Stéphane Maury

The effects of polysaccharide elicitors such as chitin, pectin, and dextran on the production of phenylpropanoids (phenolics and flavonoids) and naphtodianthrones (hypericin and pseudohypericin) in Hypericum perforatum shoot cultures were studied. Nonenzymatic antioxidant properties (NEAOP) and peroxidase (POD) activity were also observed in shoot extracts. The activities of phenylalanine ammonia lyase (PAL) and chalcone-flavanone isomerase (CHFI) were monitored to estimate channeling in phenylpropanoid/flavonoid pathways of elicited shoot cultures. A significant suppression of the production of total phenolics and flavonoids was observed in elicited shoots from day 14 to day 21 of postelicitation. This inhibition of phenylpropanoid production was probably due to the decrease in CHFI activity in elicited shoots. Pectin and dextran promoted accumulation of naphtodianthrones, particularly pseudohypericin, within 21 days of postelicitation. The enhanced accumulation of naphtodianthrone...

Tomato fruit cells are characterized by a strong increase in nuclear ploidy during fruit development. Average ploidy levels increased to similar levels (above 50C) in two distinct fruit tissues, pericarp and locular tissue. However, ploidy profiles differed significantly between these two tissues suggesting a tissue-specific control of endoreduplication in tomato fruit. To determine possible relationships between endoreduplication and epigenetic mechanisms, the methylation status of genomic DNA from pericarp and locular tissue of tomato fruit was analysed. Pericarp genomic DNA was characterized by an increase of CG and/or CNG methylation at the 5S and 18S rDNA loci and at gyspsy-like retrotransposon sequences during fruit growth. A sharp decrease of the global DNA methylation level together with a reduction of methylation at the rDNA loci was also observed in pericarp during fruit ripening. Inversely, no major variation of DNA methylation either global or locus-specific, was observed in locular tissue. Thus, tissue-specific variations of DNA methylation are unlikely to be triggered by the induction of endoreduplication in fruit tissues, but may reflect tissue-specific ploidy profiles. Expression analysis of eight putative tomato DNA methyltransferases encoding genes showed that one chromomethylase (CMT) and two rearranged methyltransferases (DRMs) are preferentially expressed in the pericarp during fruit growth and could be involved in the locus-specific increase of methylation observed at this developmental phase in the pericarp.

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.