The seco-iridoid pathway from Catharanthus roseus

The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications

Rôle des cytochromes P450 dans l’arôme du vin

A thorough annotation of the P450 superfamily in grapevine, revealed its genomic organization, phylogeny and expression. Specifically, we identified genes showing an activated expression in the ripe grape berry, the stage during which the biosynthesis of many aroma compounds takes place. Among the known oxygenated monoterpenols in grapevine, wine lactone has the lowest odor detection threshold and therefore the largest potential impact on wine aroma. We demonstrated that wine lactone is formed during wine ageing via a slow non-enzymatic reaction from the precursor (E)-8-carboxylinalool. We showed that the accumulation of this precursor in grape berries parallels the expression of several cytochrome P450 genes, among which CYP76F14 has the highest expression. While three of them catalyzed some of the oxidative steps from linalool to (E)-8-carboxylinalool, only CYP76F14 efficiently catalyzed the whole pathway. Taken together, CYP76F14 catalytic activity and expression pattern indicate that it is a prime candidate for the formation of the wine lactone precursor in grape berries.L’annotation détaillée de la superfamille des cytochromes P450 dans le génome de la vigne nous a permis d’étudier sa structure génétique, sa phylogénie et son expression, mais aussi d’identifier des gènes dont l’expression est activée dans le grain à maturité, lors de la synthèse de nombreux composés aromatiques. La lactone du vin est la molécule dont le seuil de détection olfactive est le plus bas, ce qui en fait un composant essentiel de l’arôme du vin. Nous avons pu démontrer que cette lactone se forme au cours du vieillissement du vin par une réaction lente et non-enzymatique à partir du 8-carboxylinalool. L’accumulation de ce dernier dans la baie est concomitante à l’expression de plusieurs P450s, dont CYP76F14 est le plus fortement exprimé. Trois enzymes catalysent des étapes d’oxydation conduisant du linalool au (E)-8-carboxylinalool, mais seul CYP76F14 catalyse efficacement la formation de l’acide. Tant par son activité catalytique que son profil d’expression, CYP76F14 apparaît donc comme le responsable le plus probable de la formation du précurseur de la lactone du vin

Meta-analysis of the core aroma components of grape and wine aroma

Wine aroma strongly influences wine quality, yet its composition and its evolution during the winemaking process are poorly understood. Volatile compounds that constitute wine aroma are traditionally divided into three classes according to their origin: grape, fermentation and maturation aroma. We challenge this view with meta-analysis and review of grape and wine volatiles and their precursors from 82 profiling experiments. We compiled a list of 141 common grape and wine volatiles and quantitatively compared 43 of them. Our work offers insight into complex relationships between biosynthesis of aroma in grapes and the changes during the winemaking process. Monoterpenes are one of the largest and most researched wine aroma compounds. We show that their diversity in wines is mainly due to the oxidative metabolism of linalool in grapes. Furthermore, we demonstrate that most of the linalool produced in grapes is converted to these oxidized derivatives

Formation of G‑Wires: The Role of G:C-Base Pairing and G‑Quartet Stacking

G-wireslong, continuous G-quadruplexesare a promising element for use in nanotechnology, particularly in molecular electronics. The pairing of G-quadruplex structures through complementary GC-termini was tested. We designed four analogous G-quadruplex-forming oligonucleotides with the same core sequence d­(GG­T­G₄­T­GG) and with different number and position of GC-ends. We used circular dichroism to determine the topology and relative orientation of G-quadruplexes, ¹H NMR to identify Watson–Crick base pairing, and dynamic light scattering to examine the size of the G-quadruplex assemblies. The quadruplexes associated through GC-base pairs forming (G:C:G:C)-quartets and by stacking of unhindered terminal (G:G:G:G)-quartets. These two modes of association had different specificities: stacking occurred at both the 5′- and 3′- faces of the quadruplex, whereas GC-base pairing occurred only at the 5′-face of the quadruplex. These insights may be employed to design G-quadruplex-based nanowires or more complex architectures

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

International audienceCytochromes 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

Dot matrix of segmental duplications in the physical cluster 92.

<p>Physical cluster 92 is located on chromosome 18 and comprises twenty-two CYP82 sequences, one CYP74 sequence and one CYP704 sequence. The dots and the black lines represent the sequence similarities in cluster <b>92</b> compared to itself. The red rectangles on the sides of the graph represent cytochrome P450 sequences. Complete genes are labeled with their name and pseudogenes are labeled with “p” and the P450 family. A) The similarities for the whole cluster <b>92</b>. B) A zoom of the red squared region, which contains two 30kb blocks with very high similarity. Analysis of gene expression showed that CYP82D15 and CYP82D18 are co-expressed (expression cluster <b>A</b>, expression in ripe berries) as well as CYP82D17 and CYP82D20v2 (expression cluster <b>C</b>, expression in downy mildew infected leaves). The pseudogenes of the enlarged segment are not expressed.</p

Similarity of the P450 genes between and within clusters.

<p>For each circle, the grey bars correspond to the 19 grape chromosomes and the “Unknown chromosome”. The lines connect complete P450 genes according to their similarity. The lines outside the circles show the similarity between genes of the same cluster, whereas the lines in the circle connect similar genes of different clusters. Only P450 genes that form clusters composed of at least two complete genes are illustrated here. The seven largest clusters are labeled with numbers corresponding to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199902#pone.0199902.t001" target="_blank">Table 1</a>. The lines are connecting the genes corresponding to the best BLAST hit (A), second best hit (B) or third best blast hit (C).</p

Physical map of cytochrome P450 sequences on the 19 V. vinifera chromosomes.

<p>Yellow circles represent isolated annotations, light blue circles represent physical clusters composed of members of only one P450 family and the purple circles represent physical clusters composed of members of 2–3 P450 families. The circle size is proportional to the number of sequences in the cluster. The numbers 1–19 are chromosome numbers and “Un” is “Unknown chromosome” which contains sequences with unknown chromosome location.</p

Heatmap of the P450 sequences, clustered according to their expression profile.

<p>The expression levels were averaged over the experiments classified in one of the six experimental categories: leaves, downy mildew (<i>Plasmopara viticola</i>) infected leaves, powdery mildew (<i>Erysiphe necator</i>) infected leaves, flowers, young berries and ripe berries. This heatmap includes the 457 expressed cytochrome P450 sequences. The color scale for the expression level represents FPKM values normalized by row ((FPKM value − row minimum) / row maximum). The color bars on the left are showing the eight expression clusters, which are designated by the letters on the side.</p

Molecular phylogenetic analysis of grapevine cytochrome P450.

<p>The alignment of full-length cytochrome P450 protein sequences was used to generate a maximum likelihood tree. The dark blue clade is the clan 71, which often contains genes involved in specialized metabolism. The highlighted genes belong to the seven largest physical clusters.</p