Journal of Experimental Botany, Vol. 52, No. 365, pp. 2265-2273,
December 1, 2001
© 2001 Oxford University Press
Original Papers |
Immunohistochemical study of DNA methylation dynamics during plant development
Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska str. 135, CZ-612 65 Brno, Czech Republic
Received 12 March 2001; Accepted 11 July 2001
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Abstract |
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DNA methylation represents one of the key processes that play an important role in the transcriptional control of gene expression. The role of cytosine methylation in plant development has been demonstrated by at least three different kinds of evidence: parent-specific expression of some genes in developing seeds, control of flowering time and floral morphogenesis, and correlation with silencing of intrusive DNA sequences (mobile genetic elements and transgenes). In this work global changes in DNA methylation during seed germination and shoot apical meristem development in Silene latifolia have been studied using an indirect immunohistochemical approach. The data presented show that a rapid decrease in global DNA methylation during seed germination occurs first in endosperm tissue and subsequently in the hypocotyl. Using 5-bromo-2'-deoxyuridine pulses, it has been demonstrated that these demethylation events occurred before cell division had begun. In the early post-germination period, a decrease in DNA methylation was detected in cotyledons, also before cell division was observed. Taken together, these results indicate that DNA demethylation takes place in a non-replicative way, probably by an active mechanism. The central zone of the shoot apical meristem remains highly methylated during the whole period of vegetative growth and in this region, only a low cell division activity was found. However, upon the transition of the shoot apical meristem to the floral bud, the meristem both decreased its high methylation status and its cells started to divide. These data indicate that the central zone of the shoot apical meristem can represent a relatively quiescent ‘germ-line’ which is activated upon flowering to form spores and gametes.
Key words: DNA methylation, DNA replication, immunohistochemistry, seed germination, shoot apical meristem, Silene latifolia.
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Introduction |
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Development of the multicellular body of eukaryotic organisms is derived from an early establishment of distinct cell lines and the diversification of gene functions. This seems to be connected with epigenetic chromatin modifications. While cell lineages in animals become committed during early gastrulation, plant embryos form only a rudimentary body plan which develops further during the whole life span as a consequence of apical meristem action. Shoot apical meristems represent a permanent pluripotent cell line in which a balance of proliferation and differentiation promoting genes lead to both self maintenance of meristematic cells and morphogenesis of plant body. Reproductive organs are formed by a transition of shoot meristem to floral buds. Often a hidden epigenetic change accompanies this transition (Finnegan et al., 2000
; Vyskot, 1999).
The most widely studied mechanism involved in the process of epigenetic control of gene expression in plants is DNA methylation (Finnegan et al., 1998). At least two functionally different groups of DNA methyltransferases should exist. Maintenance methyltransferases are active on hemimethylated DNA (Pradhan et al., 1998), while de novo methyltransferases are capable of modifying a non-methylated template (Okano et al., 1999). De novo methylation along with DNA demethylation is thought to establish specific methylation patterns during gametogenesis and embryogenesis in mammals (Brandeis et al., 1993; Razin and Cedar, 1994) and possibly also in plants (Gavazzi et al., 1997; LoSchiavo et al., 1989). The demethylation could be a result of DNA replication which is not followed by the action of maintenance methyltransferases on the newly synthesized DNA strand or, as recently shown in animals, it could be an active enzymatic process that is not dependent on DNA replication (Zhu et al., 2000b). DNA methylation has rather pleiotropic effects on both animal and plant development. It is often correlated with the inactivation of invasive DNA, such as transgenes (Linn et al., 1990) or transposable elements (Yoder et al., 1997), and is responsible for the mitotic maintenance of gene expression patterns (Pradhan et al., 1999). In plants, there is probably one additional function of DNA methylation—protection against possible damage during the pollen quiescent stage (Janousek et al., 2000; Oakeley et al., 1997).
Plant development is characterized by at least two specific stages of quiescence which enable plants to survive unfavourable environmental conditions and to disperse spores or embryos during sexual reproduction. These stages, pollen grains and seeds, obviously evolved in connection with the loss of plant locomotion ability. To ensure their long-time survival without any external supply of nutrients and energy, both these stages are characterized by a high degree of dehydration. It is known that water plays a significant role in DNA conformational structures. It has been proposed that the DNA in embryos of seeds is conformationally different from normal somatic nuclei (Osborne and Boubriak, 1994). The dehydrated stage of mature seeds minimizes enzymatic activity during unfavourable living conditions. Another mechanism involved in seed quiescence is transcriptional inactivity which is accompanied by DNA hypermethylation. DNA methylation was studied in wheat seeds during germination and a rapid reduction of 5-mC content was observed in connection with an increasing metabolic activity (Drozdenyuk et al., 1976). Similar results were later presented (Follmann et al., 1990) where a rapid and substantial drop in the 5-mC content during the first 30 h of seed germination (from 23.7% to 15.2%) was described. These data together demonstrate large global demethylation events during the early transmission from the quiescent seed to the young plantlet. An important question arises; are these demethylation events during seed germination organ-specific and DNA replication-dependent? To investigate global DNA methylation dynamics in plant development, temporal and spatial patterns of DNA methylation have been characterized during seed germination and post-germinative plant growth and correlated with DNA replication patterns.
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Materials and methods |
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Plant material and growing conditions
Seeds of Silene latifolia Poiret subsp. alba (Miller) Greuter et Burdet from a seed collection of the Institute of Biophysics, Brno, were surface-sterilized and germinated in distilled water on a shaker under dim-light conditions. Samples for immunohistochemical studies were taken at 5 h intervals of incubation (from 0–5 d). Some plantlets were transferred to soil and cultured in a greenhouse at 20 °C and 16 h light until flowering.
DNA methylation analysis using genomic Southern hybridization
Genomic DNA from dry seeds and 3-d-old seedlings was prepared using DNeasy Plant Mini kit (Qiagen). To study the DNA methylation pattern of 5S rDNA, the samples were digested with DNA methylation-sensitive restriction enzymes MspI (mC/CGG, methylation of the outer cytosine inhibits cleavage) or HpaII (mC/mCGG, methylation of either cytosine blocks cleavage). To analyse methylation of 25S rDNA, a second digestion was performed with EcoRI to obtain smaller DNA fragments. Digested samples were fractionated on 0.8% agarose gel and transferred to Hybond-N (Amersham) membrane. The probes, a 150 bp fragment of Nicotiana tabacum 5S rRNA gene (Fulnecek et al., 1998) and an internal 2478 bp fragment of tomato 25S rDNA (Kiss et al., 1989), were labelled using the AlkPhosTM labelling kit (Amersham) and used for hybridization. The completeness of DNA digestion with restriction enzymes was checked by rehybridization of the blots with labelled chloroplast pTB29 clone (Sugiura et al., 1986). The fluorescent signal was obtained using ECF substrate (Amersham) and visualized on a Phosporimager using ImageQuant software.
Preparation of tissue sections
The germinating seeds were fixed in an ethanol–acetic acid mixture (3:1) overnight at 4 °C, embedded in Cryomount (Histolab, Göteborg) and sectioned 10 µm thick using a cryomicrotome Leica CM 1800 (Leica Instruments GmbH). Vegetative and floral meristems were excised from plants and, after fixation, sectioned at 8 µm thickness. All samples were mounted on slides coated with poly-L-lysine. The slides were post-fixed in the mixture of ethanol–acetic acid and protein was further extracted in chloroform, 45% acetic acid and pepsin (100 µg ml-1). The slides were denatured in 2 N HCl, dehydrated in an ascending series of ethanol (50, 70, and 96%), and then air-dried.
Immunohistochemical detection of 5-methylcytosine
The antiserum against 5-mC was kindly provided by Dr Ruffini-Castiglione and its preparation and basic properties have been described (Podesta et al., 1993). Its specificity was verified using methylated and unmethylated oligonucleotides (Oakeley et al., 1997). An immunocytological evidence of its relevance has been presented on S. latifolia nuclei. DAPI-dense chromocentres in control interphase nuclei displayed strong 5-mC immunosignals: both disappeared after a 5-azacytidine treatment (Siroky et al., 1998).
After the blocking reaction (1% bovine serum albumin in PBS with 0.5% Tween 20, for 1 h), the anti-5-mC antibody diluted in the blocking solution was applied and the slides were incubated at 4 °C for 12 h. FITC-labelled goat anti-mouse IgG (Sigma) was used as the secondary antibody and the slides were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Accessibility of DNA epitopes for antisera was checked using the mouse anti-DNA monoclonal antibody (Boehringer Mannheim).
Analysis of DNA replication
The DNA replication pattern was evaluated using the incorporation of 5-bromo-2'-deoxyuridine (BrdU). BrdU (50 µg ml-1) was applied to plant samples in water for 14 h. To detect BrdU incorporation into nuclei, the primary mouse antibody raised against BrdU (Sigma) and the secondary FITC- labelled goat anti-mouse IgG (Sigma) antibody were used. It was not possible to run 5-mC and BrdU analyses simultaneously as a double-colour immunostaining since BrdU incorporation modified the DNA methylation signals, probably due to a changed DNA denaturation profile (see also Bernardino et al., 1996).
Image analysis
Fluorescence was visualized using an epifluorescence microscope (Olympus AX 70) and signals were evaluated using ISIS software (Metasystems, Sandhausen). A correlation between the DAPI counterstain fluorescence and FITC secondary antibody signal was measured by means of fluorescence intensity profiles on 10 sections from each stage examined. To study the DNA replication pattern within the apical meristem, the cumulative outlines from 20 parallel sections from each stage were prepared. Each dot stands for one dividing cell.
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Results |
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Southern blot analysis of DNA methylation
The DNA methylation patterns of two model sequences were monitored during seed germination of S. latifolia using 5-mC sensitive restriction enzymes and Southern analysis. DNA methylation analysis of 5S rRNA genes (Fig. 1a
) using MspI did not reveal any differences between seeds and 3-d-old seedlings (i.e. 3 d after the start of water imbibition). In contrast, a difference has been observed in the HpaII digested hybridization patterns implicating a high degree of methylation of the internal cytosine in CCGG sequences in seeds and its moderate decrease during germination. Similar results were obtained using the 25S rDNA probe (Fig. 1b). The combined MspI+EcoRI digests showed an almost negligible shift to lower molecular bands in seedlings when compared to seeds. While the HpaII+EcoRI treatment of seed DNA sample resulted only in one large fragment (5.2 kb), corresponding to EcoRI activity (Janousek et al., 1996), several smaller distinct fragments appeared in 3-d-old seedlings. From these data it is concluded that there is a moderate drop in global methylation during seed germination and early plantlet development.
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Control immunostaining experiments
To ensure that antibodies penetrate plant samples properly, the monoclonal anti-DNA antibody was applied to sections and a measurement of the antibody and counterstain (DAPI) profiles was made. These control experiments confirmed that the antibody penetrated well in all the samples studied. As demonstrated in the series of graphs (Fig. 2a), the anti-DNA (green) signal profiles correlated well with the counterstain (blue) profiles. No differences in epitope accessibility due to possible different chromatin configurations in the tissues were observed. Some fluctuations in the strength of the anti-DNA signal are apparently caused by different densities of cells within the plant samples. Similarly, sections of vegetative and floral meristems (Fig. 3a, b) show the homogeneous distribution of the anti-DNA antibody signal throughout the meristems. These data imply that the immunolabelling of related DNA epitopes (5-mC and BrdU) on plant cryosections is relevant.
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Analysis of dry seed
The monoclonal antibody specifically recognizing 5-methylcytosine (anti-5-mC) has been used to analyse DNA methylation patterns. When the signal profiles of the anti-5-mC antibody applied to the section of dry seed were studied (Fig. 2b
), the 5-mC labelling did not fully correlate with the counterstain DAPI signals, but some prominent peaks of the immunolabelling were visible. The high DNA methylation intensity was clearly restricted to the root (virtual section 1 in Fig. 2b) and apical (virtual section 3) meristems, and provascular tissues (virtual sections 2 and 4), while the other parts of the embryo were characterized by a relatively lower 5-mC signal (virtual section 5).
Germination events
Germination is a period characterized by the events that commence with the uptake of water by the quiescent dry seed and terminate with the elongation of the embryonic axis (Bewley, 1997). During early seed germination, a very rapid decline in DNA methylation labelling in the endosperm nuclei was observed. In the dry seed, the DNA methylation level of the endosperm nuclei was approximately the same as in the epidermis of hypocotyl (Fig. 2c). When these data were compared with the 5-mC signal of the germinating seed after 5 h of water imbibition (Fig. 2d), a strong decrease of DNA methylation in the endosperm tissue became apparent. Except for prominent demethylation in the endosperm nuclei, the DNA methylation pattern did not differ from that of the dry seed (cf. Fig. 2b). The highest antibody labelling was localized in the shoot apical meristem and the root meristem, a moderately lower level DNA methylation was observed in the cotyledons and hypocotyl (Fig. 2e).
To correlate the DNA demethylation process with DNA replication, the incorporation of 5-bromo-2'-deoxyuridine (BrdU) followed by its indirect antibody detection was used. It was found that the early DNA demethylation event occurred before DNA replication had begun, because no anti-BrdU labelling appeared until the late phase of seed germination (Fig. 2f, inserted picture). Shortly before the tip of the radicula was released from the seed (between 30 and 45 h after imbibition), a positive decrease of DNA methylation labelling occurred in the cells of the hypocotyl (Fig. 2g). The shoot and root meristems still displayed high anti-5-mC labelling and a lower intensity signal was localized to the cotyledons. Similarly, the large demethylation in the hypocotyl also occurred before DNA replication (Fig. 2f). This result (the absence of BrdU immunosignals) implicates the fact that no DNA replication or cell division occur during the whole period of germination in any part of the seed.
Early post-germination period
Upon comparison of the DNA methylation patterns in the cells of the late germination stage (cf. Fig. 2g), a prominent demethylation in the nuclei of the cotyledon cells was observed in the early post-germinative phase of the plantlet development (when the hypocotyl was approximately of the same length as the diameter of the seed, i.e. between 50 and 65 h after the start of water imbibition, Fig. 2h). The level of DNA methylation of the hypocotyl remained low, whereas the methylation in the cells of the shoot apical meristem was still very high. The demethylation in cotyledons again occurred before their cells started to replicate, as detected by the BrdU incorporation (Fig. 2i). However, in this stage of early post-germination, a massive nuclear division activity was found in the hypocotyl tissue (Fig. 2i). The DNA replication in the cotyledons was detected later (Fig. 2j), when the seedlings were approximately 15 mm long (90–100-h-old seedlings). At the time of cell division in the cotyledons, the first DNA replication events in the shoot apical meristem were observed (Fig. 3e). To detect areas of cell division within the apical meristem, a cumulative presentation from 20 sections was constructed. As demonstrated on the outline (Fig. 3f), the cells divided within the whole apical meristem, including the central zone.
Shoot apical meristem during vegetative growth and flowering
The high DNA methylation signal remained apparent in the cells of the central zone of the shoot apical meristem, while the other parts of the apical meristem displayed a relatively lower methylation level (Fig. 3c). The high level of DNA methylation in the central zone correlated well with its low cell division activity as measured by BrdU incorporation and its antibody immunodetection (Fig. 3g, h). However, when the floral meristem formed, its central part was largely demethylated and no differences in the DNA methylation level between individual parts of the floral meristem were observed (Fig. 3d). From the BrdU-analysis of DNA replication, presented in Fig. 3(i) and cumulatively outlined in Fig. 3(j), it is apparent that the cells of the central zone divided at the time of floral formation at high intensity.
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Discussion |
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DNA methylation and replication during plantlet development
Cytosine methylation plays, with a few exceptions (e.g. yeasts), a crucial role in the control of development in animals and a disruption of the methylation patterns often leads to abnormal development (Razin and Cedar, 1994
). Even though flowering plants possess a higher global level of cytosine methylation which plays a similar role in development, plants are more tolerant of methylation disturbance, including a substantially reduced degree of DNA methylation. These manipulations include 5-azacytidine treatment (Janousek et al., 1996), antisense DNA methyltransferases (Finnegan et al., 1996), and mutations (Jacobsen and Meyerowitz, 1997; Kakutani et al., 1996), but the resulting plants usually suffer only from floral malformation and related deviations. There are little data available showing the DNA methylation dynamics during embryo formation and seed germination. Treatment of tobacco seeds or seedlings with 5-azacytidine led to a global hypomethylation accompanied by the formation of aberrant flowers, only when the drug was applied at a specific stage of early plantlet development when cells in the central zone of the apical meristem divided with a high frequency (Vyskot et al., 1995). Studying the effects of a 5-azacytidine hypomethylation on carrot cells in vitro, LoSchiavo et al. have observed that somatic embryogenesis is immediately blocked (LoSchiavo et al., 1989). More recently, two DNA methyltransferases have been identified with distinct expression patterns during the development of somatic embryos (Bernacchia et al., 1998). The expression pattern of DNA methyltransferase MET2-21 in the later stages of carrot embryogenesis is very similar to the DNA methylation pattern detected in dry seeds (i.e. highly methylated regions in apical meristems).
A moderate decline of DNA methylation in the 5S and 25S rRNA genes (Fig. 1a, b) was observed during seed germination which is in accordance with data by others (Drozdenyuk et al., 1976; Follmann et al., 1990). In this work, immunohistochemical data showing the precise organ and tissue specificity and timing of DNA demethylation events during seed germination are presented. The relatively highly methylated seed (Figs 1, 2b) is hypomethylated step-by-step. This process starts in the extraembryonic cells (endosperm, Fig. 2d), then continues in the hypocotyl (Fig. 2g), and finally in the cotyledons (Fig. 2h). The genome size of S. latifolia is quite large (2C=5.6x109 bp; Siroky et al., 2001), approximately 30x higher than A. thaliana, which implies a high proportion of hypermethylated DNA repeats. According to the data from the hydrolysis of end-labelled T/CGA fragments followed by thin layer chromatography, these motifs at least are globally highly methylated in the S. latifolia genome (73%; Janousek et al., 1996). It is obvious that the presented immunohistochemical analyses reflect (at least in part) methylation dynamics which may occur in numerous repetitive DNA sequences before the onset of replication.
As shown by the simultaneous analyses of DNA replication, the demethylation always occurs before DNA replication. These data confirm that DNA methylation is a dynamic process and methylation patterns are the result of de novo methylation, demethylation, and the maintenance of existing methylation (Hsieh, 2000). The large and global hypomethylation of seeds during germination and post-germination periods obviously reflects a transition from the metabolically quiescent seed to the actively growing and developing seedling. It also corresponds to the generally accepted scheme of seed germination events: protein synthesis begins from extant mRNA followed by transcriptional activation. DNA replication and cell division start later, at the post-germination period (for a review see Bewley, 1997).
The present study's immunohistochemical results concerning the rapid DNA demethylation during the first hours of seed germination are in accordance with other results (Follmann et al., 1990) in which the decrease of 5-mC content during the first 5 h after water imbibition was also observed. At this time, probably no DNA synthesis de novo takes place in germinating seeds, because the first process touching DNA is reparation (Bewley, 1997), which can be involved in the decrease of the 5-mC level. The experiments with the incorporation of BrdU cannot confirm or disprove this. It is also not possible to deny the effect of the 5-methylcytosine-DNA glycosylase found in animal model systems (Zhu et al., 2000a, b). These data describing the DNA replication pattern correspond with the detection of elongation of the radicle cells which correlates with the rupture of the testa and emergence of the tip of the radicula; cell division was observed later, during the post-germinative phase of plantlet development.
DNA demethylation was observed prior to DNA replication in all the cases that were studied. These results implicate the presence of an active mechanism of DNA demethylation in plants. This idea can be supported by data indicating the presence of an active mechanism of DNA demethylation in the non-dividing vegetative nucleus during pollen tube germination (Janousek et al., 2000). The active mechanism of DNA demethylation based on the excise repair has been observed in chicken embryos (Fremont et al., 1997) and components of DNA demethylation complex were further characterized (Jost et al., 1999a, b; Zhu et al., 2000b). Recently, 5-methylcytosine DNA glycosylase activity was also found in the human protein MBD4 (Zhu et al., 2000a). Taken together, these data indicate that DNA demethylation based on the 5-methylcytosine DNA glycosylase is a widespread mechanism in animals and probably in plants. Even though a plant enzyme capable to demethylate DNA has not been identified yet, the data presented show that the organ-specific demethylation events occur before the beginning of DNA replication.
Developmental changes in the apical meristem
The shoot apical meristem is initiated during plant embryogenesis and acts as a region which adds new organs to the existing plant body. The cells of the shoot apical meristem are organized in two levels (Steeves and Sussex, 1989). The first level of organization is based on the spatial arrangement of cells and on the direction of cell division. From this point of view, the shoot apical meristem is subdivided into two layers—tunica and corpus. The second level of cell organization within the shoot apical meristem has been established on a model describing the frequency and spatial distribution of dividing cells. It is generally accepted that the shoot apical meristem consists of a central zone of slowly-dividing cells serving as a source of cells for the peripheral and rib zones. The peripheral zone surrounding the central zone is formed from faster-cycling cells. This apical zonation is established during the development of the seedling as demonstrated on the histone H4 transcription pattern which reflects DNA replication. The shoot apex of the young seedling showed accumulation of histone H4 mRNA throughout the apical meristem and no differentiation in mitotically active and inactive zones was observed (Brandstadter et al., 1994). In contrast, the mature vegetative meristem displayed a highly labelled (i.e. mitotically active) peripheral zone and a weakly labelled (i.e. mitotically inactive) central zone. However, this DNA replication pattern is extensively changed during floral transition when the cells of the central zone start to divide with a high frequency (Steeves and Sussex, 1989). Molecular evidence supports the common existence of both these models. Recently, genes expressed in the shoot apical meristem corresponding either to the tunica–corpus or to the zonation models have been found. An analysis of the expression pattern of the tobacco NTH20 gene showed that this gene is expressed in the peripheral zone of the vegetative shoot apex, whereas the NTH9 gene is expressed in the rib zone (Nishimura et al., 1999). In contrast to these genes, the expression of NTH1 and NTH15 genes is localized to the corpus (Nishimura et al., 1999) and the Arabidopsis gene ATML1 expressed just in the L1 tunica layer (Sessions et al., 1999).
The present results concerning DNA methylation and replication patterns during shoot apical meristem development are in accord with the zonation model. According to these data, the pattern of the cell division within the shoot apical meristem can change during plant ontogenesis. In the young plantlet, equal cell division in all parts of the meristem was observed (Fig. 3e, f). The mature vegetative apex is characterized by the zonation pattern: the very slowly dividing central zone is surrounded by the peripheral zone with a higher DNA division rate (Fig. 3g, h). After the transition to flowering, cell division in the shoot apical meristem is restored (Fig. 3i, j). The DNA replication pattern in the shoot apical meristem negatively correlates with the DNA methylation pattern, i.e. the high cell division rate corresponds with the low DNA methylation level and vice versa (Fig. 3c, d). The cells in the central zone of the vegetative meristem are probably preserved by their low division rate and high DNA methylation status against the action of unfavourable external factors which may cause possible mutations. The cells are also preserved against internal factors by the symplastic isolation of the central zone (Gisel et al., 1999; Rinne et al., 1998). The cells of the central zone do participate in the formation of flowers. During floral meristem formation, DNA demethylation and replication of the central zone were observed (Fig. 3d, i, j). These results suggest that the central zone of the vegetative apical meristem can represent a relatively quiescent ‘germ-line’ or ‘stem cells’ which are activated upon flowering to form spores and gametes. This hypothesis is supported by the fact that expression of homologues of the genes piwi from Drosophila and prg-1 and prg-2 from Caenorhabditis, which are specifically required for the self-renewal of germ-line stem cells (Cox et al., 1998), have been found in the plant apical meristem (Lynn et al., 1999; Moussian et al., 1998).
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Acknowledgments |
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We thank Drs M Ruffini-Castiglione (University of Pisa) for a generous gift of the anti-5-methylcytosine antibody, S Grant (University of North Carolina) for critical reading of the manuscript and J Siroky (Institute of Biophysics, Brno) for help in the processing of image analysis. This project was supported by the Grant Agency of the Czech Republic (521/99/0696) and the Grant Agency of the Academy of Sciences (A5004901).
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Notes |
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1 To whom correspondence should be addressed. Fax: +420 5 412 40500. E-mail: vyskot{at}ibp.cz
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