Exogenous application of a lipid transfer protein-jasmonic acid complex induces protection of grapevine towards infection by Botrytis cinerea.

Type I plant lipid transfer proteins (LTPs) are small, basic, cystein-rich proteins involved in plant defense mechanisms. Five type I LTPs isoforms, named VvLTP1, 2, 3, 4 and 5 (Vitis vinifera lipid transfer proteins 1-5) were purified to homogeneity from the culture media of 41B grapevine cell suspension. The full sequence of isoforms 1, 3, 4 and 5 could be determined from mass spectrometry measurements of the enzymatically hydrolyzed proteins and from available VvLTP sequences. Phylogenetic analysis revealed that these proteins form two subgroups, one with isoforms 1 and 4, and the second one with isoforms 3 and 5. The ability of the three most abundant ones (VvLTP1, 4 and 3) to interact with jasmonic acid (JA) was tested by fluorometric studies, showing that VvLTP4 was the most efficient to interact with this oxylipin. Exogenous application of the VvLTP4-JA complex on grapevine plantlets induced a high level (80.3+/-10.05%) of tolerance towards Botrytis cinerea, as compared with control plants (18.65+/-12.13%); whereas plants treated with JA or VvLTP4 alone exhibited a lower protection level (31.04+/-9.72% and 45.52+/-7.51% of protection, respectively). The results are discussed in the context of grapevine defense mechanisms.

In vitro tolerance to Botrytis cinerea of grapevine 41B rootstock in transgenic plants expressing the stilbene synthase Vst1 gene under the control of a pathogen-inducible PR 10 promoter.

Resveratrol is a major phytoalexin in grapevine but its synthesis in response to phytopathogen attack decreases with grape berry ripening. A chimeric gene combining an alfalfa PR 10 promoter and Vst1 (Vitis stilbene synthase 1) gene was introduced into the genome of 41B rootstock. Transgenic plants were analysed for resveratrol production in leaves infected with Botrytis using an in vitro test. Among the 50 transgenic lines analysed, some exhibited a production lower than the non-transgenic control, but others accumulated resveratrol from 5-100-fold. Moreover, in the latter clones, symptoms were highly reduced in response to infection. These results were a good indication that the combination of a pathogen-inducible promoter and a defence gene may increase tolerance against fungi in grapevine. The efficacy of this approach should be further tested by experiments conducted in the vineyard.

Cloning and expression of a hexose transporter gene expressed during the ripening of grape berry.

The ripening of grape (Vitis vinifera L.) is characterized by massive sugar import into the berries. The events triggering this process and the pathways of assimilate transport are still poorly known. A genomic clone Vvht1 (Vitis vinifera hexose transporter1) and the corresponding cDNA encoding a hexose transporter whose expression is induced during berry ripening have been isolated. Vvht1 is expressed mainly in the berries, with a first peak of expression at anthesis, and a second peak about 5 weeks after véraison (a viniculture term for the inception of ripening). Vvht is strictly conserved between two grape cultivars (Pinot Noir and Ugni-Blanc). The organization of the Vvht1 genomic sequence is homologous to that of the Arabidopsis hexose transporter, but differs strongly from that of the Chlorella kessleri hexose transporter genes. The Vvht1 promoter sequence contains several potential regulating cis elements, including ethylene-, abscisic acid-, and sugar-responsive boxes. Comparison of the Vvht1 promoter with the promoter of grape alcohol dehydrogenase, which is expressed at the same time during ripening, also allowed the identification of a 15-bp consensus sequence, which suggests a possible co-regulation of the expression of these genes. The expression of Vvht1 during ripening indicates that sucrose is at least partially cleaved before uptake into the flesh cells.

Four 9-kDa proteins excreted by somatic embryos of grapevine are isoforms of lipid-transfer proteins.

Four 9-kDa small extracellular proteins produced by embryogenic cultures in the absence of auxin have been purified from the extracellular medium of grapevine somatic embryo cultures through cation-exchange chromatography and hydrophobic-interaction chromatography. The partial amino-acid sequences reflect high similarities between the four proteins as well as with the sequences established for carrot, spinach, millet and maize nonspecific lipid-transfer proteins. All these sequences show conservation of three cysteines at positions 4, 14 and 30-32, as well as glycine, valine, tyrosine and lysine residues at positions 5, 7, 17 and 37, respectively. In-vitro lipid-transfer assays reveal that the four proteins catalyze the transfer of phosphatidylcholine from liposomes towards mitochondria with an efficiency similar or higher than that of a purified maize lipid-transfer protein.

Embryogenic and non-embryogenic cell lines of Daucus carota cloned from meristematic cell clusters: relation with cell ploidy determined by flow cytometry.

The presence of totipotent and non-totipotent cells in embryogenic carrot cell suspension cultures was examined by cloning of cell microclusters. Forty clones were isolated and the distribution of their embryogenic potential was studied. Nonembryogenic, weakly and highly embryogenic cell lines were selected. After one year of subculture a second cloning round showed that the highly embryogenic and the non-embryogenic cell lines were homogenous and stable. A measurement of ploidy levels of clones by flow cytometry showed that the embryogenic clones were all diploid whereas the non-embryogenic were diploid or tetraploid. Hence, for our strain, there was a strict relationship between the tetraploid state and the inability to produce somatic embryos.