Wild Vitis Research Articles

Overexpression of a SBP-Box Gene (VpSBP16) from Chinese Wild Vitis Species in Arabidopsis Improves Salinity and Drought Stress Tolerance.

Salinity and drought are two major abiotic stresses that limit grape productivity. Responses to stress in grape are known to be regulated by several families of transcription factors. However, little is known about the role of grape Squamosa promoter binding protein (SBP)-box transcription factor genes in response to abiotic stress. To better understand the functions of the grape SBP-box genes in abiotic stress tolerance, a full-length complementary DNA (cDNA) sequence of the putative SBP-box transcription factor gene, VpSBP16 was amplified from Chinese wild grapevine Vitis pseudoreticulata clone “Baihe-35-1”. We observed that the VpSBP16 protein fused to the green fluorescent protein (GFP) reporter accumulated in the nucleus when transiently expressed in onion epidermal cells. Moreover, VpSBP16 was shown to have transcriptional activation activity using a yeast trans-activation assay. We performed a VpSBP16 functional analysis through the characterization of transgenic Arabidopsis thaliana plants constitutively over-expressing VpSBP16. The transgenic lines had longer roots and the seeds had a higher germination rate than the wild type (WT) under osmotic stress. In addition, the accumulation of reactive oxygen species (ROS) of transgenic seedlings was significantly lower than WT in the transgenic lines, as was electrolyte leakage. VpSBP16 overexpression also elevated expression levels of stress-response genes involved in the salt overly sensitive (SOS) pathway. These results indicate that overexpression VpSBP16 in A. thaliana enhances tolerance of salt and drought stress during seed germination, as well in seedlings and mature plants, by regulating SOS and ROS signaling cascades.

Overexpression of VpPR10.1 by an efficient transformation method enhances downy mildew resistance in V. vinifera.

Putrescine and spermidine increase the transformation efficiency of Vitis vinifera L. cv. Thompson seedless. Accumulation of VpPR10.1 in transgenic V. vinifera Thompson seedless, likely increases its resistance to downy mildew. A more efficient method is described for facilitating Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless somatic embryogenesis using polyamines (PAs). The efficacies of putrescine, spermidine and spermine are identified at a range of concentrations (10µM, 100µM and 1mM) added to the culture medium during somatic embryo growth. Putrescine (PUT) and spermidine (SPD) promote the recovery of proembryonic masses (PEM) and the development of somatic embryos (SE) after co-cultivation. Judging from the importance of the time-frame in genetic transformation, PAs added at the co-cultivation stage have a stronger effect than delayed selection treatments, which are superior to antibiotic treatments in the selection stage. Best embryogenic responses are with 1mM PUT and 100µM SPD added to the co-culture medium. Using the above method, a pathogenesis-related gene (VpPR10.1) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. The transgenic line, confirmed by western blot analysis, was inoculated with Plasmopara viticola to test for downy mildew resistance. Based on observed restrictions of hyphal growth and increases in H2O2 accumulation in the transgenic plants, the accumulation of VpPR10.1 likely enhanced the transgenic plants resistance to downy mildew.

Expression of Vitis amurensis VaERF20 in Arabidopsis thaliana Improves Resistance to Botrytis cinerea and Pseudomonas syringae pv. Tomato DC3000.

Ethylene response factor (ERF) transcription factors play important roles in regulating immune responses in plants. In our study, we characterized a member of the ERF transcription factor family, VaERF20, from the Chinese wild Vitis genotype, V. amurensis Rupr “Shuangyou”. Phylogenetic analysis indicated that VaERF20 belongs to group IXc of the ERF family, in which many members are known to contribute to fighting pathogen infection. Consistent with this, expression of VaERF20 was induced by treatment with the necrotrophic fungal pathogen Botrytis cinerea (B. cinerea) in “Shuangyou” and V. vinifera “Red Globe”. Arabidopsis thaliana plants over-expressing VaERF20 displayed enhanced resistance to B. cinerea and the bacterium Pseudomonas syringae pv. tomato (Pst) DC3000. Patterns of pathogen-induced reactive oxygen species (ROS) accumulation were entirely distinct in B. cinerea and PstDC3000 inoculated plants. Examples of both salicylic acid (SA) and jasmonic acid/ethylene (JA/ET) responsive defense genes were up-regulated after B. cinerea and PstDC3000 inoculation of the VaERF20-overexpressing transgenic A. thaliana plants. Evidence of pattern-triggered immunity (PTI), callose accumulation and stomatal defense, together with increased expression of PTI genes, was also greater in the transgenic lines. These data indicate that VaERF20 participates in various signal transduction pathways and acts as an inducer of immune responses.

Genetic Diversity of Vitis davidii Accessions Revealed Using Microsatellite and Sequence-related Amplified Polymorphism Markers

In this study, simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) markers were used to analyze the genetic diversity of 48 wild Vitis davidii accessions. A total of 78 distinct alleles were amplified by 11 SSR primers, and the average allele number was 8.8. The average observed heterozygosity (Ho) and expected heterozygosity (He) values were 0.785 and 0.814, respectively. The effective allele numbers ranged from 3.92 to 9.61. The average polymorphism information content (PIC) was 0.798. Twelve of 169 SRAP primer combinations were selected for SRAP analysis. A total of 188 bands were produced, and the average was 15.7 bands per primer combination; the average percentage of polymorphic bands was 84.0%. The average PIC was 0.76. The results of the clustering analysis based on SSR markers showed that the 48 wild V. davidii accessions could be classified into five main clusters and had a genetic similarity coefficient level of 0.68. The dendrogram obtained from the SRAP data showed that 48 wild V. davidii accessions could be classified into five main clusters and had a genetic similarity coefficient of 0.72. SSR and SRAP markers differentiated all accessions studied including those with a similar pedigree. We speculated on the origin of Ciputao 0941♀, Ciputao 0940♂, and Fu’an-ci-01 using SSR markers and used both SSR and SRAP markers to resolve homonymy. The result will be valuable for further management and protection of V. davidii germplasm resources.

The GARP/MYB-related grape transcription factor AQUILO improves cold tolerance and promotes the accumulation of raffinose family oligosaccharides

Grapevine (Vitis vinifera L.) is a widely cultivated fruit crop whose growth and productivity are greatly affected by low temperatures. On the other hand, wild Vitis species represent valuable genetic resources of natural stress tolerance. We have isolated and characterized a MYB-like gene encoding a putative GARP-type transcription factor from Amur grape (V. amurensis) designated as VaAQUILO. AQUILO (AQ) is induced by cold in both V. amurensis and V. vinifera, and its overexpression results in significantly improved tolerance to cold both in transgenic Arabidopsis and in Amur grape calli. In Arabidopsis, the ectopic expression of VaAQ increased antioxidant enzyme activities and up-regulated reactive oxygen species- (ROS) scavenging-related genes. Comparative mRNA sequencing profiling of 35S:VaAQ Arabidopsis plants suggests that this transcription factor is related to phosphate homeostasis like their Arabidopsis closest homologues: AtHRS1 and AtHHO2. However, when a cold stress is imposed, AQ is tightly associated with the cold-responsive pathway and with the raffinose family oligosaccharides (RFOs), as observed by the up-regulation of galactinol synthase (GoLS) and raffinose synthase genes. Gene co-expression network (GCN) and cis-regulatory element (CRE) analyses in grapevine indicated AQ as potentially regulating VvGoLS genes. Increased RFO content was confirmed in both transgenic Arabidopsis and Amur grape calli overexpressing VaAQ. Taken together, our results imply that AQ improves cold tolerance through promoting the accumulation of osmoprotectants.

Histoire de la vigne sauvage, Vitis vinifera ssp. sylvestris, en Camargue / Wild grapevine Vitis vinifera ssp. sylvestris in Camargue, southern France

La vigne sauvage (Vitis vinifera ssp. sylvestris) est en déclin marqué depuis la fin du XIXe siècle. Depuis 2000, l’espèce est considérée en danger critique d’extinction. Des prospections dans la vallée du Rhône ont montré qu’il existait quelques micropopulations jusque dans le delta. Une population de 52 pieds de grandes dimensions et présentant un phénotype sauvage a récemment été découverte en rive gauche du Grand Rhône, dans le boisement privé du mas Saint-Georges. Une recherche pluridisciplinaire a été entreprise sur cette population de vignes afin de reconstituer leur histoire ; les outils utilisés ont été la génétique (effectuée sur 37 individus), l’écologie (dimensions des pieds, identification des porteurs), et la dendrochronologie effectuée sur 5 pieds. L’habitat forestier a également été analysé du point de vue architectural et dynamique de la lumière. Trente et un (31) individus ont été déterminés comme étant de véritables vignes sauvages, dont la diversité est légèrement inférieure à celle des autres populations européennes. Quatre sont des individus hybrides ou introgressés : deux entre vignes sauvages et vignes américaines, et deux entre vignes sauvages et vignes cultivées. Trois haplotypes typiques des vignes sauvages ont été déterminés, indiquant que ce site est à un point de convergence entre populations méditerranéennes du sud, de l’ouest et du centre de la Méditerranée. Le boisement a été suffisamment fermé pour éviter les échanges de pollen avec les vignes américaines échappées. À partir de ces données et de l’analyse des cartes anciennes, il a pu être déduit que ce boisement s’était développé au début du XXe siècle à partir d’un marais asséché par les aménagements hydrauliques. L’âge des vignes, estimé par la dendrochronologie à une fourchette de 38 à 50 ans, pourrait cependant être de l’âge de la forêt. Les vignes se seraient installées lors de la succession forestière. Les caractéristiques architecturales du boisement (à la fois dense et lumineux) ont permis à la vigne de s’y établir sur une longue durée. Actuellement, cette population ne se reproduit plus. Les conditions actuelles ne sont donc plus favorables à l’espèce, à l’inverse des populations échappées de porte-greffes, très abondantes en Camargue. Une protection forte de ces derniers pieds de vigne forestière devrait être mise en place avec l’accord des propriétaires.

Flavan-3-ols and procyanidins in grape seeds: biodiversity and relationships among wild and cultivated vines

Flavan-3-ol monomers and polymers composition of seeds from wild (17) and autochthonous (8) Vitis vinifera grapes growing in northern Tunisia were evaluated. Wild grape seeds were spherical with a small beak and relatively a high seed/berry ratio (~ 18.1%w/w). Local cultivars developed pyriform-shaped seeds with a well-developed beak representing on average 2.2% of total weight of the berry. Flavanol concentrations ranged between 40.9 and 67.5 mg/g FW in seeds from wild accessions and between 48.9 and 96.7 mg/g FW in seeds from cultivated grapes. Differences between accessions were highly significant (p (−)-epicatechin (Ec) > (−)-epicatechin-3-O-gallate (EcG). The Cat/Ec ratio was approximately 1.7 for wild grapes while it was about 2.5 for cultivated grapes. Procyanidins in wild seeds differed from cultivated ones by a lower mDP and higher proportions of galloylated derivatives, likely to affect fruit bitterness and astringency. (−)-epicatechin was the main extension subunit in grape seed procyanidins, reaching on average 52% in wild and 58% in cultivated seeds. Hierarchical cluster analysis based on seeds morphometry and procyanidin profile indicated close proximity between some wild and cultivated grapes suggesting that some cultivars derived from ancestral events of local domestication or cross hybridization with native wild plants.

A novel grablovirus from non-cultivated grapevine (Vitis sp.) in North America.

During screening of non-cultivated (wild) grapevine (Vitis sp.) from Napa County, California for the grapevine red blotch virus (GRBV; genus Glabrovirus, family Geminiviridae), an atypical polymerase chain reaction product pattern was observed. Rolling circle amplification followed by cloning and sequencing revealed the presence of a circular DNA characteristic of geminiviruses. The complete genome of nine isolates of the virus ranged from 3204 to 3278 nt in size. The genome most closely resembled that of GRBV in both sequence (57 to 59% identity) and organization. With limited sequence identity to described geminiviruses, this virus warrants designation as a new species, and the name 'Wild Vitis virus 1' is proposed.

VpUR9, a novel RING-type ubiquitin ligase gene from Vitis pseudoreticulata, is involved in powdery mildew response in transgenic V. vinifera plants

Ubiquitination plays important roles in disease resistance in plants. We report the identification and functional characterization of the RING-type ubiquitin ligase gene VpUR9 from Chinese wild Vitis pseudoreticulata accession Baihe-35-1. VpUR9, encodes 164 amino acids and possesses a RING conserved motif. It is homologously cloned from the cDNA library of the high powdery mildew (Erysiphe necator [Schw.] Burr) resistant V. pseudoreticulata accession Baihe-35-1 inoculated with E. necator. The gene is induced in response to powdery mildew and salicylic acid. VpUR9 fused with FLAG-tag controlled by 35S promoter was transformed into 15 regenerated V. vinifera L. cv. Red Globe lines via Agrobacterium tumefaciens-mediated transformation. Twelve of these lines were confirmed by Western blot of FLAG-tag. As a result, the powdery mildew-resistance of Red Globe transformed with VpUR9 was repressed. Furthermore, the expression of some disease-resistant related genes (NPR1, PR1, PR10 and PAL) of the transgenic Red Globe declined compared with wild type grapes when inoculated with powdery mildew or salicylic acid. When treated with jasmonic acid methyl ester, its PR1 gene expression decreased, while the expressions of NPR1, PR10 and PAL all increased, contrasting with the wild type grape.

Resveratrol accumulation and its involvement in stilbene synthetic pathway of Chinese wild grapes during berry development using quantitative proteome analysis

Attention has become focused on resveratrol not only because of its role in grapevine fungal resistance but also because of its benefits in human health. This report describes the Chinese wild grapevine Vitis quinquangularis accession Danfeng-2 in relation to the high resveratrol content of its ripe berries. In this study, we used isobaric tags for relative and absolute quantification (iTRAQ) tandem mass spectrometry strategy to quantify and identify proteome changes, resulting in the detection of a total of 3,751 proteins produced under natural conditions. Among the proteins quantified, a total of 578 differentially expressed proteins were detected between Danfeng-2 and Cabernet Sauvignon during berry development. Differentially expressed proteins are involved in secondary metabolism, biotic stress, abiotic stress and transport activity and indicate novel biological processes in Chinese wild grapevine. Eleven proteins involved in phenylpropanoid metabolism and stilbene synthesis were differently expressed between Danfeng-2 and Cabernet Sauvignon at the veraison stage of berry development. These findings suggest that Chinese wild V. quinquangularis accession Danfeng-2 is an extremely important genetic resource for grape breeding and especially for increasing the resveratrol content of European grape cultivars for disease resistance and for improved human nutritional benefits.

Pathogen development and host responses to Plasmopara viticola in resistant and susceptible grapevines: an ultrastructural study

The downy mildew disease in grapevines is caused by Plasmopara viticola. This disease poses a serious threat wherever viticulture is practiced. Wild Vitis species showing resistance to P. viticola offer a promising pathway to develop new grapevine cultivars resistant to P. viticola which will allow reduced use of environmentally unfriendly fungicides. Here, transmission and scanning microscopy was used to compare the resistance responses to downy mildew of three resistant genotypes of V. davidii var. cyanocarpa, V. piasesezkii and V. pseudoreticulata and the suceptible V. vinifera cultivar ‘Pinot Noir’. Following inoculation with sporangia of P. viticola isolate ‘YL’ on V. vinifera cv. ‘Pinot Noir’, the infection was characterized by a rapid spread of intercellular hyphae, a high frequency of haustorium formation within the host’s mesophyll cells, the production of sporangia and by the absence of host-cell necrosis. In contrast zoospores were collapsed in the resistant V. pseudoreticulata ‘Baihe-35-1’, or secretions appeared arround stomata at the beginning of the infection period in V. davidii var. cyanocarpa ‘Langao-5’ and V. piasezkii ‘Liuba-8’. The main characteristics of the resistance responses were the rapid depositions of callose and the appearance of empty hyphae and the plasmolysis of penetrated tissue. Moreover, collapsed haustoria were observed in V. davidii var. cyanocarpa ‘Langao-5’ at 5 days post inoculation (dpi) and in V. piasezkii ‘Liuba-8’ at 7 dpi. Lastly, necrosis extended beyond the zone of restricted colonization in all three resistant genotypes. Sporangia were absent in V. piasezkii ‘Liuba-8’ and greatly decreased in V. davidii var. cyanocarpa ‘Langao-5’ and in V. pseudoreticulata ‘Baihe-35-1’ compared with in V. vinifera cv. ‘Pinot Noir’. Overall, these results provide insights into the cellular biological basis of the incompatible interactions between the pathogen and the host. They indicate a number of several resistant Chinese wild species that could be used in developing new cultivars having good levels of downy mildew resistance.

The SBP-Box Gene VpSBP11 from Chinese Wild Vitis Is Involved in Floral Transition and Affects Leaf Development.

Flowering occurs in angiosperms during a major developmental transition from vegetative growth to the reproductive phase. Squamosa promoter binding protein (SBP)-box genes have been found to play critical roles in regulating flower and fruit development, but their roles in grapevine have remained unclear. To better understand the functions of the grape SBP-box genes in both vegetative and reproductive growth phases, a full-length complementary DNA (cDNA) sequence of the putative SBP-box transcription factor gene, VpSBP11, was obtained from Chinese wild grapevine Vitis pseudoreticulata Wen Tsai Wang (W. T. Wang) clone ‘Baihe-35-1’. VpSBP11 encoded a putative polypeptide of 170 amino acids with a highly conserved SBP-domain with two zinc-binding sites of the Cx2C-x3-H-x11-C-x6-H (C2HCH) type and a nuclear localization signal. We confirmed that the VpSBP11 protein was targeted to the nucleus and possessed transcriptional activation activity by subcellular localization and trans-activation assay. Over-expression of VpSBP11 in Arabidopsis thaliana was shown to activate the FUL gene, and subsequently the AP1 and LFY genes, all of which were floral meristem identity genes, and to cause earlier flowering than in wild type (WT) plants. The pattern of vegetative growth was also different between the transgenic and WT plants. For example, in the VpSBP11 over-expressing transgenic plants, the number of rosette leaves was less than that of WT; the petiole was significantly elongated; and the rosette and cauline leaves curled upwards or downwards. These results were consistent with VpSBP11 acting as a transcription factor during the transition from the vegetative stage to the reproductive stage.

Overexpression of VpEIFP1, a novel F-box/Kelch-repeat protein from wild Chinese Vitis pseudoreticulata, confers higher tolerance to powdery mildew by inducing thioredoxin z proteolysis

An F-box protein (VpEIFP1) induced by Erysiphe necator was isolated from Vitis pseudoreticulata, a wild Chinese grapevine species naturally resistant to powdery mildew (PM). It contains an F-box domain and two Kelch-repeat motifs. Expression profiles indicate the VpEIFP1 is strongly induced at both transcriptional and translational levels by PM infection. A subcellular localisation assay showed that VpEIFP1 is predominantly located in the nucleus and cytoplasm. Overexpression of VpEIFP1 accelerated the accumulation of hydrogen peroxide (H2O2) and up-regulated the expressions of ICS2, NPR1 and PR1 involved in defence responses, resulting in suppression of PM germination and growth. As an F-box protein, VpEIFP1 interacts with thioredoxin z (VpTrxz) in the yeast-two-hybrid (Y2H) assay and in the bimolecular fluorescence complementation (BiFC) assay. Decreased amounts of VpTrxz protein in transgenic grapevine leaves overexpressing VpEIFP1 were restored by proteasome inhibitor MG132, implying that VpEIFP1 mediated VpTrxz for degradation through the SCFVpEIFP1 (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. The RNA interference line of VpTrxz showed increased H2O2 accumulation following PM inoculation. We propose VpEIFP1 positively modulates the grapevine defence response to PM by inducing the degradation of VpTrxz via the ubiquitin/26S proteasome system.

The growing season impacts the accumulation and composition of flavonoids in grape skins in two-crop-a-year viticulture.

The influence of growing season (winter vs. summer) on the flavonoid accumulation and composition was studied in the skins of three grape cultivars for two consecutive years under a two-crop-a-year viticulture practice in Southwest China. The total anthocyanin, flavonol and flavan-3-ol contents in winter berry skins were significantly higher than those in summer berry skins for 'Kyoho' and 'Muscat Hamburg'. Reversely, the content of anthocyanin in 'NW196' winter berry was lower than summer berry. However, the percentage of diglycosylated, trihydroxylated, methylated, and acylated anthocyanins, trihydroxylated and methylated flavonols, and flavan-3-ol polymers were higher in the summer berry skins than the winter berry skins among all the three grape cultivars. Winter climatic conditions were favorable to flavonoid accumulation for the non native grapes 'Kyoho' and 'Muscat Hamburg', while the summer climatic conditions were beneficial to anthocyanin accumulation for 'NW196' that has 50% genetic background from a local wild grape species Vitis quinquangularis. These seasonal variations of flavonoid accumulations and compositions in the grape skins were primarily contributed by different climatic factors, such as temperature, solar radiation, and rainfall.

An efficient method for transgenic callus induction from Vitis amurensis petiole.

Transformation is the main platform for genetic improvement and gene function studies in plants. However, the established somatic embryo transformation system for grapevines is time-consuming and has low efficiency, which limits its utilization in functional genomics research. Vitis amurensis is a wild Vitis species with remarkable cold tolerance. The lack of an efficient genetic transformation system for it has significantly hindered the functional identification of cold stress related genes in the species. Herein, an efficient method was established to produce transformed calli of V. amurensis. Segments of petioles from micropropagated plantlets of V. amurensis exhibited better capacity to differentiate calli than leaf-discs and stem segments, and thus was chosen as target tissue for Agrobacterium-mediated transformation. Both neomycin phosphotransferase II (NPTII) and enhanced green fluorescent protein (eGFP) genes were used for simultaneous selection of transgenic calli based on kanamycin resistance and eGFP fluorescence. Several parameters affecting the transformation efficiency were optimized including the concentration of kanamycin, Agrobacterium stains, bacterial densities, infection treatments and co-cultivation time. The transgenic callus lines were verified by checking the integration of NPTII gene into calli genomes, the expression of eGFP gene and the fluorescence of eGFP. Up to 20% of the petiole segments produced transformed calli after 2 months of cultivation. This efficient transformation system will facilitate the functional analysis of agronomic characteristics and related genes not only in V. amurensis but also in other grapevine species.

Expression patterns and promoter characteristics of the Vitis quinquangularis VqSTS36 gene involved in abiotic and biotic stress response.

Resveratrol is a stilbene compound that is synthesized by plants in response to biotic stress and has been linked to health benefits associated with the consumption of certain foods and food products, such as grapes and wine. The final step in the biosynthesis of resveratrol is catalyzed by the enzyme stilbene synthase (STS). Here, we assessed the expression of two STS genes (VqSTS36 and VpSTS36) from the wild grape species Vitis quinquangularis (accession 'Shang-24'; powdery mildew (PM) resistant) and Vitis pseudoreticulata (accession 'Hunan-1'; PM susceptible) following infection by Uncinula necator (Schw.) Burr, the causal agent of PM disease. Some correlation was observed between the relative levels of STS36 transcript and disease resistance. We also cloned the 5' upstream sequence of both VpSTS36 and VqSTS36 and generated a series of 5' VqSTS36 promoter deletions fused to the GUS reporter gene in order to analyze expression in response to wounding, the application of exogenous stress-associated hormones, and biotic stress in tobacco leaves. The promoter was shown to be induced by the hormone salicylic acid (SA), inoculation with the fungal pathogen Erysiphe cichoracearum, and by wounding. These results suggest that VqSTS36 is regulated by biotic stresses and that it plays an important role in mediating disease resistance in grape.

Fungal endophytes of wild and hybrid Vitis leaves and their potential for vineyard biocontrol.

Plants are colonized by diverse assemblages of fungal endophytes that have potential as biocontrol agents for a variety of crops, including grapevine. Although the diversity of symbionts can be very high in wild plants, the fungal endophytes of wild Vitis plants have not yet been investigated. We surveyed the fungal endophytes of 6 wild populations of Vitis riparia, as well as a cold-tolerant, hybrid grapevine in 5 vineyards (1 certified organic), using 454 pyrosequencing. We detected between 43 and 235 operational taxonomic units per sample, with the highest richness and diversity in the wild, the lowest in conventional vineyards, and intermediate levels in the organic vineyard. Wild plants supported a range of taxa not seen in the conventional vineyards, and vineyards were dominated by relatively few taxa. We also isolated fungi from the wild plants and tested them for their ability to inhibit pathogens of grapevine. Several wild isolates (e.g., Ramularia spp.) were strongly inhibitory to grapevine pathogens. We show that wild Vitis supports a distinct and highly diverse community of fungal endophytes and may represent a rich repository of potential vineyard biocontrol agents.

Exploration of Elite Stilbene Synthase Alleles for Resveratrol Concentration in Wild Chinese Vitis spp. and Vitis Cultivars.

Resveratrol contributes to a plant’s tolerance of various abiotic and biotic stresses and is highly beneficial to human health. A search for elite alleles affecting resveratrol production was undertaken to find useful grapevine germplasm resources. Resveratrol levels in both berry skins and leaves were determined in 95 grapevine accessions (including 50 wild Chinese grapevine accessions and 45 cultivars) during two consecutive years. Resveratrol contents were higher in berry skins than in leaves and in wild Chinese grapevines than in grapevine cultivars. Using genotyping data, 79 simple sequence repeat (SSR) markers linked to 44 stilbene synthase (STS) genes were detected in the 95 accessions, identifying 40 SSR markers with higher polymorphisms. Eight SSR marker loci, encompassing 19 alleles, were significantly associated with resveratrol content on (P < 0.001), and 5 SSR loci showed repeated associations. Locus Sh5 had four associations: three positive for allele 232 (including leaves in the 2 years) and one negative for allele 236 in four environments. Loci Sh9 and Sh56 for a total of 7 alleles exhibited positive effects in berry skins in the 2 years. In berry skins, locus Sh56 with positive effects was closely linked to VvSTS27, and locus Sh77 with negative effects to VvSTS17, importantly, the two candidate genes both were located on Chromosome 16. The SSR marker loci and candidate genes identified in this study will provide a useful basis for future molecular breeding for increased production of natural resveratrol and its derivatives.

VaERD15, a Transcription Factor Gene Associated with Cold-Tolerance in Chinese Wild Vitis amurensis.

Early responsive to dehydration (ERD) genes can be rapidly induced to counteract abiotic stresses, such as drought, low temperatures or high salinities. Here, we report on an ERD gene (VaERD15) related to cold tolerance from Chinese wild Vitis amurensis accession ‘Heilongjiang seedling’. The full-length VaERD15 cDNA is 685 bp, including a 66 bp 5′-untranslated region (UTR), a 196 bp 3′-UTR region and a 423 bp open reading frame encoding 140 amino acids. The VaERD15 protein shares a high amino acid sequence similarity with ERD15 of Arabidopsis thaliana. In our study, VaERD15 was shown to have a nucleic localization function and a transcriptional activation function. Semi-quantitative PCR and Western blot analyses showed that VaERD15 was constitutively expressed in young leaves, stems and roots of V. amurensis accession ‘Heilongjiang seedling’ plants, and expression levels increased after low-temperature treatment. We also generated a transgenic Arabidopsis Col-0 line that over-expressed VaERD15 and carried out a cold-treatment assay. Real-time quantitative PCR (qRT-PCR) and Western blot analyses showed that as the duration of cold treatment increased, the expression of both gene and protein levels increased continuously in the transgenic plants, while almost no expression was detected in the wild type Arabidopsis. Moreover, the plants that over-expressed VaERD15 showed higher cold tolerance and accumulation of proline, soluble sugars, proteins, malondialdehyde and three antioxidases (superoxide dismutase, peroxidase, and catalase). Lower levels of relative ion leakage also occurred under cold stress. Taken together, our results indicate that the transcription factor VaERD15 was induced by cold stress and was able to enhance cold tolerance.

Ectopic Expression of the Wild Grape WRKY Transcription Factor VqWRKY52 in Arabidopsis thaliana Enhances Resistance to the Biotrophic Pathogen Powdery Mildew But Not to the Necrotrophic Pathogen Botrytis cinerea.

WRKY transcription factors are known to play important roles in plant responses to biotic stresses. We previously showed that the expression of the WRKY gene, VqWRKY52, from Chinese wild Vitis quinquangularis was strongly induced 24 h post inoculation with powdery mildew. In this study, we analyzed the expression levels of VqWRKY52 following treatment with the defense related hormones salicylic acid (SA) and methyl jasmonate, revealing that VqWRKY52 was strongly induced by SA but not JA. We characterized the VqWRKY52 gene, which encodes a WRKY III gene family member, and found that ectopic expression in Arabidopsis thaliana enhanced resistance to powdery mildew and Pseudomonas syringae pv. tomato DC3000, but increased susceptibility to Botrytis cinerea, compared with wild type (WT) plants. The transgenic A. thaliana lines displayed strong cell death induced by the biotrophic powdery mildew pathogen, the hemibiotrophic P. syringe pathogen and the necrotrophic pathogen B. cinerea. In addition, the relative expression levels of various defense-related genes were compared between the transgenic A. thaliana lines and WT plants following the infection by different pathogens. Collectively, the results indicated that VqWRKY52 plays essential roles in the SA dependent signal transduction pathway and that it can enhance the hypersensitive response cell death triggered by microbial pathogens.