes and optimal seed management protocols for preserving genetic biodiversity. Many indicators of dehydration stress, such as electrolyte leakage, TTC staining, determining rates of protein synthesis, lipid peroxidation, and the balance between pro- and antioxidative processes, have been examined for use in determining seed viability and for the development of optimal seed storage protocols. Unfortunately, these approaches have failed in their ability to be universally used as a biomarker to predict the storage response of seeds or the impact of desiccation on their viability (Sershen et al. 2016). Results of the current and previous research (Michalak et al. 2015) suggest that changes in m5C levels have the potential to be used as a viability marker in seeds subjected to stress factors or time-dependent aging. It has been suggested that many of the specific effects of desiccation and the aging of seeds are the same (Walters 2015b). However, despite a number of papers published on aging in plants, considerable ambiguity and conflicting opinions still exist on this topic (Dubrovina and Kiselev 2016). Regarding changes in DNA methylation levels, in the case of animal tissues, global hypomethylation of the genome during aging has been well documented (Gonzalo 2010). But the largest numbers of DNA methylation experiments conducted in plants have assessed changes in DNA methylation in adult vs juvenile plant tissues. Results of these studies have been contradictory, while some data indicate that the level of DNA methylation is lower in adult than in juvenile tissue (Demeulmeester et al. 1999, Baurens et al. 2004, Hasbún et al. 2005, Monteuuis et al. 2008, 2009). However, data from other studies have indicated the opposite trend (Bitoni et al. 2002, Fraga et al. 2002, Valledor et al. 2010, Guo et al. 2011, Mankessi et al. 2011, Huang et al. 2012, Yuan et al. 2014). It is worth noting that there is a lack of studies where DNA methylation has been analyzed in the same plant (Dubrovina and Kiselev 2016). Additionally, a significant difference between these investigations and our research is that they examined different stages of tissue differentiation/maturity rather than its aging per se, which is reflected as a decline in the viability of tissue. Moreover, most studies have been conducted on plants growing in a natural environment. Consequently, the observed differences in DNA methylation may have been the result of environmental conditions. Nevertheless, the effect of various stress signals on changes in m5C levels in plants have been well documented (Bilichak et al. 2012, Dowen et al. 2012), in addition to an important role of DNA methylation in plant senescence (Ay et al. 2014, Kwiatkowska et al. 2014, Cho et al. 2016). For the first time, we demonstrated that the significant differences of DNA methylation status in seeds during exposure to severe desiccation stress, followed by viability decline, are tissue- and category-specific. This statement is supported by the results of multivariate statistical analyses, which confirmed that there are two clearly separated categories of m5C results in seeds. Discriminant analyses showed that 82% of tested samples were correctly classified to one of two seed categories (Figure 7). The results from this study justify the pursuit of additional experiments that may aim to further increase our knowledge in areas such as analyses of cross talk between DNA methylation and other epigenetic processes. These future studies will be informative because even though methylation marks have been the most intensively investigated, its direct influence on gene expression has only been reported for a small subset of genes (Seymour and Becker 2017) and ‘epigenetic memory’ is controlled by complex processes that may not strictly rely on changes in DNA methylation alone (Wibowo et al. 2016). However, taken together, such investigations will enable a better understanding for the phenomenon of seed recalcitrance and may provide an opportunity to monitor seed deterioration and aging processes. Supplementary Data Supplementary Data for this article are available at Tree Physiology Online. Conflict of interest None declared. Funding National Science Centre, Poland (UMO-2012/07/B/NZ9/01312); Institute of Dendrology of the Polish Academy of Sciences. Acknowledgment The authors would like to thank Magdalena Sobczak and Elżbieta Drzewiecka for their excellent technical assistance. References Avramova Z ( 2015) Transcriptional ‘memory’ of a stress, transient chromatin and memory (epigenetic) marks at stress response genes. Plant J  83: 149– 159. Google Scholar CrossRef Search ADS PubMed  Ay N, Janack B, Humbeck K ( 2014) Epigenetic control of plant senescence and linked processes. J Exp Bot  65: 3875– 3887. Google Scholar CrossRef Search ADS PubMed  Ballesteros D, Sershen, Varghese B, Berjak P, Pammenter NW ( 2014) Uneven drying of zygotic embryos and embryonic axes of recalcitrant seeds: challenges and considerations for cryopreservation. Cryobiology  69: 100– 109. Google Scholar CrossRef Search ADS PubMed  Barbedo CJ, Centeno DDC, Ribeiro RDCLF ( 2013) Do recalcitrant seeds really exist? 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TI - Changes in genomic 5-methylcytosine level mirror the response of orthodox (Acer platanoides L.) and recalcitrant (Acer pseudoplatanus L.) seeds to severe desiccation
JF - Tree Physiology
DO - 10.1093/treephys/tpx134
DA - 2018-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/changes-in-genomic-5-methylcytosine-level-mirror-the-response-of-PGIWFEZVPO
SP - 617
EP - 629
VL - 38
IS - 4
DP - DeepDyve
ER -