Original article DNA methylating and demethylating treatments modify phenotype and cell wall differentiation state in sugarbeet cell lines Adisa Causevic a,b,c , Alain Delaunay a , Saïda Ounnar d , Michel Righezza d , Francis Delmotte a , Franck Brignolas a , Daniel Hagège a , Stéphane Maury a, * a Laboratoire de Biologie des Ligneux et des Grandes Cultures, UPRES EA, 1207, rue de Chartres, BP 6759, Faculté des Sciences, Université d’Orléans, 45067 Orléans cedex 2, France b Department of Biology, University of Sarajevo, 71000 Sarajevo, Bosnia-Herzegovina c Institute for Genetic Engineering and Biotechnology, 71000 Sarajevo, Bosnia-Herzegovina d Laboratoire de Biologie des Ligneux et des Grandes Cultures, UPRES EA 1207, Antenne Scientifique Universitaire de Chartres, 21, rue de Loigny La Bataille, Faculté des Sciences, Université d’Orléans, 28000 Chartres, France Received 19 April 2005; accepted 31 May 2005 Available online 05 July 2005 Abstract In plants organogenesis, cell differentiation and dedifferentiation are fundamental processes allowing high developmental plasticity. Such plasticity involved epigenetic mechanisms but limited knowledge is available concerning quantitative aspects. Three sugarbeet (Beta vulgaris L. altissima) cell lines originating from the same mother plant and exhibiting graduate states of morphogenesis were used to assess whether these differences could be related or not to changes in DNA methylation levels. Methylcytosine percentages from 18.3 to 28.8% and distinct levels of DNA methyltransferase (EC 2.1.1.37) activities were shown in the three cell lines. The lowest methylcytosine percentage was associated to organogenesis. In order to test the plasticity of these cell lines, various treatments causing DNA hypo or hypermethylation were performed at different times and concentrations. In this collection of treated lines with ± 10% of methylcytosine percentages, loss of organo- genic properties and cell dedifferentiation were observed. As cell wall formation fits well with cell differentiation state, the lignification process was further investigated in treated and untreated lines as a biochemical marker of the phenotypic changes. For example, peroxidase specific activities (EC 1.11.1.7) varied from 0.7 to 0.02 pkat mg –1 of protein in organogenic and dedifferentiated lines, respectively.A negative relationship between peroxidase activities, incorporation of cell wall-bound phenolic compounds as ferulate and sinapate derivatives and methylcytosine percentages was obtained. This is the first biochemical evidence that phenotypic changes in plant cells induced by DNA hypo- or hypermethylating treatments are correlated in a linear relationship to modifications of the cell wall differentiation state. © 2005 Elsevier SAS. All rights reserved. Keywords: Cell wall; Methylcytosine; Differentiation; Morphogenesis; Phenolic; Sugarbeet 1. Introduction Plant development is strongly influenced by environmen- tal signals that can induce local and systemic changes in genomic programming [21,23]. This developmental plastic- ity may be expressed phenotypically by growth modifica- tions, cell differentiation or dedifferentiation. Plants employ epigenetic regulatory strategies to maintain this plasticity. This allows relatively rapid adaptation to new conditions without changing the DNA sequence. There have been several progresses in linking cytosine methylation states to cell lin- eage or species and developmental progression in both plants and animals [10,16,35,36]. In plants, methylcytosine percent- ages varied between 5% in Arabidopsis to 37% in Helianthus [12,26] and few reports established on different plant sys- tems showed modifications of morphogenetic properties, accompanied by variations of methylcytosine percentage dur- Abbreviations: DD, dedifferentiated callus line; DNMT, DNA methyl- transferase; EDTA, ethylenediaminotetra-acetate; HPLC, high-performance liquid chromatography; LC/APCI-MS, liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry; MC, methylcytosine; NaCl, sodium chloride; NO, non organogenic callus line; O, organogenic callus line; SDS, sodium dodecyl sulfate; SE, standard error. * Corresponding author. Tel./fax: +33 2 38 41 70 22. E-mail address: stephane.maury@univ-orleans.fr (S. Maury). Plant Physiology and Biochemistry 43 (2005) 681–691 www.elsevier.com/locate/plaphy 0981-9428/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2005.05.011