Genetic analysis of the biosynthesis of 2-methoxy-3-isobutylpyrazine, a major grape-derived aroma compound impacting wine quality.

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.

Thiamine modulates metabolism of the phenylpropanoid pathway leading to enhanced resistance to Plasmopara viticola in grapevine.

BACKGROUND: Previously, we have reported the ability of thiamine (vitamin B1) to induce resistance against Plasmopara viticola in a susceptible grapevine cv. Chardonnay. However, mechanisms underlying vitamins, especially, thiamine-induced disease resistance in grapevine are still largely unknown. Here, we assessed whether thiamine could modulate phenylpropanoid pathway-derived phytoalexins in grapevine plants, as well as, the role of such secondary metabolites in thiamine-induced resistance process to P. viticola. RESULTS: Our data show that thiamine treatment elicited the expression of phenylpropanoid pathway genes in grapevine plants. The expression of these genes correlated with an accumulation of stilbenes, phenolic compounds, flavonoids and lignin. Furthermore, the total anti-oxidant potential of thiamine-treaded plants was increased by 3.5-fold higher level as compared with untreated-control plants. Four phenolic compounds are responsible of 97% of the total anti-oxidant potential of thiamine-treated plants. Among these compounds, is the caftaric acid, belonging to the hydroxy-cinnamic acids family. This element contributed, by its own, by 20% of this total anti-oxidant potential. Epifluorescence microscopy analysis revealed a concomitant presence of unbranched-altered P. viticola mycelia and stilbenes production in the leaf mesophyll of thiamine-treated inoculated plants, suggesting that stilbenes are an important component of thiamine-induced resistance in grapevine. CONCLUSION: This work is the first to show the role of thiamine, as a vitamin, in the modulation of grapevine plant secondary metabolism contributing to an enhanced resistance to P. viticola, the most destructive fungal disease in vineyards.

Structural, functional, and evolutionary analysis of the unusually large stilbene synthase gene family in grapevine.

Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (Vitis vinifera). In addition to their participation in defense mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are postulated to be involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (STSs), which catalyze the biosynthesis of the stilbene backbone, seem to have evolved from chalcone synthases (CHSs) several times independently in stilbene-producing plants. STS genes usually form small families of two to five closely related paralogs. By contrast, the sequence of grapevine reference genome (cv PN40024) has revealed an unusually large STS gene family. Here, we combine molecular evolution and structural and functional analyses to investigate further the high number of STS genes in grapevine. Our reannotation of the STS and CHS gene families yielded 48 STS genes, including at least 32 potentially functional ones. Functional characterization of nine genes representing most of the STS gene family diversity clearly indicated that these genes do encode for proteins with STS activity. Evolutionary analysis of the STS gene family revealed that both STS and CHS evolution are dominated by purifying selection, with no evidence for strong selection for new functions among STS genes. However, we found a few sites under different selection pressures in CHS and STS sequences, whose potential functional consequences are discussed using a structural model of a typical STS from grapevine that we developed.