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.The pleasure experienced while enjoying a glass of wine is the result of sophisticated sensory, neurophysiological, and psychological processes triggered by wine aroma. Wine flavor is the result of a complex mixture of volatile compounds in the headspace of the glass that induces feelings of pleasure at the brain level (Shepherd, 2006). During the last 40 years, over 800 volatile molecules have been formally identified in wines, in concentrations ranging from hundreds of milligrams per liter down to a few picograms per liter (Ebeler and Thorngate, 2009; Styger et al., 2011). Among all of them, a relatively limited number of compounds, called varietal (or primary) aromas, play a crucial role in wine flavor and typicality. These aromas, which are related to the grape variety, belong to a limited number of chemical families, including monoterpenes, C13 norisoprenoids, volatile sulfur compounds, and methoxypyrazines (MPs; Ebeler and Thorngate, 2009). Quite frequently, they exist mostly in the grape (Vitis vinifera) berry as nonvolatile, odorless, “bound” forms that can be released by chemical and enzymatic reactions occurring during the winemaking and wine aging processes, thus enhancing wine’s varietal expression (Styger et al., 2011). Two classical examples are the glycoside precursors of the monoterpenols (Strauss et al., 1986) and the cysteinylated or glutathionylated precursors of the volatile thiols (Tominaga et al., 1998; Peña-Gallego et al., 2012). Noticeable exceptions are the MPs, which are found in grape berries exclusively as free, volatile molecules.MPs are strongly odorant volatile heterocycles, with vegetable-like fragrances, that are widely occurring in the plant kingdom (Maga, 1982). In grape, they can be detected in fruits, leaves, shoots, and roots (Dunlevy et al., 2010). They are found in different grape varieties and are particularly abundant in the so-called Bordeaux cultivars (i.e. cv Cabernet Franc, Cabernet Sauvignon [CS], Sauvignon Blanc, Merlot, and Carménère [Car]; Bayonove et al., 1975; Lacey et al., 1991; Roujou de Boubée et al., 2002; Belancic and Agosin, 2007), whereas they are rarely detected in other cultivars, such as cv Pinot Noir (PN), Chardonnay, or Petit Verdot (PV). This finding indicates a strong genotype dependency of MP biosynthesis (Koch et al., 2010). MPs are accumulated in berries until bunch closure or véraison, and then their level declines after véraison (Hashizume and Samuta, 1999; Ryona et al., 2008). MP concentration in wine is highly correlated with the grape berry content at harvest (Roujou de Boubée et al., 2002). Three MPs are found in grape berries: 2-methoxy-3-isobutylpyrazine (IBMP), which is the most abundant, and two others, 2-methoxy-3-isopropylpyrazine (IPMP) and 2-methoxy-3-sec-butylpyrazine (SBMP; Ebeler and Thorngate, 2009). Both IBMP and IPMP display very low sensory detection thresholds in the wine matrix, ranging from 1 to 16 ng L^–1.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 (Dubourdieu et al., 2006; Lund et al., 2009). Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these extremely powerful odorants will reduce consumer acceptance (Parr et al., 2007). In red wine, MPs are considered as off-flavor, and red wines can be depreciated by concentrations above 10 ng L^–1 (Allen et al., 1991; Roujou de Boubée et al., 2000; Belancic and Agosin, 2007). Given the importance of MPs, either as typical varietal aromas or as detrimental off-flavors, deciphering the genetic and molecular determinism of their accumulation is of high interest for viticulture.In spite of this, until recently little was known about the MP biosynthesis pathway or the MP biosynthetic genes, either in grapevine or other plant species. Theoretical biosynthesis pathways have been proposed since the mid-1970s. They all start by the addition of an α-dicarbonyl on a branched amino acid (Leu for IBMP, Val for IPMP) to form a 2-hydroxy-3-alkylpyrazine, which is subsequently transformed into the corresponding MP, by a methoxylation reaction (Murray and Whitfield 1975; Gallois et al., 1988). While the initial addition step remains to be demonstrated in plants, an S-adenosyl-l-Met (SAM)-dependent O-methyltransferase (OMT), capable of converting 2-hydroxy-3-isobutylpyrazine (IBHP) into IBMP, has been detected in CS shoots, partially purified and sequenced (Hashizume et al., 2001a, 2001b; Fig. 1). Recently, Dunlevy et al. (2010) characterized two OMTs, VvOMT1 and VvOMT2, capable of methylating IBHP in vitro, albeit with high apparent K_m values. To investigate the genetic bases of MP biosynthesis in grape berries, we performed a quantitative trait loci (QTL) analysis, which has led to the identification of two previously uncharacterized OMTs termed VvOMT3 and VvOMT4. Functional characterization of these two OMTs showed that VvOMT3 was highly specific and efficient for IBHP methylation. Based on its differential expression in high-MP and low-MP grapevine varieties, we propose that VvOMT3 and, to a lesser extent, VvOMT4 are key genes for MP biosynthesis in grapevine berries.Open in a separate windowFigure 1.Putative biosynthesis pathway for IBMP adapted from Hashizume et al. (2001a). SAHcy, S-Adenosyl-l-homo-Cys.     

Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine

A genetic linkage map of grapevine was constructed using a pseudo-testcross strategy based upon 138 individuals derived from a cross of Vitis vinifera Cabernet Sauvignon × Vitis riparia Gloire de Montpellier. A total of 212 DNA markers including 199 single sequence repeats (SSRs), 11 single strand conformation polymorphisms (SSCPs) and two morphological markers were mapped onto 19 linkage groups (LG) which covered 1,249 cM with an average of 6.7 cM between markers. The position of SSR loci in the maps presented here is consistent with the genome sequence. Quantitative traits loci (QTLs) for several traits of inflorescence and flower morphology, and downy mildew resistance were investigated. Two novel QTLs for downy mildew resistance were mapped on linkage groups 9 and 12, they explain 26.0–34.4 and 28.9–31.5% of total variance, respectively. QTLs for inflorescence morphology with a large effect (14–70% of total variance explained) were detected close to the Sex locus on LG 2. The gene of the enzyme 1-aminocyclopropane-1-carboxylic acid synthase, involved in melon male organ development and located in the confidence interval of all QTLs detected on the LG 2, could be considered as a putative candidate gene for the control of sexual traits in grapevine. Co-localisations were found between four QTLs, detected on linkage groups 1, 14, 17 and 18, and the position of the floral organ development genes GIBBERELLIN INSENSITIVE1, FRUITFULL, LEAFY and AGAMOUS. Our results demonstrate that the sex determinism locus also determines both flower and inflorescence morphological traits.     

Multiple resistance traits control Plum pox virus infection in Arabidopsis thaliana

Twelve Arabidopsis accessions were challenged with Plum pox potyvirus (PPV) isolates representative of the four PPV strains. Each accession supported local and systemic infection by at least some of the PPV isolates, but high variability was observed in the behavior of the five PPV isolates or the 12 Arabidopsis accessions. Resistance to local infection or long-distance movement occurred in about 40% of all the accession-isolate combinations analyzed. Except for Nd-1, all accessions showed resistance to local infection by PPV-SoC; in the Landsberg erecta (Ler) accession, this resistance was compromised by sgt1 and rar1 mutations, suggesting that it could be controlled by an R gene-mediated resistance pathway. While most of the susceptible accessions were symptomless, PPV induced severe symptoms on inflorescences in C24, Ler, and Bay-0 as early as 15 days after inoculation. Genetic analyses indicated that these interaction phenotypes are controlled by different genetic systems. The restriction of long-distance movement of PPV-El Amar and of another member of genus Potyvirus, Lettuce mosaic virus, in Col-0 requires the RTM genes, indicating for the first time that the RTM system may provide a broad range, potyvirus-specific protection against systemic infection. The restriction to PPV-PS long-distance movement in Cvi-1 is controlled by a single recessive gene, designated rpv1, which was mapped to chromosome 1. The nuclear inclusion polymerase b-capsid protein region of the viral genome appears to be responsible for the ability of PPV-R to overcome rpv1-mediated resistance.     

Development and transferability of apricot and grape EST microsatellite markers across taxa

EST microsatellite markers were developed in apricot (Prunus armeniaca L.) and grape (Vitis vinifera L.). cDNA libraries from either apricot leaves or grape roots were used in an enrichment procedure for GA and CA repeats. The transferability of EST simple sequence repeat (SSR) markers from apricot and grapevine to other related and unrelated species was examined. Overall, grape primers amplified products in most of the Vitaceae accessions while the apricot primers amplified polymorphic alleles only in closely related species of the Rosaceae. In this taxonomic family, ten EST SSR loci were tested, and one single primer pair, PacB22, was amplified across species and sections in the Prunoideae and Maloideae. Sequencing of EST SSR loci in other species and genera confirmed a higher level of conservation in the microsatellite motif and flanking regions in the Vitaceae compared to the Rosaceae. Two distinct fragments of the PacB22 locus amplified across the Malus and Pyrus genera; however, while the coding region was highly conserved, the microsatellite repeat motif was no longer present. The banding pattern was explained by base substitution and insertion/deletion events in the intronic region of PacB22. This study includes the determination of the degree of polymorphism detected among species and genera in two unrelated taxonomic families and the evaluation of the information provided by the microsatellite repeats and the flanking regions.     

A case of chloroplast heteroplasmy in kiwifruit (Actinidia deliciosa) that is not transmitted during sexual reproduction

We report the first case of plastid chimera within the Actinidia genus, where plastid inheritance was believed to be paternal. The heterogeneity of chloroplast DNA observed in the hexaploid Actinidia deliciosa cultivar D uno involves the presence or absence of a particular MspI restriction site in the region between the psbC gene and the tRNA-Ser(UGA) gene. The heterogeneity was first observed using restriction fragment length polymorphism and then confirmed through cloning and sequencing. The analysis of the cloned fragments revealed the presence of two haplotypes: the most frequent type was found in 123 (88.5%) out of a total of 139 colonies screened. Partial sequences of the psbC-trnS fragment from both haplotypes revealed that the polymorphism occurs within the coding region of the psbC gene and consists of a synonymous transition. A contamination-free cross involving D uno as the male parent produced only plants characterized by the most frequent haplotype, indicating either selection bias against the rare type or more likely fixation of the frequent type in tissues leading to the formation of the male gametes. The MspI restriction profiles performed on various tissues suggest that the rarer type is absent from the histogenic layer LII and that D uno is a periclinal plastid chimera.     

A set of polymorphic microsatellites for Vochysia ferruginea,a promising tree for land reclamation in the Neotropics

Vochysia ferruginea Mart. (Vochysiaceae) is a gap colonist of Neotropical forest. As a result of its high tolerance of low‐nutrient acidic conditions and high aluminium and iron concentrations, and its high potential seed and pollen dispersal, it is a promising timber species for commercial development as regenerated or reclaimed forest on degraded land. We present here the primer sequences for 10 polymorphic simple sequence repeat loci for use with V. ferruginea to assess fine‐scale genetic structure and gene flow dynamics.     

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.     

Isolation and characterization of polymorphic simple sequence repeat loci in Armillaria ostoyae

A modified hybridization strategy was used to construct a microsatellite enriched library from DNA of Armillaria ostoyae, a serious root pathogen on pine. Sequence characterization of 19 random clones revealed 12 distinct loci harbouring a repetitive motif. Primer design from the flanking regions allowed for their development as polymerase chain reaction based markers. Polymorphic assessment at both the population and global levels revealed levels of variation useful for genetic studies. The level of cross‐species amplification observed with closely related Armillaria species was high, raising the possible exploitation of these primers across the genus.     

Linkage disequilibrium in French wild cherry germplasm and worldwide sweet cherry germplasm

A basic knowledge on linkage disequilibrium (LD) is necessary in order to determine resolution of association studies. We investigated the extent and patterns of LD in a self-incompatible species (Prunus avium L.), in 3 groups (wild cherry, sweet cherry landraces and sweet cherry modern varieties), using a set of 35 microsatellite markers and the gametophytic self-incompatibility locus. Since population structure might create spurious LD, we thus used the information provided by a structure analysis published in a previous study to perform the LD analysis. In the current study, we detected a greater LD extent in sweet cherry than in wild cherry, which is plausibly due to the bottleneck associated with domestication and breeding. Higher LD values in sweet cherry sub-groups may be explained by smaller sample sizes. We also showed that the remaining structure in the groups of sweet cherry, in particular landraces, is responsible for a part of the LD extent. Intra-group relatedness may also account for extensive LD in two sub-groups. These results demonstrate, if ever necessary, the importance of controlling the genetic structure and relatedness when estimating LD. Moreover, LD decays very rapidly with genetic linkage distance in both wild and sweet cherries, which seems promising for future association studies.