IVES Conference Series

IVES 9 Tag: IVES Conference Series ( Page 34 )

Proteomic profiling of grape berry presenting early loss of mesocarp cell vitality

From fruit set to ripening, the grape berry mesocarp experiences a wide range of dynamic physical, physiological, and biochemical changes, such as mesocarp cell death (MCD) and hydraulic isolation. The premature occurrence of such events is a characteristic of the Niagara Rosada (NR) variety, utilised as table grapes and winemaking. In our opinion, the onset of ripening would not cause MCD, but a down-regulation of respiratory enzymes during the early loss of cell viability, while maintaining membrane integrity. For this, we investigated three distinct developmental stages (green (E-L33), veraison (E-L35), and ripe (E-L39)) of NR berries by label-free proteomics, enzymatic respiratory activity and outer mesocarp imaging. Cell wall-modifying proteins were found to accumulate differently throughout ripening, while cytoplasmic membranes continue intact.

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Tracking the origin of Tempranillo Tinto through whole genome resequencing and high-throughput genotyping  

Grapevine cultivars are vegetatively propagated to maintain their varietal characteristics. This process of multiplication leads to spontaneous somatic mutations that can eventually generate a variant phenotype, of potential interest for cultivar improvement and innovation. However, regardless their phenotypic effect, somatic mutations stack in the genome, and they can be used to reveal the origin and dissemination history of ancient cultivars. Here, a stringent somatic variant calling over whole genome resequencing data from 35 ‘Tempranillo Tinto’ clones or old vines from seven Iberian winemaking regions revealed 135 single nucleotide variations (SNVs) shared by some of the clonal lines.

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Artificial intelligence (AI)-based protein modeling for the interpretation of grapevine genetic variants

Genetic variants known to produce single residue missense mutations have been associated with phenotypic traits of commercial interest in grapevine. This is the case of the K284N substitution in VviDXS1 associated with muscat aroma, or the R197L in VviAGL11 causing stenospermocarpic seedless grapes. The impact of such mutations on protein structure, stability, dynamics, interactions, or functional mechanism can be studied by computational methods, including our pyDock scoring, previously developed. For this, knowledge on the 3D structure of the protein and its complexes with other proteins and biomolecules is required, but such knowledge is not available for virtually none of the proteins and complexes in grapevine.

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Effects of hormone- and natural-based elicitors at the transcriptomic level in berries of cv. Tempranillo

One of the most important effects of climate change in wine-growing areas is the advance of phenological stages, especially concerning early berry ripening. In the hottest seasons, this results in a lack of synchrony between sugar and phenolic ripeness. In order to cope with this fact, a general effort is being made by researchers and growers aiming at delaying ripening through different strategies. One of the proposed approaches is the application of elicitors. This study aims to assess the effect at the transcriptomic level of the application of three hormone- and natural-based elicitors in Tempranillo.

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Digitalization and valorization of the genotypic and phenotypic information retained within the FEM grapevine germplasm

The maintenance and valorization of genetic diversity is an undoubtable resource for the viticulture of the future, since the climate crisis is forcing us to think of new, more resilient varieties. For this reason, the grapevine germplasm of the Fondazione Edmund Mach has been continuously expanded in the last decade to a total of 3,120 accessions, whose trueness-to-type has been verified by means of the universal set of nine microsatellites. About two thirds are V. vinifera subsp. vinifera accessions, while the rest consists of naturalized and selected hybrids, V. vinifera subsp. sylvestris, and pure species. The genetic material has also been characterized over three consecutive years for ampelographic, vine development, and biotic stress response traits to be exploited for experimental purposes.

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Haplotype-Resolved genome assembly of the Microvine

Developing a tractable genetic engineering and gene editing system is an essential tool for grapevine. We initiated a plant transformation and biotechnology program at Oregon State University using the grape microvine system (V. vinifera) in 2018 to interrogate gene-to-trait relationships using traditional genetic engineering and gene editing. The microvine model is also used for nanomaterial-assisted RNP, DNA, and RNA delivery. Most reference genomes and annotations for grapevine are collapsed assemblies of homologous chromosomes and do not represent the specific microvine cultivar ‘043023V004’ under study at our institution.

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Characterizing the molecular basis of the differences in aromatic precursors found in commercial clones of Vitis vinifera cv. Tannat

Uruguay is known for the production of Tannat wines, which is a neutral variety from an aroma point of view, but capable of providing aromatic precursors that are of interest in the production of wines for ageing. The main aromatic precursors present are glycosidic compounds and carotenoids. The contribution of carotenoid degradation by-products such as norisoprenoids to wine aroma is fundamental, as they are associated with pleasant aroma descriptors and very low olfactory perception thresholds. Several factors have been shown to influence carotenoid concentrations in grapes, such as cultivar, climatic conditions, viticultural region, plant water status, exposure to sunlight and ripening stage.

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How sensor technologies combined with artificial intelligence increase the efficiency in grapevine breeding (research): current developments and future perspectives

Viticulture and grapevine breeding programs have to face and adapt to the rapidly changing growing conditions due to the ongoing climate change, the scarcity of resources and the demand for sustainability within the whole value chain of wine production. In times of highly effective and cost-efficient genotyping technologies routinely applied in plant research and breeding, the need for comparable high-speed and high-resolution phenotyping tools has increased substantially. The disciplines of grapevine research, breeding and precision viticulture picked up this demand – mostly independent from each other – by the development, validation and establishment of different sensor technologies in order to extend management strategies or to transform labor-intensive and expensive phenotyping.

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The grapevine single-berry clock, practical tools and outcomes 

The dynamic sequence of physiological events along the three-months of berry development from anthesis to ripe stage has been thoroughly investigated. Most studies were performed on average samples, taking care to crush enough fruits to fairly represent the overall trend of the future harvest. However, phenological stages like 30% caps off (EL25) highlights the asynchronous nature of this population. Consequently, softening, onset of sugar accumulation and coloration were melted by asynchrony in a developmental mumbo jumbo, until their respective timing could be clarified by single berries approaches.

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Unveiling the Grapevine Red Blotch Virus (GRBV) host-pathogen arms-race via multi-omics for enhanced viral defense 

The Grapevine Red Blotch Virus (GRBV) poses a critical challenge to the wine sector, lacking a uniquely identified vector. Current control methods involve costly and labor-intensive vine removal, emphasizing the urgency for targeted alternatives. The limited understanding of intricate host-virus interactions underscores the need for foundational knowledge to develop innovative disease control strategies. These include efforts to boost the plant’s RNA interference (RNAi) response, including RNA-based topical applications.

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Activation of retrotransposition in grapevine

Retrotransposons, particularly of the Ty-Copia and Ty-Gypsy superfamilies, represent the most abundant and widespread transposons in many plant genomes. Grapevine is no exception and it is clear that these mobile elements have played a major role in the evolution of Vitaceae genomes. While speculation abounds around the possible role of transposons in plant genomes, outside of the rather obvious involvement of retrotransposition in fueling genome expansion, there is little clarity of the actual role these elements have in both developing new genetic variation and in modulating epigenetic responses within genomes to changing climate. To this end we have been exploring de-novo assembled Sauvignon blanc and Pinot noir genomes with a view to catalogue retrotransposon loci to determine the structural intactness and thus age of insertion variation across a small number of clonal linages of these 2 varietals in an attempt to identify ‘live’ TE loci.

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Reduced bunch compactness in a clone of Tempranillo associates with a complex reciprocal translocation detected by long-read sequencing genomics

Grapevine cultivars are vegetatively propagated to maintain their varietal attributes. However, spontaneous somatic variation emerges during prolonged periods of vegetative growth, providing an opportunity for the natural improvement of traditional grapevine cultivars. Notably, reduction in bunch compactness is a favorable trait in viticulture, offering advantages such as decreased susceptibility to bunch fungal diseases, and a more uniform ripening of berries. To unravel the genetic and developmental mechanisms behind bunch compactness variation, we examined a somatic variant of Tempranillo Tinto cultivar with loose bunches. We found that the mutant clone exhibits a ~50% reduction in pollen viability compared to typical Tempranillo clones.

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The grapevine QTLome is ripe: QTL survey, databasing, and first applications

Overarching surveys of QTL (Quantitative Trait Loci) studies in both model plants and staple crops have facilitated the access to information and boosted the impact of existing data on plant improvement activities. Today, the grapevine community is ready to take up the challenge of making the wealth of QTL information F.A.I.R.. To ensure that all valuable published data can be used more effectively, the myriad of identified QTLs have to be captured, standardised and stored in a dedicated public database.
As an outcome of the GRAPEDIA initiative, QTL-dedicated experts from around the world have gathered to compile the grapevine QTLome: the complete information (e.g., map positions, associated phenotypes) describing all experimentally supported QTLs for a specific trait.

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Genomic characterization of terpene biosynthetic genes in seven Vitis vinifera L. varieties 

Grapes (Vitis vinifera L.) are a fruit crop of high economic significance globally. Each grapevine cultivar is characterized by its distinctive grape aroma, affecting the wine quality. In several cultivars, the aroma is shaped by terpenoid (mono- and sesqui-terpenoids). Their profile is controlled by terpene synthases (TPS), which are part of a largely expanded gene family. How the variation in TPS copy number and sequence among cultivars determines terpenoid profiles of grapes remains largely unexplored. We annotated TPS in the haplotypes of seven genomes (Riesling, Albariño, Fiano, Gewürztraminer, Pinot Noir, Cabernet Sauvignon, and Viognier) using BLAST, GMAP, PFAM, and phylogenetic analyses. Further, TPS expression patterns and terpenoid accumulation during berry development and ripening were characterized using RNA-Seq and SPME/GC-MS platforms, respectively. Variation in TPS copy number exists among cultivars. Specifically, the TPS counts span a range of 251 to 150 for Riesling and Fiano, respectively, when considering combined haplotypes within each cultivar. Total terpenoid accumulation patterns throughout development were consistent among the five aromatic cultivars, marked by high concentrations in flowers, followed by a decline and subsequent rise during berry development and ripening, respectively. Conversely, non-aromatic cultivars exhibited no substantial increase in terpenoid concentration during ripening. Transcriptome and network analyses are currently employed to determine which TPS are expressed in the berry and determine the terpenoid profile of the specific cultivar. These findings shed light on the genomic determinants of grape aroma in major cultivars, and allow future studies focused on cultivar-specific responses of terpenoid biosynthesis to environmental stresses.

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Exploring the gene regulatory networks of WRKY family in grapevine (Vitis vinifera  L.) using DAP-Seq

The recent development of regulatory genomics has raised increasing interest in plant research since transcriptional regulation of genes plays a pivotal role in many biological processes. By shedding light on the target genes of the various transcription factors (TFs), it is therefore possible to infer the influence they exert on the different molecular mechanisms. In this regard, the attention was focused on WRKYs, a family of TFs almost exclusively found in plant species. In grapevine, WRKYs are involved in several biological processes, playing a key role in berry development, hormonal balance and signalling, biotic and abiotic stresses responses, and secondary metabolites biosynthesis.

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Somatic embryogenesis and polyploidy in grapevine: morphological shoot and leaf traits variations

Somatic embryogenesis (SE) has been used in a variety of biotechnology applications such as virus elimination, cryopreservation, induced mutagenesis and genetic transformation. The SE induction process may cause DNA alterations and ploidy changes, which may provide a source of genetic variability useful for the improvement of agronomic characteristics of plants. This research aims at investigating the spontaneous alterations of the genome in grapevine plants regenerated through SE. Regenerants obtained from different embryogenic events from three different grapevine genotypes (Catarratto, Frappato and Nero d’Avola) were analysed.

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Optimized protocol for high-quality RNA extraction from grape tissues using sorbitol pre-wash

Obtaining high-quality RNA from grape tissues, including berry pulp, berry skins, stems, rachis, or roots, is challenging due to their composition, which includes polysaccharides, phenolic compounds, sugars, and organic acids that can negatively affect RNA extraction. For instance, polyphenols and other secondary metabolites can bind to RNA, making it difficult to extract a pure sample. Additionally, RNA can co-precipitate with polysaccharides, leading to lower extraction yield. Also, sugars and organic acids can interfere with the pH and ionic properties of the extraction buffer. To address these challenges, we optimized a protocol for RNA isolation from grape tissues.

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Preliminary steps of a protocol to isolate transcription factors bound to a specific DNA locus in grapevine using CRISPR-dCas9 system

Cis-acting regulatory elements are DNA sequences that can be bound by transcription factors to regulate the expression of genes in a condition-dependent and tissue-specific way. It is nowadays possible to search for DNA motives and sequences that a given transcription factor is binding or at least can, but it is still hard to have a glance at all the transcription factors that are contemporaneously located at the same locus. Inspired by an existing technique that uses the CRISPR-Cas system in mammal cells, we are trying to develop a protocol to study such regulation in Vitis vinifera. Using the highly sequence-specific binding capacity of a catalytically inactive Cas9 protein (dCas9), our idea is to set up a system to target a desired sequence and precipitate all the crosslinked proteins and distantly interacting chromatin at this locus and analyze them.

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