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

Strictosidine activation in Apocynaceae: towards a "nuclear time bomb"?

<p>Abstract</p> <p>Background</p> <p>The first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine β-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine. The resulting unstable aglycon is rapidly converted into a highly reactive dialdehyde, from which more than 2,000 MIAs are derived. Many studies were conducted to elucidate the biosynthesis and regulation of pharmacologically valuable MIAs such as vinblastine and vincristine in <it>Catharanthus roseus </it>or ajmaline in <it>Rauvolfia serpentina</it>. However, very few reports focused on the MIA physiological functions.</p> <p>Results</p> <p>In this study we showed that a strictosidine pool existed <it>in planta </it>and that the strictosidine deglucosylation product(s) was (were) specifically responsible for <it>in vitro </it>protein cross-linking and precipitation suggesting a potential role for strictosidine activation in plant defence. The spatial feasibility of such an activation process was evaluated <it>in planta</it>. On the one hand, <it>in situ </it>hybridisation studies showed that CrSTR and CrSGD were coexpressed in the epidermal first barrier of <it>C. roseus </it>aerial organs. However, a combination of GFP-imaging, bimolecular fluorescence complementation and electromobility shift-zymogram experiments revealed that STR from both <it>C. roseus </it>and <it>R. serpentina </it>were localised to the vacuole whereas SGD from both species were shown to accumulate as highly stable supramolecular aggregates within the nucleus. Deletion and fusion studies allowed us to identify and to demonstrate the functionality of CrSTR and CrSGD targeting sequences.</p> <p>Conclusions</p> <p>A spatial model was drawn to explain the role of the subcellular sequestration of STR and SGD to control the MIA metabolic flux under normal physiological conditions. The model also illustrates the possible mechanism of massive activation of the strictosidine vacuolar pool upon enzyme-substrate reunion occurring during potential herbivore feeding constituting a so-called "nuclear time bomb" in reference to the "mustard oil bomb" commonly used to describe the myrosinase-glucosinolate defence system in Brassicaceae.</p

Impact of deficit irrigation on grapevine cv. ‘Touriga Nacional’ during three seasons in Douro region: an agronomical and metabolomics approach

The introduction of irrigation in vineyards of the Mediterranean basin is a matter of debate, in particular in those of the Douro Demarcated Region (DDR), due to the limited number of available studies. Here, we aimed to perform a robust analysis in three consecutive vintages (2018, 2019, and 2020) on the impact of deficit irrigation on the yield, berry quality traits, and metabolome of cv. ‘Touriga Nacional’. Results showed that in the peaks of extreme drought, irrigation at 30% crop evapotranspiration (ETc) (R30) was able to prevent a decay of up to 0.4 MPa of leaf predawn water potential (ΨPd), but irrigation at 70% ETc (R70) did not translate into additional protection against drought stress. Following three seasons of irrigation, the yield was significantly improved in vines irrigated at R30, whereas irrigation at R70 positively affected the yield only in the 2020 season. Berry quality traits at harvest were not significantly changed by irrigation, except for Total Soluble Solids (TSS) in 2018. A UPLC–MS-based targeted metabolomic analysis identified eight classes of compounds, amino acids, phenolic acids, stilbenoid DP1, stilbenoid DP2, flavonols, flavan-3-ols, di-OH- and tri-OH anthocyanins, and showed that anthocyanins and phenolic acids did not change significantly with irrigation. The present study showed that deficit irrigation partially mitigated the severe summer water deficit conditions in the DDR but did not significantly change key metabolites.This research was funded by the VISCA project (Vineyards’ Integrated Smart Climate Application), funded by European Union’s Horizon 2020 research and innovation programme under grant agreement no. 730253. The Région-Centre Val de Loire (France) supported this work under the grant agreement to Project VITI’ACTIF. The work was also supported by the “Contrato-Programa” UIDB/04050/2020 funded by Portuguese national funds through the FCT I.P. The work was also supported by FCT, CCDR-N (Norte Portugal Regional Coordination and Development Commission) and European Funds (FEDER/POCI/COMPETE2020) through the project AgriFoodXXI (NORTE01-0145-FEDER-000041) and the research projects BerryPlastid (PTDC/BIA-FBT/28165/2017 and POCI-01-0145-FEDER-028165), MitiVineDrought (PTDC/BIA-FBT/30341/2017 and POCI-01-0145- FEDER-030341), and GrapeInfectomics (PTDC/ASPHOR/28485/2017). A.T. was supported by a post-doctoral researcher contract/position within the project “BerryPlastid”. 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 Industry

Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis

Field-Based Metabolomics of Vitis vinifera L. Stems Provides New Insights for Genotype Discrimination and Polyphenol Metabolism Structuring

Grape accumulates numerous polyphenols with abundant health benefit and organoleptic properties that in planta act as key components of the plant defense system against diseases. Considerable advances have been made in the chemical characterization of wine metabolites particularly volatile and polyphenolic compounds. However, the metabotyping (metabolite-phenotype characterization) of grape varieties, from polyphenolic-rich vineyard by-product is unprecedented. As this composition might result from the complex interaction between genotype, environment and viticultural practices, a field experiment was setting up with uniform pedo-climatic factors and viticultural practices of growing vines to favor the genetic determinism of polyphenol expression. As a result, UPLC-MS-based targeted metabolomic analyses of grape stems from 8 Vitis vinifera L. cultivars allowed the determination of 42 polyphenols related to phenolic acids, flavonoids, procyanidins, and stilbenoids as resveratrol oligomers (degree of oligomerization 1–4). Using a partial least-square discriminant analysis approach, grape stem chemical profiles were discriminated according to their genotypic origin showing that polyphenol profile express a varietal signature. Furthermore, hierarchical clustering highlights various degree of polyphenol similarity between grape varieties that were in agreement with the genetic distance using clustering analyses of 22 microsatellite DNA markers. Metabolite correlation network suggested that several polyphenol subclasses were differently controlled. The present polyphenol metabotyping approach coupled to multivariate statistical analyses might assist grape selection programs to improve metabolites with both health-benefit potential and plant defense traits

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

Etude de la biosynthèse et de la production d'alcaloïdes tropaniques par les racines transgéniques de Datura

La croissance et la production en métabolites de deux clones de racines transgéniques de Datura ont été caractérisées en fioles. L'ajout combiné de l'acide (RS)-phényllactique et du Tween a permis d'amplifier la diffusion de l'hyoscyamine dans le milieu de culture et d'augmenter l'accumulation totale de l'hyoscyamine de 60%. La vitesse apparente de biosynthèse de l'hyoscyamine est deux fois inférieure à celle de la littorine suggérant que la réaction d'isomérisation pourrait être une étape limitante dans la voie de biosynthèse. La croissance et la respiration des racines transgéniques ont été déterminés en bioréacteur en utilisant un système d'acquisition en ligne des mesures de conductivité du milieu de culture, de l'oxygène et du dioxyde de carbone. Ces études ont permis de mettre en évidence qu'un rythme circadien endogène persiste chez les racines transformées de D. innoxia. Enfin, un procédé de production semi-continue de scopolamine dans un bioréacteur de 16 litres a été testé.AMIENS-BU Sciences (800212103) / SudocSudocFranceF

Illuminating Fungal Infections with Bioluminescence

International audienc

Exogenous calcium deflects grape berry metabolism towards the production of more stilbenoids and less anthocyanins

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.foodchem.2019.126123.Calcium supplements have increasingly been used at pre- and post-harvest stages for improving fruit firmness, but elevated calcium levels in grape cells were shown to reduce total anthocyanin content. In this study, we hypothesized that exogenous calcium influences specific polyphenolic compounds, and performed targeted UPLC-MS analysis in fruits collected from vines cv. Vinhão sprayed with 2% (w/v) CaCl2 throughout the fruiting season, in two consecutive vintages, and in grape cell cultures elicited with calcium. Results showed that anthocyanin content is reduced upon calcium treatment, while stilbenoid synthesis is generally stimulated, in line with UFGT and STS expression patterns. The main metabolites involved in this response were malvidin-3-O-glucoside, E-piceid, E-?-viniferin and E-resveratrol. The accumulation of phenolic acids, catechin and some quercetin derivatives was also favored by calcium, while other flavonols and flavan-3-ols were affected according to the vintage and berry developmental stage. In cell cultures, the entire flavonoid pathway was repressed.This work was supported by the Portuguese Foundation for Scienceand Technology (FCT) in the framework of the strategic funding [UID/BIA/04050/2019, UID/AGR/04033/2019] and the projects [PTDC/AGR-PRO/7028/2014, PTDCBIA-FBT/28165/2017, PTDC/BIA-FBT/30341/2017, and SFRH/BPD/107905/2015 to V.M.]. The Région-Centre Val de Loire (France) supported this work under the grantagreement to Projects VITI'ACTIF and VINODRONE to A.L and K.B.info:eu-repo/semantics/publishedVersio