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Degree of DNA methylation in chicory (Cichorium intybus L.): influence of plant age and vernalization
Plant Science 142 (1999) 101 – 108
Degree of DNA methylation in chicory (Cichorium intybus L.): influence of plant age and vernalization M.A.C. Demeulemeester *, N. Van Stallen, M.P. De Proft Faculty of Agricultural and Applied Biological Sciences, Department of Applied Plant Sciences, Laboratory for Plant Culture, Katholieke Uni6ersiteit Leu6en, Willem de Croylaan 42, B-3001 He6erlee, Belgium Received 31 August 1998; accepted 12 January 1999
Abstract Chicory plants (Cichorium intybus L. var. foliosum cv. Flash) were harvested every 14 days from 43 to 168 days after sowing. The tissue was either immediately analyzed or after a 4 week post-harvest cold treatment at 5°C. DNA methylation was measured, using high pressure liquid chromatography (HPLC), in the root tissue as well as in the shoot apices. During the growing season, the degree of methylation ranged between 10 and 16% for root tissue as well as for shoot apices. The reaction (methylation/ demethylation) to cold treatment depended on the tissue type and the plant age. For root tissue, demethylation was observed after cold treatment in the beginning of the growing season followed by methylation in the middle part and strong demethylation at the end of the season. Except for two harvest dates, a demethylation of DNA in the shoot apex tissue occurred during cold treatment. Demethylation was strongest for the two last harvest dates. This is the first report on changes in degree of DNA methylation during a whole growing season and differences in the effect of cold depending on the age of a biennial plant. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cichorium intybus L. var. foliosum cv. Flash; Chicory; DNA methylation; Methylcytosine; Plant age; Vernalization
1. Introduction Chicory is a cold requiring (thermoinduction), obligatory long day plant for flower induction [1–5]. Thermoinduction of flowering is an epigenetic process, restricted to a single sexual generation [6]. Each generation requires vernalization, i.e. the response to cold is not transmitted to the progeny, suggesting that the program is reset during gamete formation [7]. Although mechanisms for the epigenetic control of gene expression are not well understood, there is increasing evidence that the pattern of DNA methylation is involved * Corresponding author. Tel.: + 32-16-32-26-61; fax: + 32-16-3229-66. E-mail address: [email protected] (M.A.C. Demeulemeester)
[8]. Other characteristics of vernalization are consistent with the response being mediated by changes in DNA. For example, non-dividing cells do not respond to cold treatment, indicating that at least cell division and probably DNA replication are essential for cells to be receptive to cold [9]. The response is not mediated by a diffusible product because vernalization occurs only when the cells giving rise to the floral meristem are subjected to cold [10]. Evidence that DNA demethylation is involved in the process of vernalization has been obtained from the use of 5-azacytidine. Treatment of plant cells with 5-azacytidine lowers the methylation level of DNA [11]. Burn et al. [6] showed that in Arabidopsis thaliana and Thlaspi ar6ense, two plant species in which low temperature induces
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early flowering, treatment with 5-azacytidine induced nonvernalized plants to flower significantly earlier than untreated controls. Normal flowering time was reset in the progeny of plants induced to flower early with 5-azacytidine, paralleling the lack of inheritance of the vernalized condition. Arabidopsis plants, treated either with cold or 5-azacytidine, had reduced levels of 5-methylcytosine (mC) in their DNA compared to nonvernalized plants. Vernalization, through its general demethylation effect, released the blockage to flowering initiation. It was proposed that demethylation is necessary for a gene controlling flowering in order to be transcribed [6]. Research on Thlaspi showed that the control affects transcription of kaurenoic acid hydroxylase, a key enzyme in the gibberellin biosynthesis pathway [12]. In Brassica, it was found that treatment of germinating seeds with 5-azacytidine causes earlier flowering [13,14]. The purpose of the present study was to investigate the degree of DNA methylation of chicory root tissue and shoot apices during the growing season. The influence of a cold period on DNA methylation in relation to age of the plants is also investigated. As the growth pattern of chicory plants can differ significantly depending on the growing season, plant age can not be defined by the harvest date only. For that reason, the development of the plants was characterized by their growth.
2. Materials and methods
2.1. Plant material Chicory plants of the cultivar Flash (INRA, France) were raised from seeds sown in May on ridges in a local field suitable for endive production. Harvests occurred approximately every 14 days starting from 21 June until 24 October. The cultural practices were those commonly used for the commercial production of chicory plants. To characterize the plants after harvest, 30 plants were used for measuring: (a) number of leaves in the rosette; (b) length of the longest leaf in the rosette; (c) fresh weight of the rosette; and (d) fresh weight of the root (cut at 18 cm length measured from the shoot apex). Fresh weights were determined using a balance with 0.1 g precision. Lengths were measured at 0.1 cm precision.
From these data, mean 9standard error (S.E.) was calculated. Instead of using the harvest dates to present the data, ‘days after sowing’ were used in the figures. A trend curve was fitted through the data to show the development during the growing season. However, the exact data were considered to draw conclusions. At each harvest date, 800 plants were lifted. Part of the whole plants was used immediately for analysis of the degree of DNA methylation, while the other part was stripped of leaves and stored at 5°C for 4 weeks in the dark under plastic foil before being used for analysis.
2.2. Sample preparation From 5×15 plants per harvest date, the root segment 1–3 cm below the shoot apex was cut into fragments and mixed to form a large sample. This root segment never included the root apex. Samples were immediately frozen in liquid nitrogen and stored at −20°C before lyophilization. After lyophilization, the samples were ground (Janke & Kunkel KG mixer, IKA Werk, Germany) until a fine powder was obtained and stored in a box with silicagel at 20°C. To isolate the apices (from 5×60 plants per harvest date), all the leaves were excised from plants until only leaflets smaller than 2 mm were left. Then the shoot was cut from the root and the apex isolated as precisely as possible (0.5–1 mm). The apices were frozen in liquid nitrogen and stored at −20°C, lyophilized, ground (Janke & Kunkel KG mixer, IKA Werk, Germany) into a fine powder and stored in a box with silicagel at 20°C.
2.3. DNA extraction The protocol used was a modification of the cetyltrimethylammoniumbromide (CTAB) extraction method by Murray and Thompson [15] to isolate total cellular DNA. Proteins were eliminated by extraction with chloroform, whereas carbohydrates were discarded after the precipitation of the CTAB–DNA complexes by lowering the salt concentration. Concentration and purity of DNA were determined spectrophotometrically (A260 for DNA, A230 for polysaccharides and A280 for proteins). Five DNA extractions (from the five different batches of plants) were carried out for each type of plant material.
M.A.C. Demeulemeester et al. / Plant Science 142 (1999) 101–108
103
Fig. 1. Mean number of leaves per rosette9 S.E. (n= 30) ( ) and mean length of the longest leaf per rosette (in cm)9S.E. (n =30) (") for the chicory cultivar Flash during the growing season.
2.4. DNA hydrolysis Approximately 40 mg DNA was hydrolyzed to bases in 50 ml of 70% perchloric acid (100°C for 1 h). The pH was adjusted to between 3 and 5 with KOH (1 M). The resulting KClO4 precipitate was washed twice with 200 ml distilled water and the total hydrolysate reduced to dryness in a speedvac before high pressure liquid chromatography
(HPLC) analysis [6,16]. DNA hydrolysis was carried out for each of the five DNA samples extracted from each type of plant material.
2.5. HPLC The bases from the hydrolysis of 40 mg DNA were redissolved in 250 ml sodium acetate (10 mmol, pH 4). After 8 min centrifugation at 10 000
Fig. 2. Mean fresh weight of the total rosette (in g)9S.E. (n= 30) ( ) and mean fresh weight of root (in g)9S.E. (n=30) (") for the chicory cultivar Flash during the growing season.
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Fig. 3. Degree of methylation 9S.E. (n= 5) measured in the root tissue at various harvest dates, before and after vernalization, during the growing season for the chicory cultivar Flash.
rpm, the supernatant was filtered through a 4 mm, nonsterile syringe filter with pore size 0.2 mm (Alltech, Belgium). An aliquot of the filtrate (at least 200 ml) was injected on the HPLC using a 100 ml sample loop. The bases were separated on a Partisil 10 SCX (4.6 ×250 mm; Phenomenex, Sopachem, Belgium) column at 60°C with sodium acetate (10 mM, pH 4) as eluent and a flow rate of 2 ml/min [16]. The absorbance of the bases was measured at 280 nm with a UV/VIS detector (Waters, Millipore, Belgium). The chromatogram was further analyzed using WINILAB2 computer software (INTERSMAT, Belgium). The standard was a mixture of the five bases: thymine, guanine, adenine, cytosine and mC in a concentration of 2 nM/100 ml each and was used for all measurements. By comparing peak areas of similar retention times, the unknown concentrations of cytosine and mC in a sample were calculated and the percentage of mC was calculated as (concentration of mC×100)/(concentration of mC+ concentration of cytosine). All analyses were repeated five times (from five separate DNA extractions) and the mean 9S.E. was calculated. In addition, statistical analysis using a one- and twofactor analysis in combination with Duncan’s test (on a 95% confidence interval, C.I.) was carried out.
3. Results
3.1. Characterization of plants The maximum number of leaves per rosette (25) was reached around 120–140 days after sowing. At the final harvest date, the mean number of leaves had begun to decrease due to senescence of the oldest leaves. Mean length of the longest leaf per rosette was maximal (34.5 cm) 100 days after sowing and then remained constant (Fig. 1). Maximum fresh weight of the total rosette (120 g) was reached 126 days after sowing after which fresh weight decreased due to loss of leaves as a result of senescence. Mean fresh root weight reached its maximum (150 g) 140 days after sowing (Fig. 2).
3.2. Degree of DNA methylation in root tissue and shoot apices during the growing season (without cold treatment) The degree of DNA methylation during the growing season was assayed for root tissue as well as shoot apex tissue. Degree of DNA methylation of the root tissue fluctuated between 10 and 16% (Fig. 3). A relatively high degree of methylation occurred at the beginning of the season, followed by a decline 85 days after sowing and then an
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105
Table 1 Results of Duncan’s test (on a 95% confidence interval, C.I.) for the percentage of DNA methylation in root tissue during the growing season without cold treatment Days after sowing 43
58
70
85
100
112
126
140
154
168
% mCa
13.8
15.5
14.7
10.8
12.0
12.0
8.9
13.5
13.2
10.6
Duncan grouping
ab b
a
a b c
b c
a b
a b
c d a b
d
c d
mC, methylcytosine. Mean separation by Duncan’s multiple range test at a= 0.05.
increase at 140 days after sowing. A low degree of DNA methylation was observed at 126 days after sowing. Results of Duncan’s test are given in Table 1. Between 43 and 126 days after sowing, degrees of methylation in shoot apices were similar, ranging between 8 and 13% (Fig. 4). Towards the end of the season, methylation increased to 16.6%. Degree of methylation at 140 days after sowing was not measured (loss of the sample due to freezer failure). Results of Duncan’s test are given in Table 2.
3.3. Degree of DNA methylation in root tissue and shoot apices after cold treatment The changes in degree of methylation as a result of the cold (vernalization) treatment were considered for both the root and the shoot apex tissue. At the first three harvests, a slight decrease in degree of methylation in cold treated roots compared to non-cold treated roots was observed (Fig. 3). At 126 days after sowing, when the degree of methylation of non-cold treated root tissue decreased during the growing season, an increase in methylation, even up to 20% mC was measured after cold treatment. For the last two harvest dates there was a very strong decrease in degree of methylation after cold treatment. Two factor statistical analysis indicated that both the ‘cold treatment’ factor and the ‘harvest date’ factor were significant (on a 95% C.I.) in the model. The two factors showed a clear interaction. The changes in DNA methylation observed after cold treatment were statistically significant for all harvest dates except 43, 70 and 168 days after sowing.
Except at the harvests 58 and 85 days after sowing, demethylation of DNA in shoot apex tissue was observed after cold treatment compared to non-cold treated tissue (Fig. 4). Demethylation was strongest for the two last harvest dates. Degree of methylation after 4 weeks cold treatment applied at 112 days after sowing was not measured (loss of sample due to freezer failure). Two factor statistical analysis indicated that only for 43, 126, 154 and 168 days after sowing, statistical significant differences between DNA methylation before and after cold treatment of shoot apices could be detected.
4. Discussion The vegetative growth of a chicory plant can be divided into two phases. In the first part, leaf biomass starts to develop from 45 days after sowing. The number of leaves (and thus also leaf biomass) reaches a maximum around 120 days after sowing. By the formation of this rosette (assimilate tissue), sufficient photosynthetic capacity is built up to produce storage sugars (inulin) [17]. Afterwards the leaf number (and thus the leaf biomass) decreases because of senescence of the oldest leaves. In the second part (which overlaps partly the first one), especially from 2 to 5 months after sowing, an increase in fresh root weight occurs. The maximum root yield in biomass is achieved around 160 days after sowing [18]. The data obtained in this study are in agreement with these generalizations in literature.
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Fig. 4. Degree of methylation 9S.E. (n =5) measured in the shoot apex tissue at various harvest dates, before and after vernalization, during the growing season for the chicory cultivar Flash.
Chicory plants have an intermediate degree of DNA methylation (10.6% in mature root tissue and 16.6% in shoot apices of mature plants) when compared to other plant species, e.g. 33% in Secale cereale and 20% in roots of Solanum esculentum, but only 4.6% in Arabidopsis thaliana [6,8,19]. However, the degree of methylation is high enough to allow accurate measurements using HPLC. The methylation degree of shoot apices was, until 112 days after sowing, lower than in the root tissue (no cold treatment). At 126 days after sowing, the degree of methylation in root tissue was very low (8.9%). Although the S.E. of the first sampling (43 days) of the root tissue was high (Fig. 3), pair-wise differences between sampling dates were found according to Duncan’s test results (Table 1). This indicates that the degree of methylation fluctuates during the growing season, presumably due to methylation as well as demethylation of the DNA. Methylation degree can differ between tissues of a plant and between developmental stages of a certain tissue, e.g. in Solanum esculentum [19]. It has been described that environmental stress factors (e.g. heat, drought) can have an influence on the DNA methylation level [8]. As plants were grown in the field, influences of the environment cannot be excluded. However, weather conditions could be considered as normal during this growing season (data not shown) which resulted in normal plant development (Figs. 1 and 2).
The difference in methylation response to the cold treatment depending on the plant age has not been studied in other plant species. However, it seems to be an important phenomenon in chicory. The observation that the methylation/demethylation response to the cold treatment varies depending on plant age and tissue hints to the presence of a developmentally or other regulated program. The strong decrease in methylation level observed after a cold treatment at the end of the growing season was parallel with the decrease in leaf number (and thus leaf biomass) in the rosette due to senescence of the plant. This could indicate a special phase in the life cycle of the plant preparing itself to survive the winter period. It is known that the sensitivity of chicory with respect to bolting and flowering changes during the growing season. In order to be induced to flower, chicory must undergo a low temperature treatment, which can be applied to germinating seeds or to the entire plant at the end of the first growing season [1,2,4]. There is a period in the field when the chicory plant reaches physiological maturity and at that time, and not before, a cold treatment will prepare the root to initiate either a shoot (vegetable) which is the item of commerce, or bolting and flowering under natural conditions in the field. Plants harvested at 154 and 168 days showed marked reductions in methylation in response to cold. This demethylation could be ‘uncovering’ genes associated with bolting in the apex (Fig. 4)
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107
Table 2 Results of Duncan’s test (on a 95% confidence interval, C.I.) for the percentage of DNA methylation in shoot apex tissue during the growing season without cold treatment days after sowing
% mCa
43 11.6
58 9.7
70 12.8
85 8.4
100 11.0
112 10.9
126 12.0
b c
b c
b c
140 –c
154 13.5
Duncan grouping
168 16.6 a
bb c
b c d
b
d
a
mC, methylcytosine. Mean separation by Duncan’s multiple range test at a= 0.05. c –, not measured. b
and genes associated with enzymes necessary for hydrolysis and transport of sugars and possibly hormones from the root to the shoot meristem (Fig. 3). This interpretation is especially favored by the fact that roots of plants harvested at days 85, 100, 112, 126 and 140 after sowing showed just the opposite response to cold. Cold increased the degree of methylation and thus presumably ‘covered’ genes and might predictably induce a kind of rest or metabolic inactivity in the root tissue. These findings are in agreement with the research results of Burn et al. [6] who found that both cold-treated and 5-azacytidine-treated Arabidopsis plants had reduced levels of 5-mC in their DNA compared to nonvernalized plants. These treatments led to significantly earlier flowering than in untreated controls. It was proposed that demethylation is necessary for a gene critical for flowering in order to be transcribed [6]. Research on Thlaspi showed that the treatments affect transcription of kaurenoic acid hydroxylase, a key enzyme in the gibberellin biosynthesis pathway [12]. It has been shown that gibberellins are responsible for the bolting reaction in chicory [20,21]. A pattern in methylation/demethylation of shoot apex DNA after cold treatment during the growing season was not clearly observed. Similar to the root tissue, strong demethylation occurred after cold applied at the end of the growing season (Fig. 4). Cold was applied on plants after harvest and stripping of leaves, as under routine storage conditions. This includes the possibility that not only the cold treatment is responsible for the changes in the methylation level. No data about influence of harvest and storage as such were found in literature, while the influence of cold has been studied extensively [6,8,11].
By study of the degree of DNA methylation of roots and shoot apices during the growing season, in combination with a cold treatment of the chicory plant, an important factor described to influence floral expression was considered. Special attention is given to the period at the end of the growing season where the decrease in leaf biomass is observed, parallel with strong demethylation in response to a cold treatment. Although the hypothesis that demethylation of specific genes in response to cold is involved in flowering of plants requiring vernalization will not be definitively established until the genes are identified and cloned [8]; the data reported in Figs. 3 and 4 are consistent with the general hypothesis (cold treatment at the time of maximum sensitivity to vernalization caused marked demethylation) and the change in DNA methylation occurred both in the tissue giving a visible response (apex, bolting) to vernalization and in the storage tissue (root) which supplied substrate for the response. Work to identify and clone the specific genes which may regulate bolting and flowering will continue.
Acknowledgements The first author is Postdoctoral Fellow of the Fund for Scientific Research-Flanders (Belgium) (F.W.O.) and the research was supported by a N.F.W.O.-grant 2.0079.93 and a K.U.L.-grant OT/91/18. The authors thank L. Sagi for the technique of DNA extraction, J. Vanderleyden and J. Desair for the technique of DNA hydrolysis and the National Experimental Station for Chicory in Herent (Belgium) for the growing of
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the plants. The authors also wish to thank P. Morgan (Texas A&M University) and A. Limami (INRA, France) for the critical reading of the text.
[11] [12]
References [1] Th. Hartman, After effects of low temperature on leaf morphology of Cichorium intybus L., Proc. van de Koninklijke Nederlandse Akademie voor Wetenschappen 59 (1956) 677–684. [2] L. Rappaport, S.H. Wittwer, Stimulation of flowering by vernalization of endive seedlings. A preliminary report, Proc. Am. Soc. Hort. Sci. 67 (1956) 438–439. [3] J. Doorenbos, P.C. Riemens, Effect of vernalization and daylength on number and shape of leaves in chicory and endive, Acta Botanica Neerlandica 8 (1959) 63–67. [4] J.F. Harrington, K. Verkerk, J. Doorenbos, Interaction of vernalization, photoperiod and light intensity in floral initiation of endive, Neth. J. Agri. Sci. 7 (1959) 68 – 74. [5] P. Paulet, in: A.H. Halevy (Ed.), Cichorium intybus and Cichorium endi6ia, CRC Handbook of Flowering, Boca Raton, FL, 1985, pp. 265–271. [6] J.E. Burn, D.J. Bagnall, J.D. Metzger, E.S. Dennis, W.J. Peacock, DNA methylation, vernalization and the initiation of flowering, Proc. Natl. Acad. Sci. USA 90 (1993) 287–291. [7] A. Lang, Physiology of flower initiation, in: W. Ruhland (Ed.), Encyclopaedia Plant Physiology 15 (Pt.1), Springer Verlag, Berlin, 1965, pp. 1379–1536. [8] E.J. Finnegan, R.I.S. Bretell, E.S. Dennis, The role of DNA methylation in the regulation of plant gene expression, in: J.P. Jost, H.P. Saluz (Eds.), DNA Methylation: Molecular Biology and Biological Significance, Birkha¨user Verlag, Basel, 1993, pp. 218–261. [9] S.J. Wellensiek, Dividing cells as the prerequisite for vernalization, Plant Physiol. 39 (1964) 832–835. [10] J.D. Metzger, Localization of the site of perception of
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thermoinductive temperatures in Thlaspi ar6ense L., Plant Physiol. 88 (1988) 424 – 428. P.A. Jones, Altering gene expression with 5-azacytidine, Cell 40 (1985) 485 – 486. J.P. Hazebroek, J.D. Metzger, Thermoinductive regulation of gibberellin metabolism in Thlaspi ar6ense L. I. Metabolism of (2H)-ent-kaurenoic acid and (14C) gibberellin A12-aldehyde, Plant Physiol. 94 (1990) 157–165. G.J. King, The role of DNA methylation in modulation of floral initiation, Flowering Newslett. 18 (1994) 21–25. G.J. King, Morphological development in Brassica oleracea is modulated by in 6i6o treatment with 5-azacytidine, J. Horticult. Sci. 70 (1995) 333 – 342. H.G. Murray, W.F. Thompson, Rapid isolation of high molecular weight DNA, Nucleic Acids Res. 8 (1980) 4321 – 4325. D.S. Fairweather, M. Fox, G.P. Margison, The in 6itro lifespan of MRC-5 cells is shortened by 5-azacytidine-induced demethylation, Exp. Cell Res. 168 (1987) 153–159. W. Van den Acker, M. Demeulemeester and M. De Proft, Morfologie en fysiologie van de plant, in: De witloofteelt, Ministerie van Landbouw, Brussel, Dienst Inf, 1993, pp. 13 – 23. J.P. Cochet, M. Marle, Appre´ciation du Stade Physiologique de la Racine d’Endive, Bull. Liaison Endiviers 5 (1987) 27 – 32. R. Messegeur, W.G. Martin, J.C. Steffens, S.D. Tanksley, Characterization of the level, target sides and inheritance of cytosine methylation in tomato nuclear DNA, Plant Mol. Biol. 16 (1991) 753 – 770. M.A.C. Demeulemeester, W. Rademacher, A. Van de Mierop, M.P. De Proft, Influence of gibberellin biosynthesis inhibitors on stem elongation and floral initiation on in vitro chicory root explants under dark and light conditions, Plant Growth Regul. 17 (1995) 47 – 52. M.A.C. Demeulemeester, A. Voet, M.P. De Proft, Induction of stem elongation on in vitro chicory root explants under unfavorable photoperiodic conditions by gibberellin A3, Plant Growth Regul. 16 (1995) 239 – 241.
Degree of DNA methylation in chicory (Cichorium intybus L.): influence of plant age and vernalization M.A.C. Demeulemeester *, N. Van Stallen, M.P. De Proft Faculty of Agricultural and Applied Biological Sciences, Department of Applied Plant Sciences, Laboratory for Plant Culture, Katholieke Uni6ersiteit Leu6en, Willem de Croylaan 42, B-3001 He6erlee, Belgium Received 31 August 1998; accepted 12 January 1999
Abstract Chicory plants (Cichorium intybus L. var. foliosum cv. Flash) were harvested every 14 days from 43 to 168 days after sowing. The tissue was either immediately analyzed or after a 4 week post-harvest cold treatment at 5°C. DNA methylation was measured, using high pressure liquid chromatography (HPLC), in the root tissue as well as in the shoot apices. During the growing season, the degree of methylation ranged between 10 and 16% for root tissue as well as for shoot apices. The reaction (methylation/ demethylation) to cold treatment depended on the tissue type and the plant age. For root tissue, demethylation was observed after cold treatment in the beginning of the growing season followed by methylation in the middle part and strong demethylation at the end of the season. Except for two harvest dates, a demethylation of DNA in the shoot apex tissue occurred during cold treatment. Demethylation was strongest for the two last harvest dates. This is the first report on changes in degree of DNA methylation during a whole growing season and differences in the effect of cold depending on the age of a biennial plant. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cichorium intybus L. var. foliosum cv. Flash; Chicory; DNA methylation; Methylcytosine; Plant age; Vernalization
1. Introduction Chicory is a cold requiring (thermoinduction), obligatory long day plant for flower induction [1–5]. Thermoinduction of flowering is an epigenetic process, restricted to a single sexual generation [6]. Each generation requires vernalization, i.e. the response to cold is not transmitted to the progeny, suggesting that the program is reset during gamete formation [7]. Although mechanisms for the epigenetic control of gene expression are not well understood, there is increasing evidence that the pattern of DNA methylation is involved * Corresponding author. Tel.: + 32-16-32-26-61; fax: + 32-16-3229-66. E-mail address: [email protected] (M.A.C. Demeulemeester)
[8]. Other characteristics of vernalization are consistent with the response being mediated by changes in DNA. For example, non-dividing cells do not respond to cold treatment, indicating that at least cell division and probably DNA replication are essential for cells to be receptive to cold [9]. The response is not mediated by a diffusible product because vernalization occurs only when the cells giving rise to the floral meristem are subjected to cold [10]. Evidence that DNA demethylation is involved in the process of vernalization has been obtained from the use of 5-azacytidine. Treatment of plant cells with 5-azacytidine lowers the methylation level of DNA [11]. Burn et al. [6] showed that in Arabidopsis thaliana and Thlaspi ar6ense, two plant species in which low temperature induces
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early flowering, treatment with 5-azacytidine induced nonvernalized plants to flower significantly earlier than untreated controls. Normal flowering time was reset in the progeny of plants induced to flower early with 5-azacytidine, paralleling the lack of inheritance of the vernalized condition. Arabidopsis plants, treated either with cold or 5-azacytidine, had reduced levels of 5-methylcytosine (mC) in their DNA compared to nonvernalized plants. Vernalization, through its general demethylation effect, released the blockage to flowering initiation. It was proposed that demethylation is necessary for a gene controlling flowering in order to be transcribed [6]. Research on Thlaspi showed that the control affects transcription of kaurenoic acid hydroxylase, a key enzyme in the gibberellin biosynthesis pathway [12]. In Brassica, it was found that treatment of germinating seeds with 5-azacytidine causes earlier flowering [13,14]. The purpose of the present study was to investigate the degree of DNA methylation of chicory root tissue and shoot apices during the growing season. The influence of a cold period on DNA methylation in relation to age of the plants is also investigated. As the growth pattern of chicory plants can differ significantly depending on the growing season, plant age can not be defined by the harvest date only. For that reason, the development of the plants was characterized by their growth.
2. Materials and methods
2.1. Plant material Chicory plants of the cultivar Flash (INRA, France) were raised from seeds sown in May on ridges in a local field suitable for endive production. Harvests occurred approximately every 14 days starting from 21 June until 24 October. The cultural practices were those commonly used for the commercial production of chicory plants. To characterize the plants after harvest, 30 plants were used for measuring: (a) number of leaves in the rosette; (b) length of the longest leaf in the rosette; (c) fresh weight of the rosette; and (d) fresh weight of the root (cut at 18 cm length measured from the shoot apex). Fresh weights were determined using a balance with 0.1 g precision. Lengths were measured at 0.1 cm precision.
From these data, mean 9standard error (S.E.) was calculated. Instead of using the harvest dates to present the data, ‘days after sowing’ were used in the figures. A trend curve was fitted through the data to show the development during the growing season. However, the exact data were considered to draw conclusions. At each harvest date, 800 plants were lifted. Part of the whole plants was used immediately for analysis of the degree of DNA methylation, while the other part was stripped of leaves and stored at 5°C for 4 weeks in the dark under plastic foil before being used for analysis.
2.2. Sample preparation From 5×15 plants per harvest date, the root segment 1–3 cm below the shoot apex was cut into fragments and mixed to form a large sample. This root segment never included the root apex. Samples were immediately frozen in liquid nitrogen and stored at −20°C before lyophilization. After lyophilization, the samples were ground (Janke & Kunkel KG mixer, IKA Werk, Germany) until a fine powder was obtained and stored in a box with silicagel at 20°C. To isolate the apices (from 5×60 plants per harvest date), all the leaves were excised from plants until only leaflets smaller than 2 mm were left. Then the shoot was cut from the root and the apex isolated as precisely as possible (0.5–1 mm). The apices were frozen in liquid nitrogen and stored at −20°C, lyophilized, ground (Janke & Kunkel KG mixer, IKA Werk, Germany) into a fine powder and stored in a box with silicagel at 20°C.
2.3. DNA extraction The protocol used was a modification of the cetyltrimethylammoniumbromide (CTAB) extraction method by Murray and Thompson [15] to isolate total cellular DNA. Proteins were eliminated by extraction with chloroform, whereas carbohydrates were discarded after the precipitation of the CTAB–DNA complexes by lowering the salt concentration. Concentration and purity of DNA were determined spectrophotometrically (A260 for DNA, A230 for polysaccharides and A280 for proteins). Five DNA extractions (from the five different batches of plants) were carried out for each type of plant material.
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Fig. 1. Mean number of leaves per rosette9 S.E. (n= 30) ( ) and mean length of the longest leaf per rosette (in cm)9S.E. (n =30) (") for the chicory cultivar Flash during the growing season.
2.4. DNA hydrolysis Approximately 40 mg DNA was hydrolyzed to bases in 50 ml of 70% perchloric acid (100°C for 1 h). The pH was adjusted to between 3 and 5 with KOH (1 M). The resulting KClO4 precipitate was washed twice with 200 ml distilled water and the total hydrolysate reduced to dryness in a speedvac before high pressure liquid chromatography
(HPLC) analysis [6,16]. DNA hydrolysis was carried out for each of the five DNA samples extracted from each type of plant material.
2.5. HPLC The bases from the hydrolysis of 40 mg DNA were redissolved in 250 ml sodium acetate (10 mmol, pH 4). After 8 min centrifugation at 10 000
Fig. 2. Mean fresh weight of the total rosette (in g)9S.E. (n= 30) ( ) and mean fresh weight of root (in g)9S.E. (n=30) (") for the chicory cultivar Flash during the growing season.
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Fig. 3. Degree of methylation 9S.E. (n= 5) measured in the root tissue at various harvest dates, before and after vernalization, during the growing season for the chicory cultivar Flash.
rpm, the supernatant was filtered through a 4 mm, nonsterile syringe filter with pore size 0.2 mm (Alltech, Belgium). An aliquot of the filtrate (at least 200 ml) was injected on the HPLC using a 100 ml sample loop. The bases were separated on a Partisil 10 SCX (4.6 ×250 mm; Phenomenex, Sopachem, Belgium) column at 60°C with sodium acetate (10 mM, pH 4) as eluent and a flow rate of 2 ml/min [16]. The absorbance of the bases was measured at 280 nm with a UV/VIS detector (Waters, Millipore, Belgium). The chromatogram was further analyzed using WINILAB2 computer software (INTERSMAT, Belgium). The standard was a mixture of the five bases: thymine, guanine, adenine, cytosine and mC in a concentration of 2 nM/100 ml each and was used for all measurements. By comparing peak areas of similar retention times, the unknown concentrations of cytosine and mC in a sample were calculated and the percentage of mC was calculated as (concentration of mC×100)/(concentration of mC+ concentration of cytosine). All analyses were repeated five times (from five separate DNA extractions) and the mean 9S.E. was calculated. In addition, statistical analysis using a one- and twofactor analysis in combination with Duncan’s test (on a 95% confidence interval, C.I.) was carried out.
3. Results
3.1. Characterization of plants The maximum number of leaves per rosette (25) was reached around 120–140 days after sowing. At the final harvest date, the mean number of leaves had begun to decrease due to senescence of the oldest leaves. Mean length of the longest leaf per rosette was maximal (34.5 cm) 100 days after sowing and then remained constant (Fig. 1). Maximum fresh weight of the total rosette (120 g) was reached 126 days after sowing after which fresh weight decreased due to loss of leaves as a result of senescence. Mean fresh root weight reached its maximum (150 g) 140 days after sowing (Fig. 2).
3.2. Degree of DNA methylation in root tissue and shoot apices during the growing season (without cold treatment) The degree of DNA methylation during the growing season was assayed for root tissue as well as shoot apex tissue. Degree of DNA methylation of the root tissue fluctuated between 10 and 16% (Fig. 3). A relatively high degree of methylation occurred at the beginning of the season, followed by a decline 85 days after sowing and then an
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105
Table 1 Results of Duncan’s test (on a 95% confidence interval, C.I.) for the percentage of DNA methylation in root tissue during the growing season without cold treatment Days after sowing 43
58
70
85
100
112
126
140
154
168
% mCa
13.8
15.5
14.7
10.8
12.0
12.0
8.9
13.5
13.2
10.6
Duncan grouping
ab b
a
a b c
b c
a b
a b
c d a b
d
c d
mC, methylcytosine. Mean separation by Duncan’s multiple range test at a= 0.05.
increase at 140 days after sowing. A low degree of DNA methylation was observed at 126 days after sowing. Results of Duncan’s test are given in Table 1. Between 43 and 126 days after sowing, degrees of methylation in shoot apices were similar, ranging between 8 and 13% (Fig. 4). Towards the end of the season, methylation increased to 16.6%. Degree of methylation at 140 days after sowing was not measured (loss of the sample due to freezer failure). Results of Duncan’s test are given in Table 2.
3.3. Degree of DNA methylation in root tissue and shoot apices after cold treatment The changes in degree of methylation as a result of the cold (vernalization) treatment were considered for both the root and the shoot apex tissue. At the first three harvests, a slight decrease in degree of methylation in cold treated roots compared to non-cold treated roots was observed (Fig. 3). At 126 days after sowing, when the degree of methylation of non-cold treated root tissue decreased during the growing season, an increase in methylation, even up to 20% mC was measured after cold treatment. For the last two harvest dates there was a very strong decrease in degree of methylation after cold treatment. Two factor statistical analysis indicated that both the ‘cold treatment’ factor and the ‘harvest date’ factor were significant (on a 95% C.I.) in the model. The two factors showed a clear interaction. The changes in DNA methylation observed after cold treatment were statistically significant for all harvest dates except 43, 70 and 168 days after sowing.
Except at the harvests 58 and 85 days after sowing, demethylation of DNA in shoot apex tissue was observed after cold treatment compared to non-cold treated tissue (Fig. 4). Demethylation was strongest for the two last harvest dates. Degree of methylation after 4 weeks cold treatment applied at 112 days after sowing was not measured (loss of sample due to freezer failure). Two factor statistical analysis indicated that only for 43, 126, 154 and 168 days after sowing, statistical significant differences between DNA methylation before and after cold treatment of shoot apices could be detected.
4. Discussion The vegetative growth of a chicory plant can be divided into two phases. In the first part, leaf biomass starts to develop from 45 days after sowing. The number of leaves (and thus also leaf biomass) reaches a maximum around 120 days after sowing. By the formation of this rosette (assimilate tissue), sufficient photosynthetic capacity is built up to produce storage sugars (inulin) [17]. Afterwards the leaf number (and thus the leaf biomass) decreases because of senescence of the oldest leaves. In the second part (which overlaps partly the first one), especially from 2 to 5 months after sowing, an increase in fresh root weight occurs. The maximum root yield in biomass is achieved around 160 days after sowing [18]. The data obtained in this study are in agreement with these generalizations in literature.
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Fig. 4. Degree of methylation 9S.E. (n =5) measured in the shoot apex tissue at various harvest dates, before and after vernalization, during the growing season for the chicory cultivar Flash.
Chicory plants have an intermediate degree of DNA methylation (10.6% in mature root tissue and 16.6% in shoot apices of mature plants) when compared to other plant species, e.g. 33% in Secale cereale and 20% in roots of Solanum esculentum, but only 4.6% in Arabidopsis thaliana [6,8,19]. However, the degree of methylation is high enough to allow accurate measurements using HPLC. The methylation degree of shoot apices was, until 112 days after sowing, lower than in the root tissue (no cold treatment). At 126 days after sowing, the degree of methylation in root tissue was very low (8.9%). Although the S.E. of the first sampling (43 days) of the root tissue was high (Fig. 3), pair-wise differences between sampling dates were found according to Duncan’s test results (Table 1). This indicates that the degree of methylation fluctuates during the growing season, presumably due to methylation as well as demethylation of the DNA. Methylation degree can differ between tissues of a plant and between developmental stages of a certain tissue, e.g. in Solanum esculentum [19]. It has been described that environmental stress factors (e.g. heat, drought) can have an influence on the DNA methylation level [8]. As plants were grown in the field, influences of the environment cannot be excluded. However, weather conditions could be considered as normal during this growing season (data not shown) which resulted in normal plant development (Figs. 1 and 2).
The difference in methylation response to the cold treatment depending on the plant age has not been studied in other plant species. However, it seems to be an important phenomenon in chicory. The observation that the methylation/demethylation response to the cold treatment varies depending on plant age and tissue hints to the presence of a developmentally or other regulated program. The strong decrease in methylation level observed after a cold treatment at the end of the growing season was parallel with the decrease in leaf number (and thus leaf biomass) in the rosette due to senescence of the plant. This could indicate a special phase in the life cycle of the plant preparing itself to survive the winter period. It is known that the sensitivity of chicory with respect to bolting and flowering changes during the growing season. In order to be induced to flower, chicory must undergo a low temperature treatment, which can be applied to germinating seeds or to the entire plant at the end of the first growing season [1,2,4]. There is a period in the field when the chicory plant reaches physiological maturity and at that time, and not before, a cold treatment will prepare the root to initiate either a shoot (vegetable) which is the item of commerce, or bolting and flowering under natural conditions in the field. Plants harvested at 154 and 168 days showed marked reductions in methylation in response to cold. This demethylation could be ‘uncovering’ genes associated with bolting in the apex (Fig. 4)
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107
Table 2 Results of Duncan’s test (on a 95% confidence interval, C.I.) for the percentage of DNA methylation in shoot apex tissue during the growing season without cold treatment days after sowing
% mCa
43 11.6
58 9.7
70 12.8
85 8.4
100 11.0
112 10.9
126 12.0
b c
b c
b c
140 –c
154 13.5
Duncan grouping
168 16.6 a
bb c
b c d
b
d
a
mC, methylcytosine. Mean separation by Duncan’s multiple range test at a= 0.05. c –, not measured. b
and genes associated with enzymes necessary for hydrolysis and transport of sugars and possibly hormones from the root to the shoot meristem (Fig. 3). This interpretation is especially favored by the fact that roots of plants harvested at days 85, 100, 112, 126 and 140 after sowing showed just the opposite response to cold. Cold increased the degree of methylation and thus presumably ‘covered’ genes and might predictably induce a kind of rest or metabolic inactivity in the root tissue. These findings are in agreement with the research results of Burn et al. [6] who found that both cold-treated and 5-azacytidine-treated Arabidopsis plants had reduced levels of 5-mC in their DNA compared to nonvernalized plants. These treatments led to significantly earlier flowering than in untreated controls. It was proposed that demethylation is necessary for a gene critical for flowering in order to be transcribed [6]. Research on Thlaspi showed that the treatments affect transcription of kaurenoic acid hydroxylase, a key enzyme in the gibberellin biosynthesis pathway [12]. It has been shown that gibberellins are responsible for the bolting reaction in chicory [20,21]. A pattern in methylation/demethylation of shoot apex DNA after cold treatment during the growing season was not clearly observed. Similar to the root tissue, strong demethylation occurred after cold applied at the end of the growing season (Fig. 4). Cold was applied on plants after harvest and stripping of leaves, as under routine storage conditions. This includes the possibility that not only the cold treatment is responsible for the changes in the methylation level. No data about influence of harvest and storage as such were found in literature, while the influence of cold has been studied extensively [6,8,11].
By study of the degree of DNA methylation of roots and shoot apices during the growing season, in combination with a cold treatment of the chicory plant, an important factor described to influence floral expression was considered. Special attention is given to the period at the end of the growing season where the decrease in leaf biomass is observed, parallel with strong demethylation in response to a cold treatment. Although the hypothesis that demethylation of specific genes in response to cold is involved in flowering of plants requiring vernalization will not be definitively established until the genes are identified and cloned [8]; the data reported in Figs. 3 and 4 are consistent with the general hypothesis (cold treatment at the time of maximum sensitivity to vernalization caused marked demethylation) and the change in DNA methylation occurred both in the tissue giving a visible response (apex, bolting) to vernalization and in the storage tissue (root) which supplied substrate for the response. Work to identify and clone the specific genes which may regulate bolting and flowering will continue.
Acknowledgements The first author is Postdoctoral Fellow of the Fund for Scientific Research-Flanders (Belgium) (F.W.O.) and the research was supported by a N.F.W.O.-grant 2.0079.93 and a K.U.L.-grant OT/91/18. The authors thank L. Sagi for the technique of DNA extraction, J. Vanderleyden and J. Desair for the technique of DNA hydrolysis and the National Experimental Station for Chicory in Herent (Belgium) for the growing of
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the plants. The authors also wish to thank P. Morgan (Texas A&M University) and A. Limami (INRA, France) for the critical reading of the text.
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