Exogenous calcium delays grape berry maturation in the white cv. loureiro while increasing fruit firmness and flavonol content

Vineyard calcium (Ca) sprays have been increasingly used by grape growers to improve fruit firmness and thus maintain quality, particularly in periods of heavy rains and hail. The observation that Ca visibly modified berry size, texture, and color in the most prominent white cultivar of the DOC region Vinhos Verdes, cultivar (cv.) Loureiro, led us to hypothesize that Ca induced metabolic rearrangements that resulted in a substantial delay in fruit maturation. Targeted metabolomics by ultra-performance liquid chromatography coupled to mass spectrometry and directed transcriptomics were thus combined to characterize the metabolic and transcriptional profiles of cv. Loureiro berries that, together with firmness, °Brix, and fruit weight measurements, allowed to obtain an integrated picture of the biochemical and structural effects of Ca in this cultivar. Results showed that exogenous Ca decreased amino acid levels in ripe berries while upregulating PAL1 expression, and stimulated the accumulation of caftaric, coutaric, and fertaric acids. An increase in the levels of specific stilbenoids, namely E-piceid and E--viniferin, was observed, which correlated with the upregulation of STS expression. Trace amounts of anthocyanins were detected in berries of this white cultivar, but Ca treatment further inhibited their accumulation. The increased berry flavonol content upon Ca treatment confirmed that Ca delays the maturation process, which was further supported by an increase in fruit firmness and decrease in weight and °Brix at harvest. This newly reported effect may be specific to white cultivars, a topic that deserves further investigation.This work was supported by the “Contrato-Programa” UIDB/04050/2020 funded by Portuguese national funds through the FCT IP. This work was also supported by FCT, CCDR-N (Norte Portugal Regional Coordination and Development Commission), European Funds (FEDER/POCI/COMPETE2020) through the project AgriFood XXI (NORTE-01-0145- FEDER-000041) and the research projects BerryPlastid (PTDC/BIA-FBT/28165/2017 and POCI-01-0145-FEDER 028165), and MitiVineDrought (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER-030341). AT was supported by a postdoctoral researcher contract/position within the project “BerryPlastid”. The Région-Centre Val de Loire (France) supported this work under the grant agreement to Projects CEPATLAS and VINODRONE to AL. This work also benefited from the networking activities within the European COST Action CA 17111 INTEGRAPE, the CoLAB VINES & WINES, and the CoLAB 4FOOD – Collaborative Laboratory for Innovation in the Food Industryinfo:eu-repo/semantics/publishedVersio

Exogenous application of non-mature miRNA-encoded miPEP164c inhibits proanthocyanidin synthesis and stimulates anthocyanin accumulation in grape berry cells

Secondary metabolic pathways in grape berries are tightly regulated by an array of molecular mechanisms, including microRNA-mediated post-transcriptional regulation. As recently discovered, before being processed into mature microRNAs (miRNAs), the primary transcripts of miRNAs (pri-miRNAs) can encode for small miRNA-encoded peptides (micropeptides miPEPs) that ultimately lead to an accentuated downregulation of the respective miRNA-targeted genes. Although few studies about miPEPs are available, the discovery of miPEPs reveals a new layer of gene regulation at the post-transcriptional level that opens the possibility to regulate plant metabolism without resorting to gene manipulation. Here, we identified a miPEP encoded in non-mature miR164c putatively targeting grapevine transcription factor VvMYBPA1 (miPEP164c/miPEP-MYBPA1), a positive regulator of key genes in the proanthocyanidin (PA)-biosynthetic pathway, a pathway that competes directly for substrate with the anthocyanin-biosynthetic pathway. Thus, the objective of this work was to test the hypothesis that the exogenous application of miPEP164c (miPEP-MYBPA1) can modulate the secondary metabolism of grape berry cells by inhibiting PA biosynthetic pathway while simultaneously stimulating anthocyanin synthesis. The exogenous application of miPEP164c to suspension-cultured cells from grape berry (cv. Gamay) enhanced the transcription of its corresponding non-mature miR164c, with a maximum effect at 1 M and after a period of 10 days, thus leading to a more pronounced post-transcriptional silencing of its target VvMYBPA1. This led to a significant inhibition of the PA pathway, mostly via inhibition of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) enzymatic activities and VvLAR1 downregulation. In parallel, the anthocyanin-biosynthetic route was stimulated. Anthocyanin content was 31% higher in miPEP164c-treated cells, in agreement with the observed upregulation of VvUFGT1 transcripts and UFGT enzyme activity levels.This work was supported by Fundação para a Ciência e Tecnologia (FCT), under the strategic programs UID/AGR/04033/2020 and UID/BIA/04050/2020. This work was also supported by FCT and European Funds (FEDER/POCI/COMPETE2020) through the research project “MitiVineDrought—Combining “omics” with molecular, biochemical, and physiological analyses as an integrated effort to validate novel and easy-to-implement drought mitigation strategies in grapevine while reducing water use” with ref. PTDC/BIA-FBT/30341/2017 and ref. POCI-01-0145- FEDER-030341, respectively; through the research project “BerryPlastid—Biosynthesis of secondary compounds in the grape berry: unlocking the role of the plastid” with ref. POCI 01-0145-FEDER-028165 and ref. PTDC/BIA-FBT/28165/2017, respectively; and also through the FCT-funded research project “GrapeInfectomics” (PTDC/ASPHOR/28485/2017). AC was supported with a post-doctoral researcher contract/position within the project “MitiVineDrought” (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER 030341). FEDER/COMPETE also supported this work through the project “AgriFoodXXI” (ref. NORTE-01-0145-FEDER 000041). MV was supported by a Ph.D. fellowship funded by FCT (SFRH/BD/144637/2019). HB was supported by a Ph.D. fellowship funded by FCT (SFRH/BD/144638/2019). This work also benefited from the networking activities within the European COST Action CA17111 “INTEGRAPE—Data Integration to maximize the power of omics for grapevine improvement,” the CoLAB VINES & WINES, and the CoLAB 4FOOD – Collaborative Laboratory for Innovation in the Food Industry.info:eu-repo/semantics/publishedVersio

Constitutive expression of VviNAC17 transcription factor significantly induces the synthesis of flavonoids and other phenolics in transgenic grape berry cells

VviNAC17 is a grapevine transcription factor activated by ABA. Because ABA has been proposed as the main signal modulating the secondary metabolism in grape berry skins, here we postulated VviNAC17 as a positive regulator of secondary metabolism in grape cells. To validate the hypothesis, VviNAC17 was constitutively and stably overexpressed in grape berry suspension-cultured cells of Gamay Fréaux cv. by Agrobacterium-mediated transformation. Targeted transcriptional analyses by qPCR showed that several genes involved the phenylpropanoid (VviPAL1), stilbenoid (VviSTS1) and flavonoid pathways (VviDFR, VviLAR1, VviANR, VviLDOX, and VviUFGT1), as well as anthocyanin vacuolar transport and accumulation (VviGST4 and VvMATE1) were significantly upregulated in VviNAC17-overexpressing transgenic cells, which translated in the stimulation of a number of enzymatic activities in those pathways. This was the case of phenylalanine ammonia lyase (PAL) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) that were about 2-fold and 3.5-fold higher in VviNAC17-overexpressing cells than in control cells. VviNAC17-overexpressing cells accumulated significantly higher amounts of anthocyanins, proanthocyanidins, total flavonoids and total phenolics. These findings confirmed that VviNAC17 is an important positive regulator of secondary metabolism in grapevine contributing to the accumulation of important berry quality-related secondary metabolites.This work was supported by Fundação para a Ciência e Tecnologia (FCT), under the strategic program UIDB/ BIA/04050/2020. This work was also supported by FCT and European Funds (FEDER/POCI/COMPETE2020) through the research project “MitiVineDrought—Combining ‘omics’ with molecular, biochemical, and physiological analyses as an integrated effort to validate novel and easy-toimplement drought mitigation strategies in grapevine while reducing water use” with ref. PTDC/BIA-FBT/30341/2017 and ref. POCI-01-0145-FEDER-030341; AC was supported with a post-doctoral researcher contract/position within the project “Miti VineDrought” (ref. PTDC/BIA-FBT/30341/2017 and ref. POCI01-0145-FEDER-030341). HB was supported by a Ph.D. fellowship funded by FCT (SFRH/BD/144638/2019). MV was supported by a Ph.D. fellowship funded by FCT (SFRH/BD/144637/2019).info:eu-repo/semantics/publishedVersio

Kaolin foliar application has a stimulatory effect on phenylpropanoid and flavonoid pathways in grape berries

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. 01150Drought, elevated air temperature, and high evaporative demand are increasingly frequent during summer in grape growing areas like the Mediterranean basin, limiting grapevine productivity and berry quality. The foliar exogenous application of kaolin, a radiation-reflecting inert mineral, has proven effective in mitigating the negative impacts of these abiotic stresses in grapevine and other fruit crops, however, little is known about its influence on the composition of the grape berry and on key molecular mechanisms and metabolic pathways notably important for grape berry quality parameters. Here, we performed a thorough molecular and biochemical analysis to assess how foliar application of kaolin influences major secondary metabolism pathways associated with berry quality-traits, leading to biosynthesis of phenolics and anthocyanins, with a focus on the phenylpropanoid, flavonoid (both flavonol- and anthocyanin-biosynthetic) and stilbenoid pathways. In grape berries from different ripening stages, targeted transcriptional analysis by qPCR revealed that several genes involved in these pathways—VvPAL1, VvC4H1, VvSTSs, VvCHS1, VvFLS1, VvDFR, and VvUFGT—were more expressed in response to the foliar kaolin treatment, particularly in the latter maturation phases. In agreement, enzymatic activities of phenylalanine ammonia lyase (PAL), flavonol synthase (FLS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) were about two-fold higher in mature or fully mature berries from kaolin-treated plants, suggesting regulation also at a transcriptional level. The expression of the glutathione S-transferase VvGST4, and of the tonoplast anthocyanin transporters VvMATE1 and VvABCC1 were also all significantly increased at véraison and in mature berries, thus, when anthocyanins start to accumulate in the vacuole, in agreement with previously observed higher total concentrations of phenolics and anthocyanins in berries from kaolin-treated plaThe work was supported by European Union Funds (FEDER/COMPETE-Operational Competitiveness Programme —INNOVINE—ref. 311775, Enoexcel—Norte—07-0124FEDER-000032 and INTERACT - NORTE-01-0145-FEDER000017 - Linha VitalityWine - ON 0013), and by Portuguese national funds (FCT-Portuguese Foundation for Science and Technology) under the project FCOMP-01-0124FEDER-022692. AC was supported by Enoexcel—Norte— 07-0124-FEDER-000032 and INTERACT - NORTE-010145-FEDER-000017.This work also benefited from the networking activities within the European funded COST ACTION FA1106 “QualityFruit.”info:eu-repo/semantics/publishedVersio

Combined omics expose microbial niches of fungi and bacteria correlating with wine volatile profiles in Douro wine region

Wine microbial communities establish complex ecological ecosystems that modulate the formation of aroma compounds, but only a few studies sought for correlations between specific microorganisms and wine volatiles. The present study combined metabarcoding and metabolomics for identifying microbial niches of fungi and bacteria correlating with the volatile profiles of wines of 3 renowned cultivars of the emblematic Douro region. Three major microbial niches were identified throughout the spontaneous fermentation processes, and the Hanseniaspora-Saccharomyces succession timing was cultivar-dependent. The largest niche included Hanseniaspora, Aureobasidium, Alternaria, Rhodotorula, Sporobolomyces, Massilia, Bacillus, Staphylococcus and Cutibacterium, that positively correlated with 7 metabolites, namely, acetoin, isoamyl acetate, ethyl propanoate, c-3-hexenol, phenylethyl acetate and 4-ethylphenol. The fermentative yeasts S. cerevisiae, Torulaspora delbrueckii and Meyerozyma caribbica strongly correlated with γ-butyrolactone, t-whiskylactone, isoamyl alcohol, ethyl decanoate, ethyl isobutyrate, diethyl succinate, isovaleric acid, 4-ethylguaiacol and 4-propylguaiacol. Lachancea quebecensis clustered with several pathogenic fungi (Penicillium citrinum, Erysiphe necator, Sclerotinia sclerotiorum, Aspergillus, Mycosphaerella tassiana) and bacteria (Pseudomonas spp., Bacteroides acidifaciens, Pantoea, Stenotrophomonas and Enhydrobacter), correlating positively with various monoterpenols and norisoprenoids including linalool and β-ionone, besides with benzyl alcohol, diacetyl, isobutyl acetate, ethyl-vanillate and methyl vanillinate. Metabolite-microbiota correlations denoted cultivar specificities likely underlying regional aromatic signatures

Purification and functional characterization of protoplasts and intact vacuoles from grape cells

<p>Abstract</p> <p>Background</p> <p>During grape berry ripening, the vacuoles accumulate water, sugars and secondary metabolites, causing great impact in plant productivity and wine quality. However, the molecular basis of these compartmentation processes is still poorly understood. As in many species, the major bottleneck to study these aspects in grapevine is to obtain highly purified vacuoles with a good yield. The present paper describes an isolation method of protoplasts and intact vacuoles from grape berry cells and their functional characterization by transport and cytometric assays.</p> <p>Findings</p> <p>Protoplasts were prepared by enzymatic digestion of grape cells, and vacuoles were released and purified by a Ficoll step gradient centrifugation. The tonoplast stained strongly with the fluorescent dye FM1-43 and most vacuoles maintained an internal acidic pH, as assessed by Neutral Red. Flow cytometry analysis of vacuole samples incubated with the calcium-sensitive fluorescent probe Fluo-4 AM revealed a well-defined sub-population of intact vacuoles. As assessed by the pH-sensitive probe ACMA, intact vacuoles generated and maintained a pH gradient through the activity of V-ATPase and V-PPase and were able to transport Ca²⁺via a proton-dependent transport system.</p> <p>Conclusions</p> <p>Highly pure, intact and functional protoplast and vacuole populations from grape cells were obtained with the present method, which revealed to be fast and efficient. The capacity of the vacuole population to sequester protons and accumulate Ca²⁺strongly suggests the intactness and physiological integrity of these extremely fragile organelles. Grapevine protoplasts and vacuoles may be used as models for both basic research and biotechnological approaches, such as proteomics, solute uptake and compartmentation, toxicological assessments and breeding programs.</p

How can a milk protein selectively kill cancer cells? Mechanisms underlying lactoferrin-induced apoptosis

Lactoferrin (Lf) is an iron-binding protein abundant in milk that has been shown to exhibit anticancer activity. Since Lf is non-toxic to cancer cells (1) and is well tolerated in humans (2), this protein has a huge potential to be used in cancer therapy. However, the targets and mechanisms underlying its selective anticancer activity are poorly elucidated, which limits its clinical exploitation. The recruitment of the proton pump V-ATPase to the plasma membrane, where it mediates the acidification of the tumor microenvironment, is a recognized feature involved in the acquisition of a metastatic phenotype in different cancers, including breast cancer. Therefore, inhibitors of this pump have emerged as promising anticancer drugs. Here we show that bovine lactoferrin (bLf) preferentially inhibits cell proliferation and induces apoptosis in two highly metastatic breast cancer cell lines, which display a prominent localization of V-ATPase at the plasma membrane, but not in a lowly metastatic or a non-tumorigenic cell lines (3). We then characterized the mechanism underlying bLf-induced apoptosis and demonstrated that bLf selective cytotoxicity is caused by the inhibition of extracellular acidification rate and the ensuing intracellular acidification in the highly metastatic breast cancer cells. Accordingly, bLf, like the well-known proton pump inhibitors concanamycin A and bafilomycin A1, inhibits V-ATPase proton pumping and hydrolytic activities in sub-cellular fractions enriched in this proton pump. We recently also demonstrated that bLf preferentially induces apoptosis in other types of highly metastatic cancer cells other than breast (4). Altogether, our data demonstrated for the first time that bLf acts as a V-ATPase inhibitor and established a common mechanism of action of bLf against highly metastatic cancer cell exhibiting this proton pump at the plasma membrane. This study opens promising perspectives for the safer and more rational application of bLf in the therapy of these life-threatening cancers. 1. Gibbons JA et al. (2015) BMC Cancer doi: 10.1186/s12885-015-1441-4. 2. Hayes TG et al. (2010) Invest New Drugs doi: 10.1007/s10637-009-9233-9. 3. Pereira CS et al. (2016) Oncotarget doi: 10.18632/oncotarget.11394. 4. Guedes JP et al. (2018) Frontiers in Oncology doi.org/10.3389/fonc.2018.00200info:eu-repo/semantics/publishedVersio

The grapevine (Vitis vinifera) aquaporin VvNIP2;1 is a silicon channel localized at the plasma membrane highly expressed in roots

Silicon (Si) supplementation has been shown to improve plant tolerance to different stresses and its accumulation in the aerial organs is mediated by NIP2;1 aquaporins (Lsi channels) and Lsi2-type exporters in roots. In the present study, we tested the hypothesis that grapevine expresses a functional NIP2;1 that accounts for root Si uptake and, eventually, Si accumulation in leaves. Own-rooted grapevine cuttings of the cultivar Vinhão accumulated over 0.2 % Si (dw) in leaves when irrigated with 1.5 mM Si for one month, while Si was undetected in control leaves. Real-time PCR showed that VvNIP2;1 was highly expressed in roots and in green berries. The transient transformation of tobacco leaf epidermal cells mediated by Agrobacterium tumefaciens confirmed VvNIP2;1 localization at the plasma membrane. Transport experiments in oocytes showed that VvNIP2;1 mediates Si and arsenite uptake, whereas permeability studies revealed that VvNIP2;1 expressed in yeast is unable to transport water and glycerol. Si supplementation to pigmented grape cultured cells (cv. Gamay Freáux) had no impact on the total phenolic and anthocyanin content, as well as the growth rate and VvNIP2;1 expression. Long-term experiments should help determine the extent of Si uptake over time and if gapevine can benefit from Si fertilizationinfo:eu-repo/semantics/acceptedVersio

Canopy management through crop forcing impacts grapevine cv. ‘Touriga Nacional’ performance, ripening and berry metabolomics profile

Climate changes are speeding up the maturation of grapes in numerous areas of the world, including in the Mediterranean basin, but warmer temperatures often uncouple technical and phenolic maturity, resulting in unbalanced wines. We tested the efficacy of crop forcing (CF) in delaying the maturation of cv. ‘Touriga Nacional’ vines of the Douro Region, and their impacts on plant performance, berry quality attributes and metabolome were also evaluated. In two consecutive seasons (2019 and 2020), CF was conducted 15 (CF1) and 30 (CF2) days after fruit set by hedging growing shoots to five nodes and removing summer laterals, leaves and clusters. Results showed that while CF2 delayed ripening up to 51 days till first autumn rainfall, which compromised optimal sugar ripeness, CF1 delayed the technical maturation by one month, but both treatments severely impacted the production, mainly CF1, which reduced grapevine yield up to 90 %. The effect of CF in protecting vines against drought stress was not evident, judging by the values of leaf pre-dawn water potential measured along both seasons. CF1 and CF2 resulted in berries with lower pH and higher titratable acidity than controls, while total phenolics content increased by up to 48 % in 2020. A UPLC–MS-based targeted metabolomic analysis showed that CF increased the relative abundance of key metabolites like flavan-3-ols (i.e., catechin gallate increased by up to 661 %), trihydroxylated anthocyanins (i.e., delphinidin-3-O-glucoside increased by up to 656 % after CF2) and stilbenes (resveratrol increased by up to 700 % after CF2) with potential positive impacts in wine quality