МОЛЕКУЛЯРНАЯ БИОЛОГИЯ, 2010, том 44, № 5, с. 853-858
МОЛЕКУЛЯРНАЯ БИОЛОГИЯ КЛЕТКИ
UDC 577.218
DEVELOPMENTAL CHANGES IN DNA METHYLATION OF POLLEN MOTHER CELLS OF DAVID LILY DURING MEIOTIC PROPHASE I
© 2010 Junjun Huang1, Huahua Wang2, Xiaojun Xie1, Huanhuan Gao1, Guangqin Guo1*
1Institute of Cell Biology, School of Life Science, Lanzhou University, Lanzhou 730000, China 2Institute of Plant Physiology, School of Life Science, Lanzhou University, Lanzhou 730000, China
Received December 18, 2009 Accepted for publication February 15, 2010
Epigenetic marks in the form of DNA methylation are involved in the development of germ cells and are important in the maintenance of fertility. However, the controlling system of the on-off switch for DNA methylation largely remains unclear. In this study, the extent of cytosine methylation during the meiotic prophase I in David lily is assessed using high pressure liquid chromatography to evaluate the DNA methylation rates. Comparing the degree of DNA methylation before, during, and after synizesis, both de novo methylation and demethylation occurred. Mainly the methylation level decreased by 21.3% (from 54.8 to 33.5%) during synizesis in the pollen mother cells. The developmental timing of genome-wide DNA methylation acquisition during pollen mother cell development is clarified in this paper. The relative amounts of 5-methyl-deoxycytidine of global methylation in leaf DNA in David lily were also higher than in other species reported.
Key words: DNA methylation, meiotic prophase I, pollen mother cells, synizesis.
Meiosis is pivotal in the life cycle of sexual plants. It is a complex process involving a highly regulated series of cytological and biochemical events and coordinated expression of a large number of genes. It marks the transition from a diploid sporophyte to a haploid gametophyte and provides an opportunity for genetic rearrangement. The initiation of meiosis is Prophase I, during which homologous chromosomes are paired, form synapses and undergo recombination. Prophase I is further divided into six different characteristic substages: leptotene, zygotene, synizesis, pachytene, diplotene, and diakinesis. The identity of germ cell lineage may be warranted by the hy-pomethylation status of the chromosomal centric and pericentric (C/P) regions [1]. In turn, both de novo me-thylation and demethylation have been reported to occur during spermatogenesis, mainly in spermatogonia and spermatocytes in early meiotic prophase I. These modifications are progressive and are almost exclusively completed by the end of the pachytene spermatocyte stage [2]. In the gametophytic phase, MET1 is responsible for copying the mCpG patterns through DNA replication. This function is illustrated by the phenotypes of metl mutants that are severely compromised in the accuracy of epigenetic inheritance during gametogenesis, including the elimination ofimprinting at paternally silent loci such as FWA or MEDEA (MEA), and thus causing late flowering [3].
Abbreviations used: PMCs — pollen mother cells.
* E-mail: gqguo@lzu.edu.cn
Programmed gene expression is essential for the normal development of all organisms. DNA methylation in eukaryotic organisms has received considerable attention in the recent years. It has been proposed that cytosine DNA methylation plays an integral role in regulating gene expression during development and germ cell differentiation [4—7]. Further, DNA methylation has been elucidated to be implicated in gene silencing through chromatin modification and remodeling [8—11] and in the chromosomal stability [12, 13]. Histone modification is also an epigenetic factor tightly linked to DNA methylation [14, 15]. Many mutations in the methyltransferase have been isolated and characterized, and detailed cell biological studies have been conducted, revealing important details of the role of methylation in meiosis [2, 3, 16, 17]. For example, SWI1 is important for meiotic chromatin remodeling and may be a regulator of early meiotic events [18]. However, how much DNA methylation changes during meiosis is unknown. The level of5-meth-ylcytosine (5mC) varies substantially among plants and accounts for up to 33% of all cytosines in rye [19]. Oake-ley et al. [20] have used a monoclonal antibody specific to mC to follow the cytological changes in DNA methyla-tion during late male gametogenesis in tobacco. They observed a drastic reduction in the overall levels of mC in pollen generative nuclei just before pollen germination. Methylation seemed to be reduced to approximately 20% of that of a vegetative nucleus. Further, reduced lev-
Table 1. The size of the flower bud of Lilium davidii var. Will-mottiae (Wilson) Roffill at different stages of development
Flower bud, mm Developmental stage
8.5-9.5 leptotene
10.5-11 zygotene
12-12.5 synizesis
13.5-14.5 pachytene
els of methylation in the DNA have been proven to have no significant influence on either chromosome pairing or chiasma formation during meiosis [21]. In plants epige-netic inheritance appears to rely on DNA methylation that has been maintained through meiosis and postmei-otic mitoses, which result in the formation of gameto-phytes. The assessment of the role of DNA methylation during meiosis in plants has been a focus of interest.
The genus Lilium is a genetic model for plant biology and development. The studies reported here are based on the male meiotic cells (microsporocytes) of Lilium. The usefulness of Lilium for a combined biochemical and cy-tological study of meiosis has been fully discussed elsewhere [22]. The cells develop synchronously and can be easily separated from the surrounding somatic tissue. Previous studies [23] on the meiotic cycle in Lilium have indicated that unique DNA metabolic events occur during the meiotic prophase at the time when genetic recombination is assumed to occur.
Previous studies [20, 24] have assessed the different methylation patterns during development by using histochemistry and genomic sequencing. However, these studies were restricted to a short region of the genome, and the changes in the numerical value were not known. HPLC analysis of the nucleosides can be used to determine the total DNA methylation in plants and to help characterize epigenetic changes during stress, growth, and development [25]. This method can be used to monitor the genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.
In the present study, the role of DNA methylation in Lilium development is studied. To examine DNA meth-ylation relative to the meiotic cycle, HPLC is employed because of its accessibility and ease of use. The results prove that DNA methylation changes during the meiotic cycle along with the development of the cells.
EXPERIMENTAL
Plant materials. Lilium davidii var. Willmottiae (Wilson) Roffill plants were used in the study. The plants were grown from the buds in the Xiguo Garden at Lanzhou. Flower buds were collected and grouped according to their size between May and June. The exact developmen-
tal stage of each group of anthers was determined using light-microscopic examination. PMCs were squeezed from pools of anthers at defined stages of meiosis and frozen with liquid nitrogen. They were stored in a deep freezer (—80°C) until use.
DNA isolation. DNA isolation was performed as described previously [26]. The PMCs of the anthers were ground in a 2% CTAB solution (1.4 mM NaCl, 20 mM EDTA, 100 mM Tris-HCl pH 8.0, and 2% CTAB) in a scale-up of the described procedure. After RNase treatment and a second phenol/chloroform extraction, the DNA was pelleted and dissolved in 200 to 400 ^L TE (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). Three DNA extractions (from three different individuals) were carried out for each type of plant material.
DNA hydrolysis. Approximately 40 ^g of DNA were hydrolyzed to bases in 0.1 mM HC1 (incubated overnight at 80°C). The reaction was stopped by the addition of 10 ^l 0.4 mM NaOH. The samples were then centrifuged at 11000 g for 15 min. The supernatant was filtered (0.2 ^m) prior to HPLC analysis [27]. DNA hydrolysis was carried out for each type ofplant material. The samples were frozen until analysis.
Measurement of 5mC content by HPLC. Global cy-tosine methylation levels were measured using the HPLC method. The HPLC buffer was made of 5% methanol, 4.75 mM sodium hexanesulfonate, and 0.2% triethano-lamine, and adjusted to the desired pH (5.5) with phosphoric acid. The buffer was filtered through a 4 mm nonsterile syringe filter with pore size of 0.2 ^m. The position of each nucleoside was determined using commercially available standards ("Sigma"). The flow of 0.7 ml/min and the peak areas ofabsorbance at 273 nm were calibrated based on the absorption of standard deoxyribonucleo-sides in the same buffer. The 5mC content ([5mC])/ ([5mC] [C]) was normalized for absorbance difference between cytosine and 5mC.
RESULTS AND DISCUSSION
The PMCs of David lily were stained with acetocar-mine (Fig. 1), and a close relationship between the stage of gametophytogenesis and bud length was found (Table 1). The obtained data is nearly consistent with our laboratory's previous results [28]. Table 1 summarizes the relationship between bud size and stages of meiotic development. The relationship is said to be invariable during the last decade. The PMCs of 12—12.5 mm long petal midribs were at synizesis, while those of 10.5—11 mm and 13.5—14.5 mm long ones were at zytotene and pachytene, respectively. The large anthers of David lily provide thousands of cells that are undergoing synchronous development. Although there are as many as 12 pairs of large chromosomes, they can still be well spread in the squashes of PMCs. Using this material, a study on the
MO^EKyraPHAtf EHOHOraa TOM 44 № 5 2010
DEVELOPMENTAL CHANGES IN DNA METHYLATION
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Fig. 1. Photomicrographs of microsporo
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