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PubMed2002

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.

Base Sequence↗
PubMed2003

Evidence of a secondary grapevine domestication centre detected by SSR analysis.

The origin of the grapevine was investigated with archaeobotanical, cultural and historical data. A primary domestication centre was located in the Near East region but there is no agreement on the existence or role of secondary domestication centres. In this work, PCR-based microsatellite analysis has been applied to study the origin of some Italian cultivated grapevines from in situ direct domestication of the wild autoctonous grapevine. Three different Italian locations in Grosseto, Cosenza and Nuoro were identified for this study, and domesticated grapevine as well as wild local accessions growing in these location, were analysed by SSR markers. Cluster analysis performed on Cosenza and Grosseto samples showed a high value of genetic distance between domesticated and wild accessions. On the contrary two cultivars (Bovale Murru and Bovale Muristellu) recovered in Nuoro (in the Sardinia island) were very close to some wild varieties. This suggests that the latter two cultivars may have originated from wild grapevines and consequently that in this location a secondary grapevine domestication event occurred. Six Lambrusco varieties were also included in this analysis as ancient putative ancestors of the cultivated grapevines. The molecular analysis excluded this hypothesis and suggest Lambrusco as an independent Vitis taxon.

DNA, Plant↗
PubMed2005

Resveratrol glucoside (Piceid) synthesis in seeds of transgenic oilseed rape (Brassica napus L.).

Resveratrol is a phytoalexin produced in various plants like wine, peanut or pine in response to fungal infection or UV irradiation, but it is absent in members of the Brassicaceae. Moreover, resveratrol and its glucoside (piceid) are considered to have beneficial effects on human health, known to reduce heart disease, arteriosclerosis and cancer mortality. Therefore, the introduction of the gene encoding stilbene synthase for resveratrol production in rapeseed is a tempting approach to improve the quality of rapeseed products. The stilbene synthase gene isolated from grapevine (Vitis vinifera L.) was cloned under control of the seed-specific napin promotor and introduced into rapeseed (Brassica napus L.) by Agrobacterium-mediated co-transformation together with a ds-RNA-interference construct deduced from the sequence of the key enzyme for sinapate ester biosynthesis, UDP-glucose:sinapate glucosyltransferase (BnSGT1), assuming that the suppression of the sinapate ester biosynthesis may increase the resveratrol production in seeds through the increased availability of the precursor 4-coumarate. Resveratrol glucoside (piceid) was produced at levels up to 361 microg/g in the seeds of the primary transformants. This value exceeded by far piceid amounts reported from B. napus expressing VST1 in the wild type sinapine background. There was no significant difference in other important agronomic traits, like oil, protein, fatty acid and glucosinolate content in comparison to the control plants. In the third seed generation, up to 616 microg/g piceid was found in the seeds of a homozygous T3-plant with a single transgene copy integrated. The sinapate ester content in this homozygous T3-plant was reduced from 7.43 to 2.40 mg/g. These results demonstrate how the creation of a novel metabolic sink could divert the synthesis towards the production of piceid rather than sinapate ester, thereby increasing the value of oilseed products.

Acyltransferases↗
PubMed2005

Construction and characterization of BAC libraries from major grapevine cultivars.

Genome projects were initiated on grapevine (Vitis vinifera L., 2n=38, genome size 475 Mb) through the successful construction of four bacterial artificial chromosome (BAC) libraries from three major cultivars, Cabernet Sauvignon (Cabernet S), Syrah and two different clones of Pinot Noir (Pinot N). Depending on the library, the genome coverage represented 4.5-14.8 genome equivalents with clones having a mean insert size of 93-158 kb. BAC pools suitable for PCR screening were constructed for two of these BAC libraries [Cabernet S and Pinot N clone (cl) 115] and subsequently used to confirm the genome coverage of both libraries by PCR anchoring of 74 genetic markers sampled from the 19 linkage groups. For ten of these markers, two bands on separate BAC pools were differentiated that could correspond either to different alleles or to a duplication of the locus being studied. Finally, a preliminary assessment of the correspondence between genetic and physical distances was made through the anchoring of all the markers mapped along linkage group 1 of the V. vinifera genetic map. A pair of markers, 2.1 cM apart, anchored the same BAC clones, which allowed us to estimate that 1 cM corresponded in this particular region to a maximum length of 130 kb.

Chromosome Mapping↗
PubMed2006

An integrated SSR map of grapevine based on five mapping populations.

A grapevine (mainly Vitis vinifera L., 2n = 38) composite genetic map was constructed with CarthaGene using segregation data from five full-sib populations of 46, 95, 114, 139 and 153 individuals, to determine the relative position of a large set of molecular markers. This consensus map comprised 515 loci (502 SSRs and 13 other type PCR-based markers), amplified using 439 primer pairs (426 SSRs and 13 others) with 50.1% common markers shared by at least two crosses. Out of all loci, 257, 85, 74, 69 and 30 were mapped in 1, 2, 3, 4 and 5 individual mapping populations, respectively. Marker order was generally well conserved between maps of individual populations, with only a few significant differences in the recombination rate of marker pairs between two or more populations. The total length of the integrated map was 1,647 cM Kosambi covering 19 linkage groups, with a mean distance between neighbour loci of 3.3 cM. A framework-integrated map was also built, with marker order supported by a LOD of 2.0. It included 257 loci spanning 1,485 cM Kosambi with a mean inter-locus distance of 6.2 cM over 19 linkage groups. These integrated maps are the most comprehensive SSR-based maps available so far in grapevine and will serve either for choosing markers evenly scattered over the whole genome or for selecting markers that cover particular regions of interest. The framework map is also a useful starting point for the integration of the V. vinifera physical and genetic maps.

Chromosome Mapping↗
PubMed2006

Interactions between yeasts and grapevines: filamentous growth, endopolygalacturonase and phytopathogenicity of colonizing yeasts.

It has been clearly established that phytopathogenic fungi, bacteria, and viruses exert biotic stresses on plants. Much less is known, however, about the interactions between enological species of yeast and their host plants. In a previous study, we described how Saccharomyces cerevisiae, the most common enological yeast, can act as a grapevine (Vitis vinifera L.) pathogen, causing growth retardation or plant death. In the present in vitro study on 11 strains of yeast belonging to different genera, which often occur on the surfaces of vineyard grapes and V. vinifera, a link was found to exist between strain phytopathogenecity and pseudohyphal growth habits and/or endopolygalacturonase activity. The results obtained here are consistent with earlier findings showing that the phytopathogenicity of yeast strains depends on the filamentous growth process, and show that endopolygalacturonase alone is not responsible for the invasion of plants tissues. The mechanisms observed here may be of significant ecological importance and may help to explain the long periods of yeast survival found to occur in vineyards.

Ecosystem↗
PubMed2006

Molecular characterization of grapevine plants transformed with GFLV resistance genes: II.

A collection of 127 putatively transgenic individuals of Vitis vinifera cv. Russalka was characterized by PCR and Southern hybridization. Six different constructs containing the neomycin phosphotransferase (nptII) marker gene and sequences of the Grapevine Fanleaf Virus Coat Protein (GFLV CP) gene including non-translatable and truncated forms were transferred via Agrobacterium-mediated transformation. Detection of transgenic sequences by PCR was positive in all lines. Southern blot analysis revealed that the number of inserted T-DNA copies ranged from 1 to 6. More than 46% of the tested transgenic lines contain one copy of the inserted T-DNA, qualifying them as interesting candidates for further breeding programs. Southern data of one line indicate the presence of an incomplete copy of the T-DNA, thus confirming previous PCR results. Since many putative transgenic lines shared identical hybridization patterns, they were clustered into 39 lines and considered as having originated from independent transformation events. The detection of the tetracycline (TET) resistance genes in 15% of the lines shows that an integration of plasmid backbone sequences beyond the T-DNA borders occurred. Enzyme-linked immunosorbent assay (ELISA) performed on leaf tissue did not show any accumulation of the GFLV CP in the 39 transgenic lines analyzed. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot were carried out; RT-PCR analyses showed that the GFLV CP mRNA was expressed at variable levels.

Agrobacterium tumefaciens↗
PubMed2006

Biolistic transformation of grapevine using minimal gene cassette technology.

The use of minimal gene cassettes (MCs), which are linear DNA fragments (promoter+open reading frame+terminator) lacking the vector backbone sequence, was compared to the traditional use of whole circular plasmids (CPs) for transformation of grapevine. Embryogenic cell suspensions of 'Chardonnay' (Vitis vinifera L.) were transformed via particle co-bombardment using two nonlinked genes in either MCs or CPs. One construct contained the npt-II selectable marker and the second construct contained the MSI99 antimicrobial peptide gene. A total of five lines each from MC and CP treatments that showed positive signals by PCR for both the npt-II and MSI99 genes were selected. Southern blot analyses revealed up to five integration events in the DNA treatments. Transcription levels determined by semi-quantitative RT-PCR varied among transgenic lines. No significant differences were found in transgene transcription between lines from MC and CP transformation. The correlation between npt-II and MSI99 transcription levels was positive (P<0.05), however, no correlation between the transcription level and the number of integration events was observed. Transgenic lines presented a similar phenotype in leaf morphology and plant vigor compared to non-transgenic lines. Moreover, transgenic lines from both MC and CP DNA treatments produced fruit as did the non-transgenic lines in the third year of growth in the greenhouse. Our data confirm the effectiveness of the minimal cassette technology for genetic transformation of grapevine cultivars.

Biolistics↗
PubMed2002

The host guides morphogenesis and stomatal targeting in the grapevine pathogen Plasmopara viticola.

The oomycete grape downy mildew (Plasmopara viticola Berk. & Curt. Ex de Bary) is a serious pathogen of grapevine and spreads by extremely efficient cycles of asexual propagation. The high efficiency must involve efficient sensing of the host. We therefore analyzed the time course and morphology of the early development of this pathogen in a host system, by infection of leaf discs of grapevine (Vitis vinifera L. cv. Müller-Thurgau), and in a host-free system. Host factors were demonstrated to influence pathogen development in the following ways: (i) the release of zoospores from mature sporangia was accelerated, (ii) the morphogenesis of the germ tube was coordinated, and (iii) the zoospores were targeted to the stomata by factors that depended on stomata closure. The findings show that the early development of P. viticola is regulated, specifically and coordinately, by factors originating from the host plant.

Morphogenesis↗
PubMed2002

Cellular and subcellular localisation of glutamine synthetase and glutamate dehydrogenase in grapes gives new insights on the regulation of carbon and nitrogen metabolism.

The subcellular localisation of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in grapevine (Vitis vinifera L.) leaves and flowers was investigated using immunogold-labelling experiments. In mature leaf tissue or fully developed flowers, GS was visualised both in the cytosol and in the chloroplasts, a high proportion of the protein being present in the phloem companion cells. GDH was preferentially located in the mitochondria of the phloem companion cells in both leaves and flowers. This observation suggests that, in conjunction with GS, GDH plays a major role in controlling the translocation of organic carbon and nitrogen metabolites in both vegetative and reproductive organs. Significant amounts of GDH protein were also visualised in multivesicular bodies within the flower receptacle. Although the function of such organelles is still unknown, its is possible that the presence of GDH in such cellular structures is important for the recycling of carbon and nitrogen molecules in senescing tissues in which the enzyme is generally induced.

Carbon↗
PubMed2003

Oligogalacturonide signal transduction, induction of defense-related responses and protection of grapevine against Botrytis cinerea.

Grapevine ( Vitis vinifera L.) is vulnerable to a variety of pathogenic fungi, among them Botrytis cinerea, the causal agent of grey mould, is responsible for worldwide yield losses that would be even more important without a successful control that relies mainly on fungicides. In the present work we investigated an alternative way of using oligogalacturonides (OGA) to induce defense responses in grapevine and protection against B. cinerea. Kinetic experiments with grapevine cells showed that OGA induced a rapid and transient generation of H(2)O(2), followed by differential expression of nine defense-related genes and stimulation of chitinase and beta-1,3-glucanase activities. Inhibition of OGA-induced oxidative burst by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, lowered induction levels of six genes and chitinase activity. Interestingly, the induction of three other genes and beta-1,3-glucanase activity were inhibited by K252a, a protein kinase inhibitor, but not by DPI. Treatment of grapevine leaves with OGA also reduced infection by B. cinerea by about 55-65%. Accordingly, DPI or K252a with or without OGA increased the susceptibility of grapevine leaves to B. cinerea. We suggest that treatment of grapevine with OGA elicits different signalling pathways, which might act in tandem with the oxidative burst to increase grapevine defense responses required for protection against B. cinerea.

Botrytis↗
PubMed2004

Expression of the grapevine stilbene synthase gene VST1 in papaya provides increased resistance against diseases caused by Phytophthora palmivora.

The phytoalexin resveratrol (trans-3,5,4'-trihydroxy-stilbene), a natural component of resistance to fungal diseases in many plants, is synthesized by the enzyme trihydroxystilbene synthase (stilbene synthase, EC 2.3.1.95), which appears to be deficient or lacking in susceptible plants. Earlier workers isolated a stilbene synthase gene (Vst1) from grapevine (Vitis vinifera L.), which has subsequently been introduced as a transgene into a range of species to increase resistance of hosts to pathogens to which they were originally susceptible. Papaya (Carica papaya L.) is susceptible to a variety of fungal diseases, including root, stem, and fruit rot caused by the pathogen Phytophthora palmivora. Since resveratrol at 1.0 mM inhibited mycelium growth of P. palmivora in vitro, we hypothesized that papaya resistance to this pathogen might be increased by transformation with the grapevine stilbene synthase construct pVst1, containing the Vst1 gene and its pathogen-inducible promoter. Multiple transformed lines were produced, clonally propagated, and evaluated with a leaf disk bioassay and whole plant response to inoculation with P. palmivora. RNA transcripts of stilbene synthase and resveratrol glycoside were induced in plant lines transformed with the grapevine pVst1 construct shortly after pathogen inoculation, and the transformed papaya lines exhibited increased resistance to P. palmivora. The immature transformed plants appear normal and will be advanced to field trials to evaluate their utility.

Acyltransferases↗
PubMed2001

Changes in acidity and in proton transport at the tonoplast of grape berries during development.

As in many fruits, the induction of grape berry (Vitis vinifera L.) ripening results in intense breakdown of malic acid. Using membrane fractions, we tested the hypothesis that changes in acidity resulted from malate vacuolar decompartmentation. The hydrolytic activities of the two primary vacuolar pumps inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) and vacuolar ATPase (V-ATPase; EC 3.6.1.3) increased throughout development with an acceleration during ripening, as confirmed by Western blotting and analysis of transcript expression. The ratio of V-PPase activity to V-ATPase activity was always in favour of V-PPase and reached its maximum value at véraison. The rate of anion transport strongly increased during ripening. Before ripening, tonoplast passive permeability was low, but rose during ripening. Our data indicate that tonoplast leakage dramatically increased during ripening. This leakage is probably the prime cause of malate decompartmentation, amplified by the incapacity of oxidative phosphorylation to face increased energy demand.

Adenosine Triphosphate↗
PubMed2001

Expression patterns of cell wall-modifying enzymes during grape berry development.

During ripening of grape (Vitis vinifera L.) berries, softening occurs concomitantly with the second growth phase of the fruit and involves significant changes in the properties of cell wall polysaccharides. Here, the activities of enzymes that might participate in cell wall modification have been monitored throughout berry development. Alpha-galactosidase (EC 3.2.1.22), beta-galactosidase (EC 3.2.1.23) and pectin methylesterase (EC 3.1.1.11) activities were present, but no polygalacturonase (EC 3.2.1.15), cellulase (EC 3.2.1.4), xyloglucanase (xyloglucan-specific cellulase EC 3.2.1.4) or galactanase (EC 3.2.1.89) could be detected. The accumulation of mRNAs encoding wall-modifying enzymes was examined by northern hybridization analysis. Transcripts for beta-galactosidase, pectin methylesterase, polygalacturonase, pectate lyase (EC 4.2.2.2) and xyloglucan endotransglycosylase (EC 2.4.1.207) were present during ripening, although polygalacturonase activity had not been detected in berry extracts. Cellulases could not be detected in ripening berries, either at the enzyme or mRNA levels. The increase in beta-galactosidase activity and mRNA is consistent with the observed decrease in type-I arabinogalactan content of the walls during ripening, and the detection of polygalacturonase and pectate lyase mRNAs might explain the increased solubility of galacturonan in walls of ripening grapes. Thus, the modification of cell wall polysaccharides during softening of grape berries is a complex process involving subtle changes to different components of the wall, and in many cases only small amounts of enzyme activity are required to effect these changes.

Blotting, Northern↗
PubMed2005

Relative performance of European grapevine moth (Lobesia botrana) on grapes and other hosts.

The European grapevine moth, Lobesia botrana is a major grapevine pest, but despite the abundance of vineyards it is a generalist and uses either grapes or alternative species. Given the abundance and predictability of grape, L. botrana could be expected to have evolved towards monophagy. In order to understand why this species remains polyphagous, we hypothesized that larvae reared on rare wild host plants should have higher fitness than those reared on the more abundant grape host. For this, we compared larval performance and several life history traits on three alternative host plants (Daphne gnidium, Olea europaea, Tanacetum vulgare) and three Vitaceae (Vitis vinifera), two cultivars and one wild species (Ampelopsis brevipedunculata), and two control groups raised on either a low or a high nutritive value medium. Alternative hosts are more suitable than Vitaceae for the reproductive performance of L. botrana: larval mortality and development time was reduced, while pupal weight, growth rate, female longevity, female fecundity, duration of laying and mating success were increased. High quality food ingested by larvae promotes higher adult body weight and enhances female reproductive output. This suggests that alternative hosts provide greater nutritional value for L. botrana than Vitaceae. The use of alternative host plants could thus be maintained in the host range because they offer L. botrana a better fitness than on the Vitaceae. This could typically represent an advantage for moths behaving in plant diversity grape landscapes.

Animal Nutritional Physiological Phenomena↗
PubMed2004

Climate change and shifts in spring phenology of three horticultural woody perennials in northeastern USA.

We evaluated spring phenology changes from 1965 to 2001 in northeastern USA utilizing a unique data set from 72 locations with genetically identical lilac plants (Syringa chinensis, clone "Red Rothomagensis"). We also utilized a previously validated lilac-honeysuckle "spring index" model to reconstruct a more complete record of first leaf date (FLD) and first flower date (FFD) for the region from historical weather data. In addition, we examined mid-bloom dates for apple (Malus domestica) and grape (Vitis vinifera) collected at several sites in the region during approximately the same time period. Almost all lilac sites with significant linear trends for FLD or FFD versus year had negative slopes (advanced development). Regression analysis of pooled data for the 72 sites indicated an advance of -0.092 day/year for FFD (P=0.003). The slope for FLD was also negative (-0.048 day/year), but not significant (P=0.234). The simulated data from the "spring index" model, which relies on local daily temperature records, indicated highly significant (P<0.001) negative slopes of -0.210 and -0.123 day/year for FLD and FFD, respectively. Data collected for apple and grape also indicated advance spring development, with slopes for mid-bloom date versus year of -0.20 day/year (P=0.01) and -0.146 (P=0.14), respectively. Collectively, these results indicate an advance in spring phenology ranging from 2 to 8 days for these woody perennials in northeastern USA for the period 1965 to 2001, qualitatively consistent with a warming trend, and consistent with phenology shifts reported for other mid- and high-latitude regions.

Climate↗
PubMed2005

Rupestris stem pitting associated virus isolates are composed by mixtures of genomic variants which share a highly conserved coat protein.

Broad spectrum primers were used to amplify a fragment comprising the CP gene and putative ORF6 by RT-PCR from ds-RNA templates originating from 46 Portuguese varieties, totalling 190 samples, including some wild Vitis ssp sylvestris vines, and 2 vines from Slovenia. SSCP analysis was used as a preliminary screen to avoid cloning and sequencing very similar variants. Four groups of variants were recognized. In pair wise comparisons between nucleotide sequences the minimal homology found was 81%. In case of the cultivated varieties, no relationship could be seen between the phylogenetic groups and geographic origin or grape variety. Several isolates were found harbouring mixed infections with genomic variants from different groups, but the mixing did not lead to an extensive recombination between them. The deduced amino-acid sequences revealed a conserved CP subjected to strong purifying selection pressure. Analysis of the selection pressure operating on the putative ORF6 suggests that this ORF does not exist. Previously produced polyclonal antiserum raised against the recombinant CP of RSPaV expressed in Escherichia coli was shown to be able to detect all four groups of variants of RSPaV included in this study, which might enable the diagnosis of the virus on a serological basis.

Capsid Proteins↗
PubMed2006

Water and salinity stress in grapevines: early and late changes in transcript and metabolite profiles.

Grapes are grown in semiarid environments, where drought and salinity are common problems. Microarray transcript profiling, quantitative reverse transcription-PCR, and metabolite profiling were used to define genes and metabolic pathways in Vitis vinifera cv. Cabernet Sauvignon with shared and divergent responses to a gradually applied and long-term (16 days) water-deficit stress and equivalent salinity stress. In this first-of-a-kind study, distinct differences between water deficit and salinity were revealed. Water deficit caused more rapid and greater inhibition of shoot growth than did salinity at equivalent stem water potentials. One of the earliest responses to water deficit was an increase in the transcript abundance of RuBisCo activase (day 4), but this increase occurred much later in salt-stressed plants (day 12). As water deficit progressed, a greater number of affected transcripts were involved in metabolism, transport, and the biogenesis of cellular components than did salinity. Salinity affected a higher percentage of transcripts involved in transcription, protein synthesis, and protein fate than did water deficit. Metabolite profiling revealed that there were higher concentrations of glucose, malate, and proline in water-deficit-treated plants as compared to salinized plants. The metabolite differences were linked to differences in transcript abundance of many genes involved in energy metabolism and nitrogen assimilation, particularly photosynthesis, gluconeogenesis, and photorespiration. Water-deficit-treated plants appear to have a higher demand than salinized plants to adjust osmotically, detoxify free radicals (reactive oxygen species), and cope with photoinhibition.

Dehydration↗