Genetic Improvement of Grapevine: Tailoring Grape Varieties for The Third Millennium - A Review

The remarkable propagative aptitute of grapevine is one of the key factors contributing to its success as a cultivated species and to the spread of the domesticated grapevine, establishing it as one of the most important fruit species worldwide. Today there are some 8 million hectares of vineyards across the world. It is therefore titting that the successful implementation of the powerful technology of gene manipulation in grapevine is to a large extent reliant on this regenerative ability. Currently, several varieties of grapevines have been successfully genetically transformed, largely by employing somatic embryogenesis to generate highly regenerative target material. Especially attractive in the wine industry is the possibility of improving grapevine varieties by the addition of genes that confer usefultraits, such as resistances against biotic and abiotic factors and manipulation of certain metabolic functions. In principle, gene transfer technology allows for the directed manipulation of a specific trait without altering the characteristic nature of the cultivar, permitting the improvement of the traditional cultivars while maintaining their established varietal characteristics. For the most part, targeted traits currently include disease resistance andimproved berry quality. The promise of this technology is threatened by worldwide resistance to genetically modified organisms, and in the wine industry by complications surrounding the property rights and naming of transgenic vines. If it is not possible to maintain the varietal name when a transgenic vine has the same properties as the original well known variety, the significant advantages of gene technology over traditional breeding programmes are to a large extent lost. If these and other complications can be overcome, the integration of this powerful technology with traditional breeding programmes, and with other initiatives such as the study of the grapevine genome, will ensure a new era in the cultivation of this ancient species

Xiphinema index and its Relationship to Grapevines: A review

The dagger nematode, Xiphinema index, is considered a major pest in grape growing countries. Xiphinemaindex is especially important because of its ability to transmit Grapevine fanleaf virus when feeding ongrapevine roots. This paper provides a comprehensive and updated review of the classification, genetics andbiology of Xiphinema index, and its relationship with grapevine fanleaf virus. Current control measures, aswell as past and present efforts to breed resistant grapevine rootstocks, are presented

Identification of a functional TATA element in the STA2 glucoamylase gene promoter from Saccharomyces cerevisiae

Accurate transcription by RNA polymerase II is usually dependent on the presence of a TATA element, and/or an initiator element, in the promoters of protein-encoding genes. The STA1-3 genes, encoding three glucoamylase isozymes (Sta1p, Sta2p and Sta3p, respectively) responsible for starch hydrolysis in the yeast Saccharomyces cerevisiae, have been shown to contain long and complex promoters with several regulatory regions. These promoters are also virtually identical to the yeast MUC1 gene promoter; this gene encodes a mucin-like protein and is evolutionary linked to, and transcriptionally co-regulated with, STA1-3. The STA1-3 genes contain two putative TATA sequences, one conforming to the typical TATA box sequence, TATAAA, and another with the sequence of TATAAT. Here we present a study into the functional relevance of these putative TATA sequences and their effects on the transcription of the STA2 gene (as a representative model of the STA1-3 multigene family) and, by analogy, the MUC1 gene. We show that the TATAAA motif is the functional TATA box for STA2 and influences transcript levels, transcript initiation sites, and glucoamylase activities.Articl

Improved cryopreservation procedure for long term storage of synchronised culture of grapevine

Anther-derived pre-embryogenic masses (PEMs) of callus, established via suspension cultures, were encapsulated to form synthetic seeds suitable for cryopreservation. The synchronised suspension culture proliferation necessitated the optimisation of plant growth regulators for different cultivars. The growth phase and density of the culture were also important as well as the exposure of cells to vitrification solution containing 0.75 M sucrose with 0.1 M CaCl2 and 2.0 % sodium alginate (pH 5.7). Pre-treatment of the encapsulated cells for 2 d with Nitsch and Nitsch (NN) medium containing 0.75 M sucrose solution followed by dehydration for 4 h in a laminar flow box provided maximum cell viability, which varied from 0 to 40 %. The embryo proliferation from the cryopreserved beads involved warming them and then transfer to NN medium containing glutamine (50 mg dm-3) and activated charcoal (2.5 %). The maximum number of embryos obtained was 31-53 per bead. Subculture into the same medium induced secondary embryogenesis, which was initiated from the meristematic region, radicle, and root cap. Proliferation and maturation of secondary embryos was faster than of primary embryos. No phenotypic variation or abnormal structures compared to the control were observed in the regenerated plantlets. © 2011 Springer Science+Business Media B.V.Articl

High-performance liquid chromatography profiling of the major carotenoids in Arabidopsis thaliana leaf tissue

Carotenoids are extremely sensitive to a variety of physico-chemical attacks which may have a profound effect on their characteristic properties, thereby influencing the accurate identification and quantification of individual compounds. In this light, a comprehensive summary of the pitfalls encountered and precautions to be administered during handling and storage of authentic standards and samples was found to be incomplete. Furthermore, acceptable baseline separation of trans-lutein from trans-zeaxanthin and between the cis- and trans-forms of neoxanthin and violaxanthin has not been satisfactorily demonstrated. Hence the most optimal sample preparation and analytical steps were determined and a sensitive and reproducible method for the quantitative HPLC profiling of the principal carotenoids found in plant leaf tissue was developed. A reverse-phase C30 column with a binary mobile solvent system was used for the baseline separation of eight of the major carotenoids and the two chlorophylls (a and b) within 18 min. These compounds were identified via the use of authentic standards, their spectral characteristics and HPLC-atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) confirmation. This method has been successfully applied for the quantification of plant pigments in Arabidopsis thaliana wild-type (WT) leaf tissue and in two A. thaliana non-photochemical mutants, namely npq1 and npq2. These mutants have previously been well-characterised and provided valuable reference data as well as acting as internal controls for the assessment of our new method. © 2006 Elsevier B.V. All rights reserved.Articl

Overexpression, secretion and antifungal activity of the Saccharomyces cerevisiae chitinase

The excessive use of pesticides to prevent the outbreak of plant diseases holds various disadvantages for the cost-effectiveness of agricultural production practices, the quality of the end products, the environment and the labour force. The mounting public resistance to the use of hazardous pesticides on agricultural crops has hastened the search for natural antimicrobial peptides, enzymes and other types of biological control agents. The objective of this study was to investigate the potential of the antifungal activity of a yeast-derived chitinase for possible future agricultural applications. When plants are exposed to fungal pathogens, they produce pathogenesis-related (PR) defence proteins, such as chitinases, in order to degrade the chitin in the cell walls of the attacking fungi, thereby inhibiting further fungal growth and the development of hyphae. We have cloned the Saccharomyces cerevisiae chitinase gene (CTS1-2) into a multicopy 2μ-based plasmid and overexpressed it under the control of the phosphoglycerate kinase I gene (PGK1) promoter (PGK1P) and terminator (PGK1T) sequences. Secretion of the recombinant CTS1-2-encoded chitinases was directed by the native leader peptide, or by the secretion signals of either the yeast mating pheromone α-factor (MFα1S) or the Trichoderma reesei β-xylanase 2 (XYN2S). Northern blot analysis confirmed the PGK1PT-directed expression of CTS1-2 and an indirect enzyme assay was used to compare the efficiency of secretion of the three different recombinant chitinases. These recombinant chitinases were also shown to effectively inhibit spore germination and hyphal growth of Botrytis cinerea, an economically important fungal pathogen of several agricultural crop plants, including grapevine. This study could lead to the development of yeast chitinases and/or chitinolytic yeasts as biocontrol agents to combat plant diseases and reduce the use of chemical pesticides in the agricultural industry.Articl

The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae

The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end-products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast-based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1.0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA-1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA-1 transport protein) and pedC(D (encoding a protein involved in the transport and processing of PA-1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1(p)-MFa1(s)-pedA-ADH1(T), designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1(P)) and terminator (ADH1(T)). Secretion of the PED1-encoded pediocin PA-1 was directed by the yeast mating pheromone α-factor's secretion signal (MFa1(S)). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4.6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the development of bactericidal yeast strains where S. cerevisiae starter cultures not only conduct the fermentations in the wine, brewing and baking industries but also act as biological control agents to inhibit the growth of spoilage bacteria.Articl

Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrol

The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p-coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p-coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p-coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following β-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol β-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol. © 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.Articl