Active turnover of genomic methylcytosine in pluripotent cells.

Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known, and the underlying pathways are poorly understood. Here we use metabolic labeling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic 5-methyl-2'-deoxycytidine (mdC), 5-hydroxymethyl-2'-deoxycytidine (hmdC) and 5-formyl-2'-deoxycytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells, the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on ten-eleven translocation (Tet)-mediated mdC oxidation, we unveil additional oxidation-independent mdC turnover, possibly through DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage-committed cells. Thus, in pluripotent cells, active mdC turnover involves both mdC oxidation-dependent and oxidation-independent processes.

Annotation, classification, genomic organization and expression of the Vitis vinifera CYPome.

Cytochromes P450 are enzymes that participate in a wide range of functions in plants, from hormonal signaling and biosynthesis of structural polymers, to defense or communication with other organisms. They represent one of the largest gene/protein families in the plant kingdom. The manual annotation of cytochrome P450 genes in the genome of Vitis vinifera PN40024 revealed 579 P450 sequences, including 279 complete genes. Most of the P450 sequences in grapevine genome are organized in physical clusters, resulting from tandem or segmental duplications. Although most of these clusters are small (2 to 35, median = 3), some P450 families, such as CYP76 and CYP82, underwent multiple duplications and form large clusters of homologous sequences. Analysis of gene expression revealed highly specific expression patterns, which are often the same within the genes in large physical clusters. Some of these genes are induced upon biotic stress, which points to their role in plant defense, whereas others are specifically activated during grape berry ripening and might be responsible for the production of berry-specific metabolites, such as aroma compounds. Our work provides an exhaustive and robust annotation including clear identification, structural organization, evolutionary dynamics and expression patterns for the grapevine cytochrome P450 families, paving the way to efficient functional characterization of genes involved in grapevine defense pathways and aroma biosynthesis.

Identification of a Vitis vinifera endo-ß-1,3-glucanase with antimicrobial activity against Plasmopara viticola.

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis-related (PR) proteins. Endo-ß-1,3-glucanases (EGases) belong to the PR-2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti-P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR-2 family from Vitis.