Development of a transient expression system in grapevine via agro-infiltration.

Transient expression of genes using Agrobacterium is a powerful tool for the analysis of gene function in plants. We have developed this method for the analysis of genes involved in disease resistance in grapevine leaves. Our research showed that the quality of the plant material, the plant genotype used for agro-infiltration and the presence of additional virulence factors (carried on plasmid pCH32) in the Agrobacterium strain are all important factors for success of the procedure. After optimising these factors, we consistently achieve sufficient acceptable levels of expression of the markers beta-glucuronidase (GUS) and green fluorescent protein (GFP) using vacuum infiltration of grapevine leaves from plants grown in vitro. We used this procedure to investigate the proposed role of stilbenes in defense against grapevine downy mildew (Plasmopara viticola) by transiently overexpressing stilbene synthase in grapevine leaves, before infection with P. viticola. We found that agro-infiltration itself induces the synthesis of stilbenes in grapevine leaves, thus preventing us to test the effect of the overexpression of stilbene synthase in defense. However, our results revealed that agro-infiltration before P. viticola inoculation had an effect on the development of the infection. Further research is required to show whether stilbenes or some other factor are the causal agent restricting pathogen development. The method described here provides and excellent tool to exploit at the many grapevine genomic resources now available, and will contribute to a better understanding of many areas of grapevine biology.

Characterisation of a new potyvirus species infecting meadow saffron Colchicum autumnale).

The alkaloids contained in Colchicum autumnale seeds are used in numerous medicines. Good quality seeds are difficult to obtain from this undomesticated plant. Therefore, a research program was set up aiming to cultivate C. autumnale in order to improve alkaloid contents and seed yields. In this context, a collection was established in 1999 by transplanting corms from twelve different locations in Eastern France. However, serious symptoms of necrosis and decay have appeared in this collection since 2001. Electron microscopic observations of plants showing symptoms revealed the presence of filamentous particles and pinwheel-like structures characteristic of the Potyviridae family. Leaves and corms from symptomatic plants were assayed with potyvirus-specific Enzyme-Linked Immunosorbent Assay test. Positive reactions were obtained with plants from all the geographic origins, which exhibited flower breaking symptoms on petals. RT-PCR tests with family Potyviridae-specific primers confirmed the ELISA results and showed that the virus can be detected in corms, roots and flowers of symptomatic plants. The 3' region of the genome was cloned, sequenced and compared to other potyvirus species. Phylogenetic analyses suggest the presence of a new viral species tentatively named Meadow saffron breaking virus (MSBV) in C. autumnale.

Effect of light on hypericins contents in fresh flowering top parts and in an extract of St. John's Wort (Hypericum perforatum).

St. John's Wort is a medicinal plant increasingly used for its antidepressive activity. Hypericins are considered as one of the compounds contributing to the activity of the extract. These naphthodianthrones exist in various forms in Hyperici herba. Protopseudohypericin and protohypericin (protopigments) are converted into pseudohypericin and hypericin (pigments) under the action of light. The aim of this work is to study the influence of light on the phototransformation of protopigments into pigments. Two experiments were carried out. The studies were performed on one hand, on plant material in order to know the proportion of these substances in various plant parts and the possibility of transforming the protopigments into pigments under the action of sunlight; on the other hand, in the extract to determine the optimal wavelength allowing this transformation. Three parts of the fresh plant (buds, flowers, leaves) were treated with sunlight on three levels of exposure. Liquid extracts were exposed to various types of light with wavelengths ranging between 480 and 660 nm by means of diodes. The flowering tops of St. John's Wort contain a share of approximately 30% hypericins in the form of protopseudohypericin and protohypericin: buds (48%), flowers (30%), leaves (17%). After an exposure of fresh buds to sunlight for 16 hours the share of protopigments was then 32%. In the extract, the transformation of the protopigments is total and requires less energy than in the plant material. The optimal wavelength for the transformation of the protopigments in the extract is around 515 nm (green), close to the optimum absorption level of protopigments.

Improved procedure for the quality control of Hypericum perforatum L.

A new, fast and reliable procedure for the quantification of the major compounds of Hypericum perforatum L. has been developed. Four naphthodianthrones (protopseudohypericin, pseudohypericin, protohypericin, hypericin) and two phloroglucinols (hyperforin, adhyperforin) were assayed by HPLC using a short (17 min) linear gradient, with hypericin and hyperforin as external standards. Extraction of dried plant material with methanol in the dark at room temperature for 2 h led to a complete recovery of phloroglucinols but only a partial recovery of the naphthodianthrone derivatives. Treatment of plant material with water:ethanol (40:60, v/v) in a water bath shaker at 80 degrees C led to the total extraction of hypericins, but a 10% loss of total hyperforins was also observed. The two extraction methods, applied successively to the same sample, allowed the complete extraction of all compounds of interest. A 5 min exposure of the crude extract of H. perforatum to sunlight (1 E/m2) induced a 96% loss of hyperforins, whereas the dry plant material lost only 20% of hyperforins after 2 h exposure to sunlight (24 E/m2).