DNA methylating and demethylating treatments modify phenotype and cell wall differentiation state in sugarbeet cell lines.

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 organogenic 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.

Identification and quantification of hypericin and pseudohypericin in different Hypericum perforatum L. in vitro cultures.

Investigations have been made to develop an efficient protocol for micropropagation allowing to improve hypericin and pseudohypericin productions in Hypericum perforatum L. in vitro cultures. The role of growth regulator treatments has been particularly studied. Three in vitro culture lines with different morphological characteristics were obtained during H. perforatum micropropagation and referred to shoots, calli and plantlets according to their appearance. Multiplication and callogenesis from apical segments from sterile germinated seedlings were obtained on solid MS/B5 culture medium in the presence of N6-benzyladenine (BA) (0.1-5.0 mg/l BA). Regenerative potential of shoots was assessed on medium supplemented with auxins (0.05-1.0 mg/l), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). The main goal of the research was to summarize the influence of plant growth regulators on hypericin and pseudohypericin productions in in vitro cultures of Hypericum. A rapid method for naphtodianthrone quantification was developed. The use of a reversed-phase high performance liquid chromatography (HPLC) method with fluorescence detection was used. Identification of the compounds was confirmed by electrospray ionization-mass spectrometry (ESI-MS) with electrospray in negative ion mode [M-H] . Calli, shoots and plantlets of H. perforatum produced hypericin and pseudohypericin. The concentration range of BA from 0.1 to 2.0 mg/l improved the production of hypericin (25-50 microg/g dry mass (DM)) and pseudohypericin (170-350 microg/g DM) in shoots. In callus cultures, BA (4.0-5.0 mg/l) did not changed hypericin contents (15-20 microg/g DM) but influenced pseudohypericin productions (120-180 microg/g DM). In the presence of auxins (IAA and IBA), Hypericum plantlets produced hypericin (30-100 microg/g DM) and pseudohypericin (120-400 microg/g DM). The presence of IAA did not influence naphtodianthrone productions in plantlets, but IBA decreased hypericin and pseudohypericin amounts in plantlets. The specific accumulation of the naphtodianthrones in in vitro cultures was influenced by phytohormonal supplementation of the medium. Results indicated that the production of hypericin and pseudohypericin could be increased by carefully adapted in vitro cultures. Hypericum in vitro cultures represent promising systems for hypericin and pseudohypericin productions.