Grapevine leaf MALDI-MS imaging reveals the localisation of a putatively identified sucrose metabolite associated to Plasmopara viticola development.

Despite well-established pathways and metabolites involved in grapevine-Plasmopara viticola interaction, information on the molecules involved in the first moments of pathogen contact with the leaf surface and their specific location is still missing. To understand and localise these molecules, we analysed grapevine leaf discs infected with P. viticola with MSI. Plant material preparation was optimised, and different matrices and solvents were tested. Our data shows that trichomes hamper matrix deposition and the ion signal. Results show that putatively identified sucrose presents a higher accumulation and a non-homogeneous distribution in the infected leaf discs in comparison with the controls. This accumulation was mainly on the veins, leading to the hypothesis that sucrose metabolism is being manipulated by the development structures of P. viticola. Up to our knowledge this is the first time that the localisation of a putatively identified sucrose metabolite was shown to be associated to P. viticola infection sites.

More than Just Wine: The Nutritional Benefits of Grapevine Leaves.

The domesticated species Vitis vinifera L. harbours many cultivars throughout the world that present distinctive phenology and grape quality. Not only have the grapes been used for human consumption, but the leaves are also used as a source of bioactive compounds and are present in the diets of several Mediterranean countries. We have selected seven different cultivars and performed elemental, fatty acid (FA) and pigment profiling. Total reflection X-ray fluorescence (TXRF) enabled the identification of 21 elements. Among them, Na, Ca and K were highly represented in all the cultivars and Zn was prevalent in V. vinifera cv. 'Pinot noir' and 'Cabernet sauvignon'. Through gas chromatography, six FAs were identified, including omega-3 and omega-6 FA, the most abundant mainly in V. vinifera cv. 'Tinta barroca', 'Pinot noir' and 'Cabernet sauvignon'. FA composition was used to determine nutritional quality parameters, namely atherogenic, thrombogenic, hypocholesterolemic/hypercholesterolemic and peroxidisability indexes as well as oxidability and oxidative susceptibility. Grapevine leaves were highlighted as a suitable source of health-promoting lipids. Given the popularity of "green" diets, we have also performed a leaf pigment analysis. Seventeen pigments including chlorophylls, trans-lutein, b-carotene and zeaxanthins were detected. 'C19' presented the highest content of most of the detected pigments.

Fatty acid modulation and desaturase gene expression are differentially triggered in grapevine incompatible interaction with biotrophs and necrotrophs.

Grapevine (Vitis vinifera L.) is prone to fungal and oomycete diseases. Downy and powdery mildews and grey mold, are caused by Plasmopara viticola, Erisiphe necator and Botrytis cinerea, respectively. P. viticola and E. necator are obligatory biotrophs whereas B. cinerea is a necrotroph. In tolerant grapevine cultivars, plant-pathogen interaction induces defence responses, including metabolite and protein accumulation and hypersensitive reaction. Lipid and lipid-derived molecules may have a key role in the activation of defence mechanisms. Previous results suggest that V. vinifera cv Regent tolerance to P. viticola may be mediated in the first hours post inoculation by fatty acid (FA) associated signalling. In the present study we characterized FA modulation in V. vinifera cv Regent leaves upon inoculation with P. viticola, E. necator and B. cinerea and correlated FA modulation with the expression profiles of genes encoding the FA desaturases FAD6 and FAD8. In all the interactions, a progressive desaturation of stearic acid to α-linolenic acid, precursor of jasmonic acid, occurred, which was observed for a longer period against B. cinerea. Our results provide evidence of a distinct FA meditated signalling pattern in grapevine interaction with biotrophs and necrotrophs. While the interaction with the biotrophs may trigger a higher synthesis of polyunsaturated FA (PUFA) at early time-points with a tendency to return to basal levels, the interaction with B. cinerea may trigger a later and more durable induction of PUFA synthesis. In all interactions, membrane fluidity modulation occurred, which may be crucial to maintain cellular function during infection.

An apoplastic fluid extraction method for the characterization of grapevine leaves proteome and metabolome from a single sample.

The analysis of complex biological systems keeps challenging researchers. The main goal of systems biology is to decipher interactions within cells, by integrating datasets from large scale analytical approaches including transcriptomics, proteomics and metabolomics and more specialized 'OMICS' such as epigenomics and lipidomics. Studying different cellular compartments allows a broader understanding of cell dynamics. Plant apoplast, the cellular compartment external to the plasma membrane including the cell wall, is particularly demanding to analyze. Despite our knowledge on apoplast involvement on several processes from cell growth to stress responses, its dynamics is still poorly known due to the lack of efficient extraction processes adequate to each plant system. Analyzing woody plants such as grapevine raises even more challenges. Grapevine is among the most important fruit crops worldwide and a wider characterization of its apoplast is essential for a deeper understanding of its physiology and cellular mechanisms. Here, we describe, for the first time, a vacuum-infiltration-centrifugation method that allows a simultaneous extraction of grapevine apoplastic proteins and metabolites from leaves on a single sample, compatible with high-throughput mass spectrometry analyses. The extracted apoplast from two grapevine cultivars, Vitis vinifera cv 'Trincadeira' and 'Regent', was directly used for proteomics and metabolomics analysis. The proteome was analyzed by nanoLC-MS/MS and more than 700 common proteins were identified, with highly diverse biological functions. The metabolome profile through FT-ICR-MS allowed the identification of 514 unique putative compounds revealing a broad spectrum of molecular classes.

Integrating metabolomics and targeted gene expression to uncover potential biomarkers of fungal/oomycetes-associated disease susceptibility in grapevine.

Vitis vinifera, one of the most cultivated fruit crops, is susceptible to several diseases particularly caused by fungus and oomycete pathogens. In contrast, other Vitis species (American, Asian) display different degrees of tolerance/resistance to these pathogens, being widely used in breeding programs to introgress resistance traits in elite V. vinifera cultivars. Secondary metabolites are important players in plant defence responses. Therefore, the characterization of the metabolic profiles associated with disease resistance and susceptibility traits in grapevine is a promising approach to identify trait-related biomarkers. In this work, the leaf metabolic composition of eleven Vitis genotypes was analysed using an untargeted metabolomics approach. A total of 190 putative metabolites were found to discriminate resistant/partial resistant from susceptible genotypes. The biological relevance of discriminative compounds was assessed by pathway analysis. Several compounds were selected as promising biomarkers and the expression of genes coding for enzymes associated with their metabolic pathways was analysed. Reference genes for these grapevine genotypes were established for normalisation of candidate gene expression. The leucoanthocyanidin reductase 2 gene (LAR2) presented a significant increase of expression in susceptible genotypes, in accordance with catechin accumulation in this analysis group. Up to our knowledge this is the first time that metabolic constitutive biomarkers are proposed, opening new insights into plant selection on breeding programs.

Genetic Relationships Among Portuguese Cultivated and Wild Vitis vinifera L. Germplasm.

The domesticated grapevine spread along the Mediterranean basin from the primary Near East domestication area, where the greatest genetic diversity is found in its ancestor, the wild vine populations. Portuguese wild populations are on the southwestern fringe of the distribution of the Vitis vinifera L. ssp. sylvestris (C.C. Gmel.) Hegi in Europe. During the last Glacial Period they became isolated from the previous continuum that had been the territory of wild vine populations. Archaeological remains of domesticated vinifera grapevines in Portugal date back from 795 Before Common Era (BCE) in the lower Tagus river basin. In this work, 258 Portuguese vinifera varieties and sylvestris plants were characterized using 261 single nucleotide polymorphism (SNP) markers. The study of the genetic diversity of this local germplasm, its population structure and kinship, all framed in their historical and geographical backgrounds, revealed a complex network of first-degree relationships, where only Iberian varieties are involved. Some Iberian genotypes, like Alfrocheiro (Bruñal, in Spain), Sarigo (Cayetana Blanca), Mourisco Branco (Hebén), Amaral (Caiño Bravo), and Marufo (Moravia Dulce) are ancestors of a considerable fraction of all the autochthonous analyzed varieties. A part of the diversity developed was mostly local in some cases as shown by the closeness of several varieties (Vinhos Verdes) to the wild cluster in different analyses. Besides, several evidences of introgression of domesticated germplasm into wild vines was found, substantiating the high risk of genetic contamination of the sylvestris subspecies. All these findings together to the known matching between the wild maternal lineage of the Iberian Peninsula and an important number of Portuguese grapevine varieties (chlorotype A), point out that some of these varieties derive, directly or indirectly, from originally local wild populations, supporting the possible occurrence of secondary events of local domestication, or, at least, of an introgression process of wild into cultivated grapevines.

Using Rapid Chlorophyll Fluorescence Transients to Classify Vitis Genotypes.

When a dark-adapted leaf is illuminated with saturating light, a fast polyphasic rise of fluorescence emission (Kautsky effect) is observed. The shape of the curve is dependent on the molecular organization of the photochemical apparatus, which in turn is a function of the interaction between genotype and environment. In this paper, we evaluate the potential of rapid fluorescence transients, aided by machine learning techniques, to classify plant genotypes. We present results of the application of several machine learning algorithms (k-nearest neighbors, decision trees, artificial neural networks, genetic programming) to rapid induction curves recorded in different species and cultivars of vine grown in the same environmental conditions. The phylogenetic relations between the selected Vitis species and Vitis vinifera cultivars were established with molecular markers. Both neural networks (71.8%) and genetic programming (75.3%) presented much higher global classification success rates than k-nearest neighbors (58.5%) or decision trees (51.6%), genetic programming performing slightly better than neural networks. However, compared with a random classifier (success rate = 14%), even the less successful algorithms were good at the task of classifying. The use of rapid fluorescence transients, handled by genetic programming, for rapid preliminary classification of Vitis genotypes is foreseen as feasible.

Vitis flower types: from the wild to crop plants.

Vitis vinifera can be divided into two subspecies, V. vinifera subsp. vinifera, one of the most important agricultural crops in the world, and its wild ancestor, V. vinifera subsp. sylvestris. Three flower types can be observed: hermaphrodite and female (on some varieties) in vinifera, and male or female flowers in sylvestris. It is assumed that the different flower types in the wild ancestor arose through specific floral patterns of organ abortion. A considerable amount of data about the diversity of sexual systems in grapevines has been collected over the past century. Several grapevine breeding studies led to the hypothesis that dioecy in vinifera is derived from a hermaphrodite ancestor and could be controlled by either, one or two linked genetic determinants following Mendelian inherence. More recently, experiments using molecular approaches suggested that these loci were located in a specific region of the chromosome 2 of vinifera. Based on the works published so far, its seems evident that a putative sex locus is present in chromosome 2. However, it is still not fully elucidated whether flower types are regulated by two linked loci or by one locus with three alleles. Nevertheless, several genes could contribute to sex determination in grapevine. This review presents the results from early studies, combined with the recent molecular approaches, which may contribute to the design of new experiments towards a better understanding of the sex inheritance in grapevine.

Label free DNA-based optical biosensor as a potential system for wine authenticity.

The diversity found among the Vitis vinifera L. species allows the production of wines with very different characteristics. The development of platforms suitable for food composition analysis is currently an emerging area. Among these, DNA biosensors have been developed for a wide variety of applications, ranging from food safety to authenticity. The main aim of this work was to study the detection capacity of the DNA-based optical biosensor using different V. vinifera matrices (leaf, must and wine). Genomic DNA was extracted from leaf, must and wine of three V. vinifera varieties and was tested on the long-period grating (LPG) DNA-based biosensor developed within our group. The biosensor was able to distinguish the varieties even using DNA extracted from complex matrices, revealing its potential to be applied in wine authenticity.

High Resolution Melting (HRM) applied to wine authenticity.

Wine authenticity methods are in increasing demand mainly in Denomination of Origin designations. The DNA-based methodologies are a reliable means of tracking food/wine varietal composition. The main aim of this work was the study of High Resolution Melting (HRM) application as a screening method for must and wine authenticity. Three sample types (leaf, must and wine) were used to validate the three developed HRM assays (Vv1-705bp; Vv2-375bp; and Vv3-119bp). The Vv1 HRM assay was only successful when applied to leaf and must samples. The Vv2 HRM assay successfully amplified all sample types, allowing genotype discrimination based on melting temperature values. The smallest amplicon, Vv3, produced a coincident melting curve shape in all sample types (leaf and wine) with corresponding genotypes. This study presents sensitive, rapid and efficient HRM assays applied for the first time to wine samples suitable for wine authenticity purposes.

Phenolic compounds involved in grafting incompatibility of Vitis spp: development and validation of an analytical method for their quantification.

INTRODUCTION: Graft incompatibility of Vitis spp is an unresolved worldwide problem with important economic consequences. Grafting comprises a complex set of morphological and physiological alterations, in which the phenolic compounds seem to be strongly involved. Therefore, a detailed analysis and recognition of structural phenolic compounds diversity in the two partners of a Vitis graft is of great importance to evaluate their role as markers of graft establishment. OBJECTIVE: To optimise a sample extraction method, and to develop and validate a high-performance liquid chromatography (HPLC) method for the simultaneous determination of phenolic acids and flavonols in the graft union so as to understand their behaviour in the metabolism of the scion-rootstock system, using compatible and incompatible combinations of a Syrah cultivar and two rootstocks (R110 and SO4). METHODS: Sixty extracts of Vitis grafting tissues were prepared and analysed by HPLC for the qualitative and quantitative determination of their phenolic profile. RESULTS: Among the phenolic compounds identified in the samples, one benzoic acid (gallic acid), three cinnamic acids (caffeic acid, ferulic acid and sinapic acid) and two flavonols (catechin and epicatechin) are potentially suitable as markers of graft incompatibility. CONCLUSION: The method developed presents good performance and lends itself readily for application in routine analysis of the phenolic composition of Vitis grafting tissues to distinguish compatible and incompatible combinations in the graft callusing stage.

The first insight into the metabolite profiling of grapes from three Vitis vinifera L. cultivars of two controlled appellation (DOC) regions.

The characterization of the metabolites accumulated in the grapes of specific cultivars grown in different climates is of particular importance for viticulturists and enologists. In the present study, the metabolite profiling of grapes from the cultivars, Alvarinho, Arinto and Padeiro de Basto, of two Portuguese Controlled Denomination of Origin (DOC) regions (Vinho Verde and Lisboa) was investigated by gas chromatography-coupled time-of-flight mass spectrometry (GC-TOF-MS) and an amino acid analyzer. Primary metabolites, including sugars, organic acids and amino acids, and some secondary metabolites were identified. Tartaric and malic acids and free amino acids accumulated more in grapes from vines of the DOC region of Vinho Verde than DOC Lisboa, but a principal component analysis (PCA) plot showed that besides the DOC region, the grape cultivar also accounted for the variance in the relative abundance of metabolites. Grapes from the cultivar, Alvarinho, were particularly rich in malic acid and tartaric acids in both DOC regions, but sucrose accumulated more in the DOC region of Vinho Verde.

Berry phenolics of grapevine under challenging environments.

Plant phenolics have been for many years a theme of major scientific and applied interest. Grape berry phenolics contribute to organoleptic properties, color and protection against environmental challenges. Climate change has already caused significant warming in most grape-growing areas of the world, and the climatic conditions determine, to a large degree, the grape varieties that can be cultivated as well as wine quality. In particular, heat, drought and light/UV intensity severely affect phenolic metabolism and, thus, grape composition and development. In the variety Chardonnay, water stress increases the content of flavonols and decreases the expression of genes involved in biosynthesis of stilbene precursors. Also, polyphenolic profile is greatly dependent on genotype and environmental interactions. This review deals with the diversity and biosynthesis of phenolic compounds in the grape berry, from a general overview to a more detailed level, where the influence of environmental challenges on key phenolic metabolism pathways is approached. The full understanding of how and when specific phenolic compounds accumulate in the berry, and how the varietal grape berry metabolism responds to the environment is of utmost importance to adjust agricultural practices and thus, modify wine profile.

Molecular data mining to improve antibody-based detection of Grapevine leafroll-associated virus 1 (GLRaV-1).

Testing for Grapevine leafroll-associated virus 1 (GLRaV-1) is mandatory in certification schemes of propagation material within the EU. Accurate and reliable diagnostic assays are necessary for implementation of this measure. During routine detection of GLRaV-1, using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription (RT) followed by polymerase chain reaction (PCR), evidence was obtained that positive samples could be overlooked by either or both detection methods. With the aim of improving serological detection tools for GLRaV-1, a total of 20 isolates were analyzed and 83 new complete capsid protein (CP) gene sequences were obtained. This set, together with the CP sequences available at GenBank was used for a comprehensive in silico analysis. To obtain a specific antibody able to recognize all known CP variants, conserved regions with suitable antigenicity profile were identified along the deduced CP AA sequences and a 14 AA sequence was chosen for commercial peptide synthesis and immunization. Initially polyclonal antibodies were produced and tested, followed by purification of the respective monospecific antibody and conjugation with alkaline phosphatase or fluorescein isothiocyanate (FITC). These serological tools were tested successfully on all the available positive samples and proved adequate for in situ immunoassay (ISIA). Further testing showed that the monospecific antibody could also be used in tissue print immunoblotting (TPIB), a technique that allows rapid processing of large sample sets, which is highly suitable to implement protocols ensuring that, for each vine analyzed, enough random samples are taken and processed, before certification.

A candidate-gene association study for berry colour and anthocyanin content in Vitis vinifera L.

Anthocyanin content is a trait of major interest in Vitis vinifera L. These compounds affect grape and wine quality, and have beneficial effects on human health. A candidate-gene approach was used to identify genetic variants associated with anthocyanin content in grape berries. A total of 445 polymorphisms were identified in 5 genes encoding transcription factors and 10 genes involved in either the biosynthetic pathway or transport of anthocyanins. A total of 124 SNPs were selected to examine association with a wide range of phenotypes based on RP-HPLC analysis and visual characterization. The phenotypes were total skin anthocyanin (TSA) concentration but also specific types of anthocyanins and relative abundance. The visual assessment was based on OIV (Organisation Internationale de la Vigne et du Vin) descriptors for berry and skin colour. The genes encoding the transcription factors MYB11, MYBCC and MYC(B) were significantly associated with TSA concentration. UFGT and MRP were associated with several different types of anthocyanins. Skin and pulp colour were associated with nine genes (MYB11, MYBCC, MYC(B), UFGT, MRP, DFR, LDOX, CHI and GST). Pulp colour was associated with a similar group of 11 genes (MYB11, MYBCC, MYC(B), MYC(A), UFGT, MRP, GST, DFR, LDOX, CHI and CHS(A)). Statistical interactions were observed between SNPs within the transcription factors MYB11, MYBCC and MYC(B). SNPs within LDOX interacted with MYB11 and MYC(B), while SNPs within CHI interacted with MYB11 only. Together, these findings suggest the involvement of these genes in anthocyanin content and on the regulation of anthocyanin biosynthesis. This work forms a benchmark for replication and functional studies.

Occurrence of grapevine leafroll-associated virus 5 in Portugal: genetic variability and population structure in field-grown grapevines.

A comparison of 15 field isolates of grapevine leafroll-associated virus 5 (GLRaV-5) was conducted, based on the analysis of nucleotide sequences of two viral ORFs: the coat protein (CP) and the heat shock protein 90 homolog (HSP90h). After amplification and cloning, the population of viral sequences was analyzed for each isolate, revealing the within-isolate presence of sequence variants for both genes, with one or more major CP variants. Phylogenetic analysis showed the gene sequence variants detected to be exclusive for each isolate. These data, together with estimates of genetic diversity and positive selection, did not reveal evidence of vector-mediated transfer of GLRaV-5. Conversely, a strong effect of host vegetative propagation on divergence dynamics of GLRaV-5 variants was suggested by the isolates from this work The phylogeny of the CP gene further revealed clustering of GLRaV-5 isolates into eight lineages, four of which were detected in our study, revealing a higher diversity than previously described. The information gathered also contributes to firmly establishing GLRaV-5 as a cohesive taxonomic group within the ampeloviruses.

Five phylogenetic groups identified in the coat protein gene of grapevine leafroll-associated virus 3 obtained from Portuguese grapevine varieties.

The genetic variability and population structure of grapevine leafroll-associated virus 3 (GLRaV-3) variants were updated by examining the diversity within the viral coat protein (CP) gene among 174 isolates belonging to a collection of Vitis vinifera representing most of the Portuguese varieties. Phylogenetic analysis revealed the existence of five well-defined clusters. Three of these correspond to previously defined groups, another corresponds to variants from Chile for which only one sequence has been previously identified, and an additional new group includes only Portuguese variants. A typing tool based on asymmetric PCR-ELISA (APET) was developed within the frame of this population structure. This tool was used to assess the prevalence of each phylogenetic group among the infected grapevine varieties. Although most of the isolates harbour variants from groups 1 and 2, variants from the remaining three groups exist in a number of varieties, reinforcing the notion that they are genuine genomic variants and are not isolated, atypical cases.