Christophe Bertsch

Fruit cuticular waxes as a source of biologically active triterpenoids

The health benefits associated with a diet rich in fruit and vegetables include reduction of the risk of chronic diseases such as cardiovascular disease, diabetes and cancer, that are becoming prevalent in the aging human population. Triterpenoids, polycyclic compounds derived from the linear hydrocarbon squalene, are widely distributed in edible and medicinal plants and are an integral part of the human diet. As an important group of phytochemicals that exert numerous biological effects and display various pharmacological activities, triterpenoids are being evaluated for use in new functional foods, drugs, cosmetics and healthcare products. Screening plant material in the search for triterpenoid-rich plant tissues has identified fruit peel and especially fruit cuticular waxes as promising and highly available sources. The chemical composition, abundance and biological activities of triterpenoids occurring in cuticular waxes of some economically important fruits, like apple, grape berry, olive, tomato and others, are described in this review. The need for environmentally valuable and potentially profitable technologies for the recovery, recycling and upgrading of residues from fruit processing is also discussed.

The SWEET family of sugar transporters in grapevine: VvSWEET4 is involved in the interaction with Botrytis cinerea

During plant development, sugar export is determinant in multiple processes such as nectar production, pollen development and long-distance sucrose transport. The plant SWEET family of sugar transporters is a recently identified protein family of sugar uniporters. In rice, SWEET transporters are the target of extracellular bacteria, which have evolved sophisticated mechanisms to modify their expression and acquire sugars to sustain their growth. Here we report the characterization of the SWEET family of sugar transporters in Vitis vinifera. We identified 17 SWEET genes in the V. vinifera 40024 genome and show that they are differentially expressed in vegetative and reproductive organs. Inoculation with the biotrophic pathogens Erysiphe necator and Plasmopara viticola did not result in significant induction of VvSWEET gene expression. However, infection with the necrotroph Botrytis cinerea triggered a strong up-regulation of VvSWEET4 expression. Further characterization of VvSWEET4 revealed that it is a glucose transporter localized in the plasma membrane that is up-regulated by inducers of reactive oxygen species and virulence factors from necrotizing pathogens. Finally, Arabidopsis knockout mutants in the orthologous AtSWEET4 were found to be less susceptible to B. cinerea. We propose that stimulation of expression of a developmentally regulated glucose uniporter by reactive oxygen species production and extensive cell death after necrotrophic fungal infection could facilitate sugar acquisition from plant cells by the pathogen.

Esca et Black Dead Arm : deux acteurs majeurs des maladies du bois chez la Vigne

Grapevine trunk diseases are very harmful to wine-growing heritage durability because the fungi responsible, by attacking perennial organs, cause at a more or less long-term the death of the vine stock. Esca and BDA are the two main pathogens inducing such decaying diseases. The infection can be diagnosed by the presence in the wood forming tissues of sectorial and/or central necrosis, which revealed itself by brown stripes or canker, and at the foliar level by discoloration and withering. This review presents an overview of both Esca and BDA, their symptomatology, the characteristics of the associated fungi and host-pathogen interactions. To conclude, a progress report on the control of both diseases is discussed.

Vitis vinifera VvNPR1.1 is the functional ortholog of AtNPR1 and its overexpression in grapevine triggers constitutive activation of PR genes and enhanced resistance to powdery mildew

Studying grapevine (Vitis vinifera) innate defense mechanisms is a prerequisite to the development of new protection strategies, based on the stimulation of plant signaling pathways to trigger pathogen resistance. Two transcriptional coactivators (VvNPR1.1 and VvNPR1.2) with similarity to Arabidopsis thaliana NPR1 (Non-Expressor of PR genes 1), a well-characterized and key signaling element of the salicylic acid (SA) pathway, were recently isolated in Vitis vinifera. In this study, functional characterization of VvNPR1.1 and VvNPR1.2, including complementation of the Arabidopsis npr1 mutant, revealed that VvNPR1.1 is a functional ortholog of AtNPR1, whereas VvNPR1.2 likely has a different function. Ectopic overexpression of VvNPR1.1 in the Arabidopsis npr1-2 mutant restored plant growth at a high SA concentration, Pathogenesis Related 1 (PR1) gene expression after treatment with SA or bacterial inoculation, and resistance to virulent Pseudomonas syringae pv. maculicola bacteria. Moreover, stable overexpression of VvNPR1.1-GFP in V. vinifera resulted in constitutive nuclear localization of the fusion protein and enhanced PR gene expression in uninfected plants. Furthermore, grapevine plants overexpressing VvNPR1.1-GFP exhibited an enhanced resistance to powdery mildew infection. This work highlights the importance of the conserved SA/NPR1 signaling pathway for resistance to biotrophic pathogens in V. vinifera.

Genetic chimerism of Vitis vinifera cv. Chardonnay 96 is maintained through organogenesis but not somatic embryogenesis

BackgroundGrapevine can be a periclinal chimera plant which is composed at least of two distinct cell layers (L1, L2). When the cell layers of this plant are separated by passage through somatic embryogenesis, regenerated plants could show distinct DNA profiles and a novel phenotype which proved different from that of the parent plant.ResultsGenetically Chardonnay clone 96 is a periclinal chimera plant in which is L1 and L2 cell layers are distinct. Plants obtained via organogenesis through meristematic bulks are shown to be composed of both cell layers. However, plants regenerated through somatic embryogenesis starting from anthers or nodal explants are composed only of L1 cells. These somaclones do not show phenotypic differences to the parental clone up to three years after regeneration. Interestingly, the only somaclone showing an atypical phenotype (asymmetric leave) shows a genotypic modification.ConclusionThese results suggest that the phenotype of Chardonnay 96 does not result from an interaction between the two distinct cell layers L1 and L2. If phenotype conformity is further confirmed, somatic embryogenesis will result in true-to-type somaclones of Chardonnay 96 and would be well suitable for gene transfer.

Extracellular compounds produced by fungi associated with Botryosphaeria dieback induce differential defence gene expression patterns and necrosis in Vitis vinifera cv. Chardonnay cells

Three major grapevine trunk diseases, esca, botryosphaeria dieback and eutypa dieback, pose important economic problems for vineyards worldwide, and currently, no efficient treatment is available to control these diseases. The different fungi associated with grapevine trunk diseases can be isolated in the necrotic wood, but not in the symptomatic leaves. Other factors seem to be responsible for the foliar symptoms and may represent the link between wood and foliar symptoms. One hypothesis is that the extracellular compounds produced by the fungi associated with grapevine trunk diseases are responsible for pathogenicity.In the present work, we used Vitis vinifera cv. Chardonnay cells to test the aggressiveness of total extracellular compounds produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with botryosphaeria dieback. Additionally, the toxicity of purified mellein, a characteristic toxin present in the extracellular compounds of Botryosphaeriaceae, was assessed.Our results show that the total extracellular compounds produced by N. parvum induce more necrosis on Chardonnay calli and induce a different defence gene expression pattern than those of D. seriata. Mellein was produced by both fungi in amounts proportional to its aggressiveness. However, when purified mellein was added to the culture medium of calli, only a delayed necrosis and a lower-level expression of defence genes were observed. Extracellular compounds seem to be involved in the pathogenicity of the fungi associated with botryosphaeria dieback. However, the doses of mellein used in this study are 100 times higher than those found in the liquid fungal cultures: therefore, the possible function of this toxin is discussed.

Toxicity of extracellular proteins from Diplodia seriata and Neofusicoccum parvum involved in grapevine Botryosphaeria dieback

Botryosphaeria dieback, esca and Eutypa dieback are three economic major grapevine trunk diseases that cause severe yield reduction in vineyards worldwide. The frequency of disease symptoms has increased considerably over the past decade, and no efficient treatment is currently available to control these diseases. The different fungi associated with grapevine trunk diseases mainly induce necrotic wood and characteristic foliar symptoms. In this context, fungi virulence factors and host invasion are not well understood. We hypothesise that extracellular proteins produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with Botryosphaeria dieback, are virulence factors responsible for the pathogenicity. In our previous work, we demonstrated that the total extracellular compounds produced by N. parvum induced more necrosis on Chardonnay calli and triggered a different defence gene expression pattern than those produced by D. seriata. Furthermore, this aggressiveness was not clearly correlated with the production of mellein, a characteristic phytotoxin of Botryosphaeriaceae, in our in vitro calli model. To characterise other potential virulence factors and to understand the mechanisms of host invasion by the fungus, we evaluated the profile, quantity and the impact of extracellular proteins produced by these fungi on Vitis vinifera calli necrosis and defence gene expression. Our results reveal that, under the same conditions, N. parvum produces more extracellular proteins and in higher concentrations than D. seriata. With Vitis vinifera cv. Chardonnay cells, we showed that equivalent concentrations of proteins secreted by N. parvum were more aggressive than those of D. seriata in producing necrosis and that they clearly induced more grapevine defence genes.

Low level of polymorphism in two putative NPR1 homologs in the Vitaceae family.

BackgroundGrapevine is subjected to numerous pests and diseases resulting in the use of phytochemicals in large quantities. The will to decrease the use of phytochemicals leads to attempts to find alternative strategies, implying knowledge of defence mechanisms. Numerous studies have led to the identification of signalling pathways and regulatory elements involved in defence in various plant species. Nonexpressor of Pathogenesis Related 1 (NPR1) is an important regulatory component of systemic acquired resistance (SAR) in Arabidopsis thaliana.ResultsTwo putative homologs of NPR1 gene were found in the two sequenced grapevine genomes available in the Genoscope database for line 40024 and in the IASMA database for Pinot noir ENTAV 115. We named these two NPR1 genes of Vitis vinifera : VvNPR1.1 and VvNPR1.2. A PCR-based strategy with primers designed on exons was used to successfully amplify NPR1 gene fragments from different Vitaceae accessions. Sequence analyses show that NPR1.1 and NPR1.2 are highly conserved among the different accessions not only V. vinifera cultivars but also other species. We report nucleotide polymorphisms in NPR1.1 and NPR1.2 from fifteen accessions belonging to the Vitaceae family. The ratio of nonsynonymous to synonymous nucleotide substitutions determines the evolutionary pressures acting on the Vitaceae NPR1 genes. These genes appear to be experiencing purifying selection. In some of the species we have analysed one of the two alleles of NPR1.1 contains a premature stop codon. The deduced amino acid sequences share structural features with known NPR1-like proteins: ankyrin repeats, BTB/POZ domains, nuclear localization signature and cysteines. Phylogenetic analyses of deduced amino acid sequences show that VvNPR1.1 belongs to a first group of NPR1 proteins known as positive regulators of SAR and VvNPR1.2 belongs to a second group of NPR1 proteins whose principal members are AtNPR3 and AtNPR4 defined as negative regulators of SAR.ConclusionOur study shows that NPR1.1 and NPR1.2 are highly conserved among different accessions in the Vitaceae family. VvNPR1.1 and VvNPR1.2 are phylogenetically closer to the group of positive or negative SAR regulators respectively.ReviewersThis article was reviewed by Fyodor Kondrashov, Purificación López-García and George V. Shpakovski.

Impact of Quillaja saponaria saponins on grapevine ecosystem organisms

The control of grapevine pathogens is a rising concern in Vitis vinifera culture. The current international trend is toward banning chemicals that are highly toxic to the environment and human workers, and adopting tighter regulations. We evaluated the impact of saponins on three kinds of organisms found in grapevine culture. The ectoparasitic nematode Xiphinema index, the parasitic fungus Botrytis cinerea and various yeast strains representative of the must fermentation population were incubated on synthetic media supplemented with variable concentrations of Quillaja saponaria saponins. Saponins induced reduction in the growth of B. cinerea and showed nematicide effects on X. index. The control of X. index and Botrytis cinerea is discussed in the context of the potential use of these chemicals as environmentally-friendly grapevine treatments. With Saccharomyces cerevisiae and other yeasts, saponins showed higher toxicity against S. cerevisiae strains isolated from wine or palm wine whereas laboratory strains or strains isolated from oak exhibited better resistance. This indicates that Q. saponaria saponins effects against yeast microflora should be assessed in the field before they can be considered an environmentally-safe new molecule against B. cinerea and X. index.

Characterization of triterpenoid profiles and triterpene synthase expression in the leaves of eight Vitis vinifera cultivars grown in the Upper Rhine Valley

Plant triterpenoids are a diverse group of secondary metabolites with wide distribution, high chemical diversity and interesting pharmacological and antimicrobial properties. The first step in the biosynthesis of all triterpenoids is the cyclization of the 2,3-oxidosqualene precursor, catalyzed by oxidosqualene cyclases (OSCs), which have characteristic product specificities. Biosynthesis and functions of pentacyclic triterpenes have been poorly studied in grapevine. In this study, we first investigated the profile of triterpenoids present in leaf cuticular waxes from eight Vitis vinifera cultivars cultivated in the Upper Rhine Valley. Further quantification of triterpenoids showed that these cultivars can be divided into two groups, characterized by high levels of lupeol (e.g., Pinot noir) or taraxerol (e.g., Gewurztraminer) respectively. We further analyzed the OSC family involved in the synthesis of pentacyclic triterpenes (called VvTTPSs) in the sequenced V. vinifera 40024 genome and found nine genes with similarity to previously characterized triterpene synthases. Phylogenetic analysis further showed that VvTTPS1–VvTTPS3 and VvTTPS5–VvTTPS9 belong to the β-amyrin synthase and multifunctional triterpene synthase clade, whereas VvTTPS10 belongs to the lupeol synthase clade. We studied the expression of several members of the VvTTPS family following biotic and abiotic stresses in V. vinifera 40024 as well as in the eight healthy cultivars. This study further revealed that one candidate gene, VvTTPS5, which does not belong to the lupeol synthase clade, is highly expressed in lupeol-rich cultivars. VvTTPS3, VvTTPS5, VvTTPS6, VvTTPS7 and VvTTPS10 were highly upregulated by UV stress, but only VvTTPS3, VvTTPS5, VvTTPS6 and VvTTPS10 were upregulated following downy mildew and gray mold infections respectively. These results suggest differential roles of VvTTPS against environmental stresses in grape leaves.