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.

Mapping genetic loci for tolerance to lime-induced iron deficiency chlorosis in grapevine rootstocks (Vitis sp.).

Iron is essential to plants for chlorophyll formation as well as for the functioning of various iron-containing enzymes. Iron deficiency chlorosis is a wide-spread disorder of plants, in particular, of those growing on calcareous soils. Among the different ways to control iron deficiency problems for crops, plant material and especially rootstock breeding is a suitable and reliable method, especially for fruit trees and grapes. The aim of the experiment was to characterize the genetic basis of grapevine chlorosis tolerance under lime stress conditions. A segregating population of 138 F1 genotypes issued from an inter-specific cross between Vitis vinifera Cabernet Sauvignon (tolerant) × V. riparia Gloire de Montpellier (sensitive) was developed and phenotyped both as cuttings and as rootstock grafted with Cabernet Sauvignon scions in pots containing non-chlorosing and chlorosing soils. Tolerance was evaluated by chlorosis score, leaf chlorophyll content and growth parameters of the shoots and roots. The experiments were performed in 2001, 2003 and 2006. The plants analysed in 2006 were reassessed in 2007. The most significant findings of the trial were: (a) the soil properties strongly affect plant development, (b) there are differences in tolerance among segregating genotypes when grown as cuttings or as rootstocks on calcareous soil, (c) calcareous conditions induced chlorosis and revealed quantitative trait loci (QTLs) implicated in polygenic control of tolerance, (d) rootstock strongly contributes to lime-induced chlorosis response, and (e) a QTL with strong effect (from 10 to 25 % of the chlorotic symptom variance) was identified on chromosome 13. This QTL colocalized with a QTL for chlorophyll content (R (2) = 22 %) and a major QTL for plant development that explains about 50 % of both aerial and root system biomass variation. These findings were supported by stable results among the different years of experiment. These results open new insights into the genetic control of chlorosis tolerance and could aid the development of iron chlorosis-tolerant rootstocks.

Anthocyanin phytochemical profiles and anti-oxidant activities of Vitis candicans and Vitis doaniana.

INTRODUCTION: Grapes are one of the most important fruit crops in the world. The quality of red grape berries greatly depends on skin colour, mainly due to the anthocyanin profile. Today, the American Vitis species have the greatest potential for breeding work. They have multiple resistance properties in comparison with Vitis vinifera but little is known about their anthocyanin content. OBJECTIVE: To determine the anti-oxidant properties and anthocyanin profile of two American species, Vitis candicans and Vitis doaniana, by using LC-MS(n) and LC-NMR. METHODS: Grape extracts were prepared by extraction of berry skins with acidified methanol. The complete structure elucidation of the individual anthocyanins was performed with LC-MS(n) , LC-NMR and NMR experiments. Individual anthocyanins in the extracts were quantified by using malvidin glucoside as external standard. The anti-oxidant activities of grape skin extracts were evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical scavenging and oxygen radical absorbance capacity (ORAC) assays. RESULTS: By using LC-MS(n) and LC-NMR experiments, 30 anthocyanins were identified and quantified in the two Vitis species, including two new cis-p-coumaroyl derivatives. Vitis candicans and V. doaniana showed significant differences in their anthocyanin profile. These two Vitis species possess low-to-medium anti-oxidant activities in comparison with V. vinifera. CONCLUSION: The profiles of 30 anthocyanins were established unambiguously in two American Vitis species.

Contrasting Susceptibilities to Flavescence Dorée in Vitis vinifera, Rootstocks and Wild Vitis Species.

Flavescence dorée (FD) is a quarantine disease of grapevine, involving interactions between the plants, leafhopper vectors, and FD phytoplasma. Characterizing the susceptibility of vine varieties could limit disease propagation. After extensive surveys in vineyards, we showed that Cabernet Sauvignon (CS) is highly susceptible, with a high proportion of symptomatic branches and phytoplasma titers, in contrast to Merlot (M). Localized insect transmissions and grafting showed that phytoplasma circulate in the whole plant in the CS cultivar, but in M they are restricted to the transmission point. Insect-mediated transmission under high confinement mimicking natural conditions confirmed these phenotypes and allowed the classification of 28 Vitis accessions into three distinct categories, according to the percentage of infected plants and their phytoplasma titers. Reduced symptoms, low phytoplasma titers, and low percentages of infected plants were found to be associated in the Vitis vinifera cultivars tested. Interestingly, the low susceptibility of M was observed for one of its parents, i.e., Magdeleine Noire des Charentes. Rootstocks and their Vitis parents, although having high percentages of infected plants and intermediate to high phytoplasma titers, shared a symptomless response. This is troubling, because rootstocks can constitute a silent reservoir of contamination in mother plants or when they grow wild nearby vineyards. Altogether, data suggest distribution of genetic traits within the Vitis genus involved in insect-mediated phytoplasma transmission, multiplication, circulation, and symptom development.

Flavonol profiles in berries of wild Vitis accessions using liquid chromatography coupled to mass spectrometry and nuclear magnetic resonance spectrometry.

The flavonol profiles of grape berry skins were analysed in order to assess phenotypic variation between six grapevines belonging to six different species: Vitis vinifera, Vitiscandicans, Vitischampinii, Vitisamurensis, Vitiscinerea and Vitisdoaniana. High-performance liquid chromatography coupled to mass spectrometry (LC-MS) and NMR spectrometry (LC-NMR) were used to separate and identify the flavonols present in these species. The combination of LC-MS and LC-NMR data resulted in the identification of eighteen flavonols. In particular, the new flavonol diglycoside and pentoside derivatives were determined. In addition, the antioxidant capacities of flavonol grape skin extracts were evaluated by using an oxygen radical absorbance capacity method (ORAC).

Comparative analyses of stilbenoids in canes of major Vitis vinifera L. cultivars.

Grapevine canes are rich in resveratrol and its complex derivatives. These compounds have many biological activities and are needed mainly for health purposes. Canes, which are often wasted, can be used to produce these high-value compounds at low cost. We studied sixteen Vitis vinifera L. cultivars among the most widely cultivated ones worldwide. Polyphenols were extracted from their canes and identified by liquid chromatography-nuclear magnetic resonance spectroscopy. We accurately determined the content of E-ε-viniferin, E-resveratrol, E-piceatannol, and vitisin B and, for the first time, that of hopeaphenol and miyabenol C. The canes did not contain these major stilbene compounds in similar proportions, and their abundance and order of abundance varied according to the cultivar. For instance, Pinot noir has very high levels of E-resveratrol and E-ε-viniferin; Gewurztraminer has very high levels of vitisin B, and Carignan and Riesling have very high levels of hopeaphenol. These findings suggest that the right cultivar should be used to obtain the highest yield of a polyphenol of interest.

Stilbenoid profiles of canes from Vitis and Muscadinia species.

We present stilbenoid profiles of canes from 16 grapevines. Fifteen stilbenoids were obtained through isolation and structure identification using MS, NMR, and [α](D) or as commercial standards. An HPLC-UV method for the simultaneous quantification of nine of these stilbenoids was developed and applied to canes of Vitis amurensis, Vitis arizonica, Vitis berlandieri, Vitis betulifolia, Vitis cinerea, Vitis × champini, Vitis × doaniana, Vitis labrusca, Vitis candicans (syn. Vitis mustangensis), Vitis riparia, Vitis rupestris, Vitis vinifera, Muscadinia rotundifolia, and a V. vinifera × M. rotundifolia hybrid. In these species, E-ampelopsin E, E-amurensin B, E-piceid, E-piceatannol, E-resveratrol, E-resveratroloside, E-ε-viniferin, E-ω-viniferin, and E-vitisin B were quantified, when found in sufficient amounts. Total concentrations ranged from ~2.2 to 19.5 g/kg of dry weight. Additional stilbenoids, E-3,5,4'-trihydroxystilbene 2-C-glucoside, Z-ampelopsin E, Z-trans-miyabenol C, E-trans-miyabenol C, scirpusin A, and Z-vitisin B, were identified but not quantified. Our results indicate that canes, particularly those of non-vinifera species, have substantial quantities of valuable, health-promoting stilbenoids.

Anthocyanin identification and composition of wild Vitis spp. accessions by using LC-MS and LC-NMR.

The composition and concentration of anthocyanins of grape berry skins were analyzed in order to assess phenotypic variation between four grape wine varieties belonging to 4 different species: Vitis vinifera, Vitis amurensis, Vitis cinerea and Vitis X champinii. High-performance liquid chromatography coupled to mass spectrometry (LC-MS) and NMR spectroscopy (LC-NMR) were used to separate and identify the structure of anthocyanins present in these species. Combination of LC-MS and LC-NMR data resulted in the identification of 33 anthocyanins. In particular, newly reported cis isomers of p-coumaric-derivatives were identified (petunidin-, peonidin- and malvidin-3-(6-p-coumaroyl)-5-diglucoside). In V. cinerea and V. vinifera, anthocyanins were monoglucoside derivatives whereas in V. amurensis and V. X champinii, both mono- and diglucoside derivatives were identified. Malvidin-, delphinidin- and petunidin-derivatives were, respectively, the most abundant components in V. cinerea and V. vinifera, V. amurensis and V. X champinii.