A skin color mutation of grapevine, from black-skinned Pinot Noir to white-skinned Pinot Blanc, is caused by deletion of the functional VvmybA1 allele

A white-wine grape, Pinot Blanc, is thought to be a white-skinned mutant of a red-wine grape, Pinot Noir. Pinot Noir was heterozygous for VvmybA1. One allele was the non-functional VvmybA1a, and the other was the functional VvmybA1c. In Pinot Blanc, however, only VvmybA1a was observed, and the amount of VvmybA1 DNA in Pinot Blanc was half that in Pinot Noir. These findings suggest that deletion of VvmybA1c from Pinot Noir resulted in Pinot Blanc.     

Two new grape cultivars, bud sports of Cabernet Sauvignon bearing pale-coloured berries, are the result of deletion of two regulatory genes of the berry colour locus

Bud sports are infrequent changes in phenotype affecting shoots of woody perennials but the molecular basis of these mutations has rarely been identified. In this report, we show that the bronze-coloured berries of the Malian cultivar, a documented bud sport of the wine grape Cabernet Sauvignon (Vitis vinifera L.), lack anthocyanins in the subepidermal cells compared to the red/black berried Cabernet Sauvignon in which both the epidermis and several subepidermal cell layers contain anthocyanin. The Malian phenotype is correlated with an alteration in the genome indicated by a reduction of hybridisation signal using a MYBA probe. In Shalistin, a white-berried bud sport of Malian, the red allele at the berry colour locus appears to have been deleted completely. These data suggest that Malian could be a L1/L2 periclinal chimera, which gave rise to Shalistin by an invasion of epidermal cells (L1) by the mutated subepidermal cells (L2). The red grape Pinot Noir has given rise to a number of pale coloured sports, although the provenance of the extant sports is not known. We show that a clone of Pinot Blanc (white-berried) does not have a deletion of the red allele of the same dimensions as that in Shalistin, though a small deletion is a likely explanation for the altered phenotype. However, the mechanism of deletion of the red allele of the berry colour locus is a possible means by which other red to white clonal mutations of grapevines have occurred.     

Study of the relationship between the cultivars of Vitis vinifera and the white-fruited and hermaphrodite Chinese wild grapes

Chinese wild grapes are almost exclusively dioecious and black-fruited, with rare reports of white and hermaphrodite types in V. davidii. To reveal the molecular mechanisms of these phenotypic variations, specific primers were designed to detect the genotypes of mybA-related genes in Vitis species, including the Chinese wild Vitis species, V. riparia, V. rupestris, cultivars of Vitis vinifera and its hybrids. We report here that three mybA-related genes, VvmybA1a, VvmybA2 and VvmybA3, were only detected in cultivars of V. vinifera and its hybrids, but not in V. riparia, V. rupestris or Chinese wild Vitis species, indicating that these genes could be used to test the genetic relationship to V. vinifera. On the other hand, the genes were not detected in the dioecious varieties of V. davidii, but were in the hermaphrodites. In particular, the white-fruited varieties were homozygous for VvmybA1a and showed a low expression of mybA-related genes and UFGT during the entire maturation period. Simple sequence repeat analysis showed that the hermaphrodite varieties of V. davidii, including the white-fruited varieties, were more closely related to V. vinifera cv. Pinot Noir and V. labruscana cv. Kyoho. These results suggested that the white-fruited and hermaphrodite varieties of V. davidii could be the result of its crossing with V. vinifera. It provides a new approach to identify truly Chinese wild varieties and to search for possible hybridization events.     

Genome-wide Identification,Phylogenetic Analysis,and Expression Profiling of CONSTANS-like (COL) Genes in Vitis vinifera

The CONSTANS (CO) gene plays an important role in the flowering of plants. However, the other precise roles of the CO gene are poorly understood. We carried out a genomic census and analysis of expression patterns for CONSTANS-like genes in Vitis vinifera (VviCOLs) to reveal the molecular characteristics of VviCOLs. Twelve VviCOLs were identified and 11 of their full-length complementary DNAs were cloned. Multiple sequence alignment suggested the VviCOLs contained B-box and CCT conserved domains. We further classified the VviCOLs into three groups according to the variability of the second B-box domain. Synteny analysis showed that eight orthologous gene pairs were identified between grapevine and Arabidopsis, suggesting that eight pairs may descend from a common evolutionary ancestor. Tissue expression analysis of COL genes in cv. Pinot Noir showed VviCOL11a and VviCOL11b were specifically expressed in flower bud, whereas VviCOL16b was only expressed in leaves. Ten VviCOLs were expressed in the developing ovule and six of them showed higher expression in the ovule of cv. Thompson Seedless than that of cv. Pinot Noir, indicating that VviCOLs were involved in the process of seed development or ovule abortion. Furthermore, nine of twelve VviCOLs were expressed in cv. Pinot Noir leaves and all of these nine genes had a response to exogenous hormone application. In summary, our findings provide a new insight into the further studies of VviCOLs, especially in terms of seed development and hormone response.

     

Sequencing and assembly of highly heterozygous genome of Vitis vinifera L. cv Pinot Noir: problems and solutions

A new approach to sequencing and assembling a highly heterozygous genome, that of grape, species Vitis vinifera cv Pinot Noir, is described. The combining of genome shotgun of paired reads produced by Sanger sequencing and sequencing by synthesis of unpaired reads was shown to be an efficient procedure for decoding a complex genome. About 2 million SNPs and more than a million heterozygous gaps have been identified in the 500Mb genome of grape. More than 91% of the sequence assembled into 58,611 contigs is now anchored to the 19 linkage groups of V. vinifera.     

Influences of blackberry margins on population dynamics of Drosophila suzukii and grape infestation in adjacent vineyards

Understanding the dynamics of pest insect populations in relation to the presence of non‐crop habitats and infestation levels of adjacent crops is essential to develop sustainable pest management strategies. The invasive pest species Drosophila suzukii (Diptera: Drosophilidae) is able to utilize a broad range of host plants. In viticulture, scientific risk assessment for D. suzukii has only recently started and studies assessing the effects of field margins containing wild host plants on D. suzukii population dynamics and on infestation risks in adjacent vineyards are lacking. Thus, in a one‐year field study, the role of different field margins on fly abundance and crop infestation in adjacent vineyards of Vitis vinifera, variety “Pinot Noir,” were investigated. Different monitoring methods were conducted to assess fly distribution, sex ratio and grape infestation in 14 vineyards adjacent to field margins containing either blackberry (BB) Rubus spp. or non‐host (NH) plants. Our results show that blackberries strongly enhanced D. suzukii abundance within field margin vegetation all year long, whereas fly abundance in vineyards adjacent to BB margins was just enhanced in some seasonal periods. Moreover, the influence of BB margins was limited by distance. However, high fly numbers in BB field margins did result in zero egg infestation of “Pinot Noir” berries. These results may have important implications for winegrowers to make efficient management decisions: regardless of high abundance of adult D. suzukii, only grape berry monitoring can assess the actual infestation risk and the potential need to take management action.     

Malate as substrate for catabolism and gluconeogenesis during ripening in the pericarp of different grape cultivars

Malate is accumulated in grape pericarp until the start of ripening and then it is dissimilated. One aim of this study was to determine if the potential contribution of stored malate to the substrate requirements of metabolism in ripening grape pericarp is dependent on the cultivar. Two Vitis vinifera L. cultivars which accumulated different amounts of malate and had ripening periods of a different length were compared. The potential contribution of stored malate over the whole period of ripening was around 20 % in the cv. Sagrantino and 29 % in the cv. Pinot Noir. The contribution was higher in Pinot Noir because it contained more malate and had a shorter ripening period. A second aim of this study was to evaluate the contribution of gluconeogenesis to the amount of sugar accumulated in the pericarp. If all the dissimilated malate was utilized by gluconeogenesis, then the maximum contribution of stored malate to the total amount of sugar accumulated in the pericarp over the whole period of ripening was around 2.4 % in Sagrantino and 2.9 % in Pinot Noir. However, the actual contribution was only about 0.1–0.6 % in both cultivars because the majority of stored malate was not utilized by gluconeogenesis. However, it is likely that the actual contribution is much lower. This suggests that the function of gluconeogenesis is not to support accumulation of sugars in the fruits, but probably it plays other roles.     

The metacaspase gene family of Vitis vinifera L.: Characterization and differential expression during ovule abortion in stenospermocarpic seedless grapes

In both plants and animals, programmed cell death (PCD) is an indispensable process that removes redundant cells. In seedless grapes (Vitis vinifera), abnormal PCD in ovule cells and subsequent ovule abortion play key roles in stenospermocarpy. Metacaspase, a type of cysteine-dependent protease, plays an essential role in PCD. To reveal the characteristics of the metacaspase (MC) gene family and the relationship between metacaspases and the seedless trait, we identified the 6 V. vinifera metacaspases VvMC1–VvMC6, from the grape genome, using BLASTN against the 9 known Arabidopsis metacaspases. We also obtained full-length cDNAs by RT-PCR. Each of the 6 grape metacaspases contains small (p10-like) and a large (p20-like) conserved structural domains. Phylogenetic analysis of 6 grape and 9 Arabidopsis metacaspases showed that all metacaspases could be grouped into two classes: Type I and Type II. Each phylogenetic branch shares a similar exon/intron structure. Furthermore, the putative promoters of the grape metacaspases contained cis-elements that are involved in grape endosperm development. Moreover, expression analysis of metacaspases using real-time quantitative PCR demonstrated that VvMC1 and VvMC2 were able to be detected in any tissue, and VvMC3, VvMC4, VvMC5 and VvMC6 exhibited tissue-specific expression. Lastly, in cv. Thompson seedless grapes VvMC1, VvMC3, and VvMC4 were significantly up-regulated at the 35 DAF during ovule development, roughly same stage as endosperm abortion. In addition, the expression trend of VvMC2 and VvMC5 was similar between cv. Pinot Noir and cv. Thompson grape ovule development and that of VvMC6 was sustained in a relatively low level except the expression of cv. Pinot Noir significantly up-regulated in 25 DAF. Our data provided new insights into PCD by identifying the grape metacaspase gene family and provide a useful reference for further functional analysis of metacaspases in grape.     

葡萄枝条水分含量变化与抗寒性关系

以12个葡萄品种1年生枝条为试材,通过0、-15、-18、-21、-24、-27、-30、-133℃8个低温处理24h后,测定不同低温胁迫下葡萄枝条的,6-含水量、束缚水含量、自由水含量和束缚水与自由水的比值。结果表明：随着处理温度的降低,同一品种枝条的总含水量基本恒定,自由水含量呈先下降再升高的趋势,束缚水含量和束缚水与自由水的比值均呈现为先升高再下降的趋势;束缚水与自由水的比值随低温胁迫的变化呈Cubic方程,不同品种束缚水与自由水的比值达到最高点的温度不同,‘双红’、‘北冰红’、‘左优红’的拐点温度在-25℃以下,‘775’、‘巨峰’的拐点温度在-20℃左右,‘雷司令’、‘黑比诺’、‘霞多丽’的拐点温度在-18℃左右,‘赤霞珠’、‘梅鹿特’、‘白比诺’、‘红地球’拐点温度在-16～17℃。对葡萄休眠枝条进行低温处理后,通过测定束缚水与自由水含水量变化鉴定葡萄品种的抗寒性是可行的。     

MAST2-like proteinkinase from grape Vitis vinifera: Cloning of catalytic domain cDNA

During the work on the project on the identification of proteinkinases that phosphorylate protein microtubules of plants, we revealed with the help of bioinformatics the genes of assumed homologues of proteinkinase MAST2 that is associated with microtubules in animal cells. Respectively, the gene of the closest homologue of MAST2, the assumed protein that we have named GMLK (Grape MAST2-Like Kinase, A7NTE9_VITVI) was identified in the genome of grape Vitis vinifera. This study presents the results of the successful cloning of protein GMLK (A7NTE9_VITVI) cDNA from the leaves of the Pinot Noir grape.     

Changes in the chlorophyll content of grape leaves could provide a physiological index for responses and adaptation to UV‐C radiation

Stilbenes are a group of phytoalexins that play an important role in grapevine (Vitis) basal immunity and can be induced by biotic and abiotic stresses. The levels of chlorophylls, the main pigments in plant cells, can also indicate the tolerance of plants to various stresses. Here, the response of different grapevine genotypes to UV‐C radiation treatment was tested and the abundance of chlorophyll in the Hoe29 and Ke53 genotypes was observed to increase significantly within 6 h of UV‐C treatment. Conversely, chlorophyll levels decreased markedly in the Augster Weiss and Müller–Thurgau genotypes. Furthermore, stilbene abundance increased substantially in the Hoe29, Ke53, Ke83 and Pinot Blanc genotypes, but increased only slightly in Augster Weiss and Müller–Thurgau. The expression of resveratrol synthase, which encodes a key enzyme in the stilbene synthesis pathway, increased in Hoe29, Ke53, Ke83 and Pinot Blanc following UV‐C treatment, in a manner consistent with stilbene accumulation. In addition, we observed that reactive oxygen species (ROS) provide a key trigger in physiological responses and changes in secondary metabolite contents. In summary, the results from this study support a link between ROS, chlorophyll levels and genetic diversity for stilbene abundance in different grape genotypes, providing insights into mechanisms for plant physiological and biochemical responses and adaptations to stress.     

The heterologous expression of polysaccharidase-encoding genes with oenological relevance in Saccharomyces cerevisiae

AIMS: The main objective of this study was to develop polysaccharide-degrading wine strains of Saccharomyces cerevisiae, which are able to improve aspects of wine processing and clarification, as well as colour extraction and stabilization during winemaking. METHODS AND RESULTS: Two yeast expression/secretion gene cassettes were constructed, namely (i) a pectinase gene cassette (pPPK) consisting of the endo-polygalacturonase gene (pelE) from Erwinia chrysanthemi and the pectate lyase gene (peh1) from Erwinia carotovora and (ii) a glucanase/xylanase gene cassette (pEXS) containing the endo-beta-1,4-glucanase gene (end1) from Butyrivibrio fibrisolvens and the endo-beta-1,4-xylanase gene (xynC) from Aspergillus niger. The commercial wine yeast strain, VIN13, was transformed separately with these two gene cassettes and checked for the production of pectinase, glucanase and xylanase activities. Pinot Noir, Cinsaut and Muscat d'Alexandria grape juices were fermented using the VIN13[pPPK] pectinase- and the VIN13[pEXS] glucanase/xylanase-producing transformants. Chemical analyses of the resultant wines indicated that (i) the pectinase-producing strain caused a decrease in the concentration of phenolic compounds in Pinot Noir whereas the glucanase/xylanase-producing strain caused an increase in phenolic compounds presumably because of the degradation of the grape skins; (ii) the glucanase/xylanase-producing strain caused a decrease in wine turbidity, especially in Pinot Noir wine, as well as a clear increase in colour intensity and (iii) in the Muscat d'Alexandria and Cinsaut wines, the differences between the control wines (fermented with the untransformed VIN3 strain) and the wines produced by the two transformed strains were less prominent showing that the effect of these polysaccharide-degrading enzymes is cultivar-dependent. CONCLUSIONS: The recombinant wine yeasts producing pectinase, glucanase and xylanase activities during the fermentation of Pinot Noir, Cinsaut and Muscat d'Alexandria grape juice altered the chemical composition of the resultant wines in a way that such yeasts could potentially be used to improve the clarity, colour intensity and stability and aroma of wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspects of commercial-scale wine processing and clarification, colour extraction and stabilization, and aroma enhancement could potentially be improved by the use of polysaccharide-degrading wine yeasts without the addition of expensive commercial enzyme preparations. This offers the potential to further improve the price:quality ratio of wine according to consumer expectations.     

葡萄初级核心种质资源MybA基因型分析

探究MybA类基因在不同类型葡萄品种中的分布,可为葡萄品种鉴定,以及有色葡萄育种的亲本选择提供依据。本研究以欧亚种、欧美杂种、法美杂种、山欧杂种以及美洲种在内的118个葡萄初级核心种质为材料,对其MybA基因型进行分析。结果表明:欧亚种及其杂种普遍具有VvmybA1基因的等位基因VvmybA1a,仅10个欧亚种及其杂种品种中没有检测到VvmybA1a基因;欧亚种、欧美杂种以及法美杂种中普遍同时具有VvmybA1、VvmybA2和VvmybA3基因,仅少数品种未检测到VvmybA2或VvmybA3基因;山欧杂种中北玫、公酿1号和熊岳白葡萄同时具有VvmybA1、VvmybA2和VvmybA3基因,北醇和北红中仅检测到VvmybA1和VvmybA3基因;仅在具有美洲种血缘的葡萄品种中检测到VlmybA2基因,而5个认为是美洲种的品种未检测到VlmybA2基因,且检测到了欧亚种特有的VvmybA1a等位基因,推测它们为含美洲种血缘较多的欧美杂种,而非纯美洲种。     

The effect of polysaccharide-degrading wine yeast transformants on the efficiency of wine processing and wine flavour

Commercial polysaccharase preparations are applied to winemaking to improve wine processing and quality. Expression of polysaccharase-encoding genes in Saccharomyces cerevisiae allows for the recombinant strains to degrade polysaccharides that traditional commercial yeast strains cannot. In this study, we constructed recombinant wine yeast strains that were able to degrade the problem-causing grape polysaccharides, glucan and xylan, by separately integrating the Trichoderma reesei XYN2 xylanase gene construct and the Butyrivibrio fibrisolvens END1 glucanase gene cassette into the genome of the commercial wine yeast strain S. cerevisiae VIN13. These genes were also combined in S. cerevisiae VIN13 under the control of different promoters. The strains that were constructed were compared under winemaking conditions with each other and with a recombinant wine yeast strain expressing the endo-beta-1,4-glucanase gene cassette (END1) from B. fibrisolvens and the endo-beta-1,4-xylanase gene cassette (XYN4) from Aspergillus niger, a recombinant strain expressing the pectate lyase gene cassette (PEL5) from Erwinia chrysanthemi and the polygalacturonase-encoding gene cassette (PEH1) from Erwinia carotovora. Wine was made with the recombinant strains using different grape cultivars. Fermentations with the recombinant VIN13 strains resulted in significant increases in free-flow wine when Ruby Cabernet must was fermented. After 6 months of bottle ageing significant differences in colour intensity and colour stability could be detected in Pinot Noir and Ruby Cabernet wines fermented with different recombinant strains. After this period the volatile composition of Muscat d'Alexandria, Ruby Cabernet and Pinot Noir wines fermented with different recombinant strains also showed significant differences. The Pinot Noir wines were also sensorial evaluated and the tasting panel preferred the wines fermented with the recombinant strains.     

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety

Background

Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.

Principal Findings

We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).

Conclusions

Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.     

Yeasts present during spontaneous fermentation of Lake Erie Chardonnay,Pinot Gris and Riesling

The composition of wine yeast populations, present during spontaneous fermentation of Chardonnay, Pinot Gris and Riesling from the Lake Erie Region was studied. A combination of biochemical and molecular techniques was used to identify non-Saccharomyces and Saccharomyces yeast isolates. The biochemical techniques included analysis of yeast isolates by sugar fermentation and carbon and nitrogen assimilation. Molecular techniques involved ribotyping of a highly variable segment in the 26S rRNA gene using DNA sequence analysis and restriction fragment length polymorphism of amplified DNA. The results show that of the non-Saccharomyces yeasts, several related species of Hanseniaspora, were the most abundant yeasts present during early stages of fermentation. Later in fermentation S. cerevisiae dominated, and based on biochemical analyses consisted of a heterogeneous group of genotypes. There were no major differences in yeast populations among the three types of juice analyzed. This revised version was published online in June 2006 with corrections to the Cover Date.     

A SNP in the promoter region of the<Emphasis Type="Italic">VvmybA1</Emphasis> gene is responsible for differences in grape berry color between two related bud sports of grape

The VvmybA1 gene in grape (Vitis vinifera) plays a key role in the biosynthesis of anthocyanin. The grape cultivars, ‘Benitaka’ (red color) and ‘Brazil’ (black color) were the result of a bud mutation. ‘Benetika’ was derived from ‘Italia’ (green color) and ‘Brazil’ was developed from ‘Benetika’. Single sequence repeat (SSR) molecular marker analysis was performed in order to demonstrate that the three cultivars have a common pedigree. A sequence analysis of the promoter region and coding sequence of VvmybA1 revealed a base substitution between ‘Benitaka’ and ‘Brazil’ in the promoter region and a deletion of a large DNA fragment in the promoter region of ‘Italia’. Anthocyanin content and expression of the VvmybA1 and UFGT genes in ‘Brazil’ were higher than in ‘Benitaka’ and barely detectable in ‘Italia’. A transient expression system was used to introduce VvmybA1 driven by the three different promoters present in ‘Italia’, ‘Brazil’, and ‘Benitaka’ into somatic embryos of ‘Centennial Seedless’ (Vitis vinifera L.) by Agrobacterium-mediated transformation. This resulted in the production of red cells in the embryos transformed with the constructs of VvmybA1 from ‘Brazil’ and ‘Benitaka’ and no color production in embryos transformed with the VvmybA1 construct from ‘Italia’. In addition, the embryos transformed with the ‘Brazil’ construct had more red color than the embryos transformed with the ‘Benitaka’ construct. These results suggested that a SNP mutation in the promoter region of VvmybA1 in ‘Benitaka’ (red color) was responsible for the color change displayed by ‘Brazil’ (black color).     

Assessing the influence of inflorescence traits on the susceptibility of grape to vine moths

In north‐eastern Italy during 1994–2006, studies were carried out on the susceptibility of grapevine cultivars to the first generations of the European vine moth [Lobesia botrana (Den. and Shiff.); Lep., Tortricidae] and the European grape berry moth [Eupoecilia ambiguella (Hb.); Lep., Cochylidae]. In five different years, the larval population density of both moth species and the larval age composition of L. botrana were recorded on 11 grapevine cultivars (Cabernet Sauvignon, Carmenère, Chardonnay, Pinot Gris, Merlot, Refosco dal Peduncolo Rosso, Rhine Riesling, Sauvignon Blanc, Terrano, Tocai Friulano, Verduzzo Friulano), grown in the same vineyard. The influence of inflorescence traits on these demographic parameters was also evaluated. On a 5‐year average, L. botrana significantly prevailed over E. ambiguella in nine of the 11 cultivars. Chardonnay and Pinot Gris were the most infested cultivars. Age composition of L. botrana larvae varied with cultivar type, being older on Chardonnay and Tocai Friulano than the other cultivars. Inflorescence earliness and hairiness explained the majority of the variability in cultivar susceptibility. In particular, the larval population level of the two vine moths was positively correlated with inflorescence earliness and negatively correlated with inflorescence hairiness. Lobesia botrana larval age composition was negatively correlated with inflorescence hairiness. Inflorescence earliness and hairiness could be used to predict in each grape‐growing area which cultivars are potentially more infested in the first generation. Inflorescences without pubescence, favouring an older age composition of first generation larvae, could induce an earlier second generation.     

Hexokinase from grape berries: its prokaryotic expression, polyclonal antibody preparation and biochemical property analyses

Two full-length hexokinase (HXK, EC 2.7.1.1) cDNAs, VvHXK1 with 1,413 bp and VvHXK2 with 1,458 bp were cloned from grape berries (Vitis vinifera L. Cabernet Sauvignon). VvHXK1 and VvHXK2 genes sequence from grape berries were deposited in GenBank under the accession number JN118544 and JN118545, respectively. The homology of the amino acid of VvHXK1 or VvHXK2 was very similar to ‘Pinot Noir’ grape HXK sequence, their similarties were 99.36 % and 98.97 %, respectively. More intuitive phylogenetic tree showed that the homology of amino acid sequence VvHXK1 with melon CmHXK1 was 86 %, and VvHXK2 homology with rice OsHXK3 was 83 %. The HXK proteins were successfully expressed in plasmid pET-30a (+) vectors in Escherichia coli BL21 (DE3) pLysS. The expressed proteins were purified using Ni-NTA agarose column and used to produce HXK1 antibody and conducted HXK protein blotting analysis. The results,suggested that one polypeptide band of about 51 kDa HXK protein can be detected in grape berries, HXK protein level was the highest during early grape berry development, but the lowest from 50d to 60d during development. Biochemical analysis of two hexokinase isozymes indicated that glucose was the optimal substrate of HXK, The isoelectric points of the two isozymes were 5.8 and 5.6, respectively. And the optimum pH was about 8.0. These results provide a substantial basis for the further studies of functions of grape HXKs to manipulate sugar content of grape berries.     

Performance and diacetyl production of commercial strains of malolactic bacteria in wine

This study compares 11 commercial cultures of Leuconostoc oenos and Lactobacillus plantarum in Cabernet Sauvignon, Pinot Noir and Chardonnay wines. Performance of the cultures was found to be greatly influenced by wine type. Better survival of the bacteria was observed in Cabernet Sauvignon and Pinot Noir wines. The time necessary to complete malolactic fermentation (MLF) was 65 ± 14 d for Chardonnay, 71 ± 3 d for Cabernet Sauvignon, and 25 ± 8 d for Pinot Noir. The maximal rate of malate utilization was 0·4 g d^-1 for Pinot Noir, and 0·2 g d^-1 for the two other wine types. Final diacetyl concentration was lower in Chardonnay wines (highest 0·58 mg l^-1) compared to the other wines (highest 5·8 mg l^-1). Malic and citric acid were co-metabolized by all strains. None of the strains metabolized glycerol. Significant differences in final diacetyl concentration of wine vinified with the different strains were found. Panelists could reliably differentiate MLF wines from non-MLF wines, irrespective of their diacetyl content, indicating that diacetyl is not the only important MLF flavour.