Effect of water deficit on fruit metabolism in white and red grape varieties

Peterlunger, EnricoCastellarin, Simone DiegoGMPF1openPlants are sessile organisms and often they have to cope with environmental stresses (abiotic factors) such as drought, cold, heat, extreme light, excessive soil salinity, or several combinations of them. The genotype x environment (GxE) interaction is the source of the main variability in the responses to these constrains. Among the abiotic factors that can influence plant physiology, drought is the most relevant because it can influence plant growth and yield, and affects fruits composition. Secondary metabolism contributes to the adaptation of a plant to its environment. In fruit crops such as grapevine (Vitis vinifera L.), secondary metabolism also largely determines fruit quality. Grapevine is considered a drought-tolerant plant and traditionally is not irrigated, especially in Europe. Mediterranean climate, with warm and dry summers and cold and wet winters, is considered optimal for viticulture. Climate change is predicted to exacerbate drought events in several viticultural areas, potentially affecting the accumulation of secondary metabolites in the grapes, thus affecting wine quality. We adopted a multidisciplinary approach that considered a two-years field trial, high-throughput transcripts profiling (RNA-sequencing) and large-scale targeted metabolite analyses to investigate the effect of drought events on the berry metabolism during fruit development and ripening in white and red grape varieties. An open field experiment was therefore conducted on Tocai friulano (white grape variety) and Merlot (red grape variety) vines in 2011 and 2012, in a North Italian viticultural area characterized by transient drought events during the summer. Two irrigation treatments were applied to the vines: (i) control vines were weekly irrigated, in order to keep their stem water potential (a sensitive indicator of grapevine water status) between -0.4 and -0.6 MPa; (ii) deficit irrigated vines were not irrigated from fruit set to harvest except in case of acute water deficit. Merlot vines were sheltered with an open-sided transparent cover at the beginning of the seasons, while Tocai friulano vines were cultivated without any cover and hence subjected to the natural precipitations. In Merlot, deficit irrigated vines experienced water deficit from 40-50 days after anthesis in both seasons. In Tocai friulano, deficit irrigated vines manifested a late deficit (from 80-90 days after anthesis) in 2011, and a prolonged water deficit from early stages of fruit development (from 30-40 days after anthesis) to harvest in 2012. For both varieties, berries were sampled for transcript and metabolite analyses during berry development and ripening. Furthermore, at harvest, grapes were microvinificated and wines composition was evaluated focusing on the secondary metabolites that largely contribute to the final color, taste, and aromatic features. A large effect of water deficit on fruit secondary metabolism of the white grape variety Tocai friulano was observed in the season when the deficit occurred from early stages of berry development to harvest. In this particularly season, increased concentrations of phenylpropanoids, monoterpenes, and tocopherols were observed, while carotenoid and flavonoid accumulations were differentially modulated by water deficit accordingly to the berry developmental stage. In parallel, RNA sequencing analysis revealed that many key genes of the phenylpropanoid, flavonoid, and terpenoid pathways were modulated by water deficit indicating a transcriptional regulation of these specific pathways in the berry under drought. The higher co-regulation of several terpenoid transcripts with monoterpene accumulation under water deficit and the enrichment of drought-responsive elements in the promoter region of many terpenoid genes highlight that drought can enhance the production of these flavour components in grapes with potential effects on wine composition and sensory features. The wines produced from grapes subjected to water deficit revealed a more pronounced accumulation of monoterpenes and C13-norisoprenoids, and especially glycosidically-bound compounds. However, differences in the metabolic response between seasons suggest that the endurance of water deficit and the timing of application strongly impact this response. In Merlot berries, the drought stress response encompassed both ABA-dependent and ABA-independent signal transduction pathways with several VviAREB/ABFs, VvibZIP, and VviAP2/ERF-DREB transcription factors that were up-regulated by water deficit at one or more developmental stages. These transcription factors can play critical roles in the drought response by modulating a large suite of genes. Analyses of the central and specialized berry metabolism was conducted both at the transcript and metabolite levels by investigating several metabolic pathways (glycolysis and sugar accumulation, tricarboxylic acid cycle and amino acid biosynthesis, phenylpropanoid, flavonoid, terpenes, carotenoids, and fatty acid degradation pathways). The study revealed that water deficit enhanced the accumulation of several osmoprotectants (proline, sucrose, and raffinose) and of secondary metabolites such as anthocyanins and C5, C7, C8, and C9 volatile organic compounds. Furthermore, a weighted gene co-expression network analysis clustered in a module several genes involved in the branched chain amino acids biosynthesis, phenylpropanoid and flavonoid pathways, and sugar derivative metabolism together with the transcription factors mentioned above involved in the drought-stress signal, indicating a putative role of these transcription factors on the regulation of the response of the fruit metabolism to drought in Merlot berries. The wines produced from grapes subjected to water deficit revealed a higher concentration of anthocyanins that determined higher color intensity and a bluer coloration of the wines. A comparison between the two genotypes can be done only for the 2012 season, when levels and endurances of water deficit were similar between Tocai friulano and Merlot deficit irrigated vines. The data indicate that water deficit modulated the accumulation of several secondary compounds; however, the modulation of the secondary metabolism varied between cultivars indicating a genotype x environment interaction. In the Tocai friulano berry, water deficit specifically stimulated the synthesis of phenolic acids, such as gallic acid, ellagic acid, and caftaric acid, tocopherols, and monoterpenes, such as linalool, α-terpineol, nerol, and hotrienol. In Merlot, the response included an overproduction of anthocyanins, such as the tri-substituted delphinidin, petunidin, and malvidin both in the glycosylated and acylated form, and C5, C7, C8 and C9 volatile organic, such as 1-penten-3-ol, (E)-2-heptenal, (E)-2-octenal, 1-octen-3-ol, and nonanol. A common response between the two varieties included a higher accumulation of gallic acid, zeaxanthin, (E)-2-heptenal, (E)-2-octenal, 1-octen-3-ol, and nonanol in the berry. Furthermore, these results indicate that drought events can affect the composition and sensory features of white and red wines by increasing the accumulation of benzoic and cinnamic acids, pigments in the red grape variety and monoterpenes in the white grape variety.embargoed_20170407Savoi, S.Savoi, S

Transcriptome and metabolite profiling reveals that prolonged drought modulates the phenylpropanoid and terpenoid pathway in white grapes (Vitis vinifera L.)

Background: Secondary metabolism contributes to the adaptation of a plant to its environment. In wine grapes, fruit secondary metabolism largely determines wine quality. Climate change is predicted to exacerbate drought events in several viticultural areas, potentially affecting the wine quality. In red grapes, water deficit modulates flavonoid accumulation, leading to major quantitative and compositional changes in the profile of the anthocyanin pigments; in white grapes, the effect of water deficit on secondary metabolism is still largely unknown Results: In this study we investigated the impact of water deficit on the secondary metabolism of white grapes using a large scale metabolite and transcript profiling approach in a season characterized by prolonged drought. Irrigated grapevines were compared to non-irrigated grapevines that suffered from water deficit from early stages of berry development to harvest. A large effect of water deficit on fruit secondary metabolism was observed. Increased concentrations of phenylpropanoids, monoterpenes, and tocopherols were detected, while carotenoid and flavonoid accumulations were differentially modulated by water deficit according to the berry developmental stage. The RNA- sequencing analysis carried out on berries collected at three developmental stages—before, at the onset, and at late ripening—indicated that water deficit affected the expression of 4,889 genes. The Gene Ontology category secondary metabolic process was overrepresented within up-regulated genes at all the stages of fruit development considered, and within down-regulated genes before ripening. Eighteen phenylpropanoid, 16 flavonoid, 9 carotenoid, and 16 terpenoid structural genes were modulated by water deficit, indicating the transcriptional regulation of these metabolic pathways in fruit exposed to water deficit. An integrated network and promoter analyses identified a transcriptional regulatory module that encompasses terpenoid genes, transcription factors, and enriched drought- responsive elements in the promoter regions of those genes as part of the grapes response to drought Conclusion: Our study reveals that grapevine berries respond to drought by modulating several secondary metabolic pathways, and particularly, by stimulating the production of phenylpropanoids, the carotenoid zeaxanthin, and of volatile organic compounds such as monoterpenes, with potential effects on grape and wine antioxidant potential, composition, and sensory feature

Multi-Omics and Integrated Network Analyses Reveal New Insights into the Systems Relationships between Metabolites, Structural Genes, and Transcriptional Regulators in Developing Grape Berries (Vitis vinifera L.) Exposed to Water Deficit

Grapes are one of the major fruit crops and they are cultivated in many dry environments. This study comprehensively characterizes the metabolic response of grape berries exposed to water deficit at different developmental stages. Increases of proline, branched-chain amino acids, phenylpropanoids, anthocyanins, and free volatile organic compounds have been previously observed in grape berries exposed to water deficit. Integrating RNA-sequencing analysis of the transcriptome with large-scale analysis of central and specialized metabolites, we reveal that these increases occur via a coordinated regulation of key structural pathway genes. Water deficit-induced up-regulation of flavonoid genes is also coordinated with the down-regulation of many stilbene synthases and a consistent decrease in stilbenoid concentration. Water deficit activated both ABA-dependent and ABA-independent signal transduction pathways by modulating the expression of several transcription factors. Gene-gene and gene-metabolite network analyses showed that water deficit-responsive transcription factors such as bZIPs, AP2/ERFs, MYBs, and NACs are implicated in the regulation of stress-responsive metabolites. Enrichment of known and novel cis-regulatory elements in the promoters of several ripening-specific/water deficit-induced modules further affirms the involvement of a transcription factor cross-talk in the berry response to water deficit. Together, our integrated approaches show that water deficit-regulated gene modules are strongly linked to key fruit-quality metabolites and multiple signal transduction pathways may be critical to achieve a balance between the regulation of the stress-response and the berry ripening program. This study constitutes an invaluable resource for future discoveries and comparative studies, in grapes and other fruits, centered on reproductive tissue metabolism under abiotic stress.This study was funded by the European Territorial Cooperation program (Sustainable viticulture and improvement of the territorial resources of the grape and wine industry), the Fondazione Edmund Mach (GMPF Program), the COST Action FA1106 Quality Fruit, Genome British Columbia (10R21188), and the Natural Sciences and Engineering Research Council of Canada (10R23082)

Effects of initial-state dynamics on collective flow within a coupled transport and viscous hydrodynamic approach

We evaluate the effects of preequilibrium dynamics on observables in ultrarelativistic heavy-ion collisions. We simulate the initial nonequilibrium phase within A MultiPhase Transport (AMPT) model, while the subsequent near-equilibrium evolution is modeled using (2+1)-dimensional relativistic viscous hydrodynamics. We match the two stages of evolution carefully by calculating the full energy-momentum tensor from AMPT and using it as input for the hydrodynamic evolution. We find that when the preequilibrium evolution is taken into account, final-state observables are insensitive to the switching time from AMPT to hydrodynamics. Unlike some earlier treatments of preequilibrium dynamics, we do not find the initial shear viscous tensor to be large. With a shear viscosity to entropy density ratio of $0.12$, our model describes quantitatively a large set of experimental data on Pb+Pb collisions at the Large Hadron Collider(LHC) over a wide range of centrality: differential anisotropic flow $v_n(p_T) ~(n=2-6)$, event-plane correlations, correlation between $v_2$ and $v_3$, and cumulant ratio $v_2\{4\}/v_2\{2\}$.Comment: 10 pages, v2: minor revisio

Comparative Transcriptome Analysis of Two Root-Feeding Grape Phylloxera (D. vitifoliae) Lineages Feeding on a Rootstock and V. vinifera

Grape phylloxera is one of the most dangerous insect pests for worldwide viticulture. The leaf- and root-galling phylloxerid has been managed by grafting European grapevines onto American rootstock hybrids. Recent reports pinpoint the appearance of host-adapted biotypes, but information about the biomolecular characteristics underlying grape phylloxera biotypisation and its role in host performance is scarce. Using RNA-sequencing, we sequenced the transcriptome of two larval stages: L1 (probing) and L2-3 (feeding) larvae of two root-feeding grape phylloxera lineages feeding on the rootstock Teleki 5C (biotype C) and V. vinifera Riesling (biotype A). In total, 7501 differentially expressed genes (DEGs) were commonly modulated by the two biotypes. For the probing larvae, we found an increased number of DEGs functionally associated with insect chemoreception traits, such as odorant-binding proteins, chemosensory proteins, ionotropic, odorant, and gustatory receptors. The transcriptomic profile of feeding larvae was enriched with DEGs associated with the primary metabolism. Larvae feeding on the tolerant rootstock Teleki 5C exhibited higher numbers of plant defense suppression-associated DEGs than larvae feeding on the susceptible host. Based on the identified DEGs, we discuss their potential role for the compatible grape phylloxera–Vitis interaction belowground. This study was the first to compare the transcriptomes of two grape phylloxera lineages feeding on a tolerant and susceptible host, respectively, and to identify DEGs involved in the molecular interaction with these hosts. Our data provide a source for future studies on host adaptation mechanisms of grape phylloxera and help to elucidate grape phylloxera resistance further

Transcripts switched off at the stop of phloem unloading highlight the energy efficiency of sugar import in the ripening V. vinifera fruit

International audienceAbstract Transcriptomic changes at the cessation of sugar accumulation in the pericarp of Vitis vinifera were addressed on single berries re-synchronised according to their individual growth patterns. The net rates of water, sugars and K + accumulation inferred from individual growth and solute concentration confirmed that these inflows stopped simultaneously in the ripe berry, while the small amount of malic acid remaining at this stage was still being oxidised at low rate. Re-synchronised individual berries displayed negligible variations in gene expression among triplicates. RNA-seq studies revealed sharp reprogramming of cell-wall enzymes and structural proteins at the stop of phloem unloading, associated with an 80% repression of multiple sugar transporters and aquaporins on the plasma or tonoplast membranes, with the noticeable exception of H + /sugar symporters, which were rather weakly and constitutively expressed. This was verified in three genotypes placed in contrasted thermo-hydric conditions. The prevalence of SWEET suggests that electrogenic transporters would play a minor role on the plasma membranes of SE/CC complex and the one of the flesh, while sucrose/H + exchangers dominate on its tonoplast. Cis -regulatory elements present in their promoters allowed to sort these transporters in different groups, also including specific TIPs and PIPs paralogs, and cohorts of cell wall-related genes. Together with simple thermodynamic considerations, these results lead to propose that H + /sugar exchangers at the tonoplast, associated with a considerably acidic vacuolar pH, may exhaust cytosolic sugars in the flesh and alleviate the need for supplementary energisation of sugar transport at the plasma membrane

First quantitative assessment of growth, sugar accumulation and malate breakdown in a single ripening berry

International audienceBackground: Most approaches to grape physiology accept that the berry and the future harvest should display identical developmental features, which obviously requires synchronised fruits. Aims: Rejecting this assumption compels to revisit the kinetic and metabolic bases of berry ripening. Methods and Results: Two to three thousand berries were individually analysed for sugar, malate and weight. The huge heterogeneity in sugar and malic acid concentrations among fruits was mostly explained by time lags in the onset of sugar storage, which proved nearly as long as the second growth phase. Individual berries from different cultivars displayed similar kinetics following the normalisation of their maximal volume. Phloem sucrose unloading started at its maximum speed at softening, but growth resumed one week later. Four hexoses accumulated per malic acid, which was oxidised during the first two weeks of ripening, and then malate breakdown stopped without affecting sugar accumulation. Sugar and water accumulation were simultaneously arrested four weeks after softening, at 0.9 M hexose, at which point sugar concentration continued through water losses. Conclusions: The accepted sequential random sampling methods representative of average fruit and future wine compositions have led to a scrambled vision of grape developmental biology, presenting serious kinetic and composition biases. Single berry composition provides first quantitative evidence for the induction of a dominant H + / sucrose exchange on the tonoplast, which is first electro-neutralised by malate breakdown, then by ATP demanding H + recirculation, in line with functional and molecular studies. Significance of the Study: The kinetics of single berry ripening are presented for the first time. A more reliable and reproducible model of berry growth, sugar import and malate breakdown is shown here, which have definitively been improved from a quantitative point of view. It illustrates that the temporal structure of a berry population may largely contribute to future wine quality, in addition to metabolic plasticity, thereby providing another target for the impact of GxE interaction. In this respect, addressing the structure of berry cohorts may provide a new approach regarding the developmental biology/terroir nexus

Transcriptomic and metabolomic integration as a resource in grapevine to study fruit metabolite quality traits

Transcriptomics and metabolomics are methodologies being increasingly chosen to perform molecular studies in grapevine (Vitis vinifera L.), focusing either on plant and fruit development or on interaction with abiotic or biotic factors. Currently, the integration of these approaches has become of utmost relevance when studying key plant physiological and metabolic processes. The results from these analyses can undoubtedly be incorporated in breeding programs whereby genes associated with better fruit quality (e.g., those enhancing the accumulation of health-promoting compounds) or with stress resistance (e.g., those regulating beneficial responses to environmental transition) can be used as selection markers in crop improvement programs. Despite the vast amount of data being generated, integrative transcriptome/metabolome meta-analyses (i.e., the joint analysis of several studies) have not yet been fully accomplished in this species, mainly due to particular specificities of metabolomic studies, such as differences in data acquisition (i.e., different compounds being investigated), unappropriated and unstandardized metadata, or simply no deposition of data in public repositories. These meta-analyses require a high computational capacity for data mining a priori, but they also need appropriate tools to explore and visualize the integrated results. This perspective article explores the universe of omics studies conducted in V. vinifera, focusing on fruit-transcriptome and metabolome analyses as leading approaches to understand berry physiology, secondary metabolism, and quality. Moreover, we show how omics data can be integrated in a simple format and offered to the research community as a web resource, giving the chance to inspect potential gene-to-gene and gene-to-metabolite relationships that can later be tested in hypothesis-driven research. In the frame of the activities promoted by the COST Action CA17111 INTEGRAPE, we present the first grapevine transcriptomic and metabolomic integrated database (TransMetaDb) developed within the Vitis Visualization (VitViz) platform (https://tomsbiolab.com/vitviz). This tool also enables the user to conduct and explore meta-analyses utilizing different experiments, therefore hopefully motivating the community to generate Findable, Accessible, Interoperable and Reusable (F.A.I.R.) data to be included in the future.This publication is based upon work from COST Action CA17111 INTEGRAPE, supported by COST (European Cooperation in Science and Technology) and COST grant ECOST-STSM-Request-CA17111-48997 awarded to SS. This work was also supported by Grants PGC2018-099449-A-I00, PID2021-128865NB-I00 and by the Ramón y Cajal program grant RYC-2017-23645, all awarded to JM, and to the FPI scholarship PRE2019-088044 granted to LO from the Ministerio de Ciencia, Innovación y Universidades (MCIU, Spain), Agencia Estatal de Investigación (AEI, Spain), and Fondo Europeo de Desarrollo Regional (FEDER, European Union).Peer reviewe