Grape seed residues represent the raw material to produce several value-added products including polyphenol-rich extracts with nutritional and health attributes. Although the impact of variety and environmental conditions on the polyphenol composition in fresh berries is recognized, no data are available regarding grape seed residues. The chemical composition of grape seed residues from wine distilleries in France, Spain and Italy was characterized by mass spectrometry. Forty-two metabolites were identified belonging to non-galloylated and galloylated procyanidins as well as amino acids. Polyphenol concentrations in the red varieties originated from Champagne or Veneto were twice higher than in white varieties from the Loire Valley. The chemical profiles of grape seed residues were mainly classified according to the color variety with galloylated procyanidins as biomarkers of white varieties and non-galloylated procyanidins as biomarkers of red ones. The present approach might assist the selection of grape seed residues as quality raw materials for the production of polyphenol-rich extracts.

Helminth infections of livestock result in economic losses around the globe. The majority of the people in West Africa treats such infections with medicinal plants related to the local traditional medicine. In a previous ethnomedicinal survey carried out in the north of Togo, traditional healers frequently cited Cassia sieberiana DC to treat helminth infections. The aim of the present study was to validate the traditional application of C. sieberiana root bark as a dewormer and to analyse the metabolite composition in preparations with this activity. Two tannin-rich fractions (FrE14 and FrE15) obtained from ethyl acetate extract were submitted to UPLC-MS analysis. The anthelmintic activity of C. sieberiana extracts and tannins-rich fractions was investigated on the small ruminant parasite Haemonchus contortus using Larval Migration Inhibition tests. The ethyl acetate, butanol and aqueous extracts significantly disrupted larval migration of Haemonchus contortus L3 with inhibition of 30.3%, 22.2% and 26.5% respectively at 2.5 mg/mL, as compared to negative control. The two tannin-rich fractions, FrE14 and FrE15 presented larval migration inhibition of 6.5% and 20.7% respectively at the same concentration. From these two fractions, 28 flavan-3-ols were identified including 3 monomers (catechin, epicatechin and epiafzelechin), 22 dimers (7 homodimers and 15 heterodimers) as well as 3 newly described trimers consisting of (epi)afzelechin, (ent)cassiaflavan or (epi)guibourtidinol subunits.

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.

Trihydroxycinnamoyl spermidines (THCSpd) are plant specialized metabolites with promising pharmacological activities as antifungals, antibacterial, antiviral, and antidepressant drugs. However, their characterization and potential pharmaceutical exploitation are greatly impaired by the sourcing of these compounds, restricted to the pollen of core Eudicot plant species. In this work, we developed a precursor-directed biosynthesis of THCSpd in yeast using a dual enzymatic system based on 4-coumarate-CoA ligases (4CL) and spermidine N-hydroxycinnamoyltransferases (SHT). The system relies on the yeast endogenous spermidine pool and only requires hydroxycinnamic acids as exogenous precursors. By exploring 4CL isoforms and SHT diversity among plants, we have driven the production of 8 natural THCSpd, using single or mixed hydroxycinnamic acid precursors. Substrate promiscuities of 4CL and SHT were genuinely exploited to produce 8 new-to-nature THCSpd from exotic hydroxycinnamic and dihydrohydroxycinnamic acids, together with 3 new-to-nature THCSpd containing halogenated hydroxycinnamoyl moieties. In this work, we established a versatile and modular biotechnological production platform allowing the tailor-made THCSpd synthesis, constituting pioneer metabolic engineering for access to these valuable natural products.

Aphanomyces euteiches is an oomycete pathogen that causes the pea root rot. We investigated the potential role of early belowground defense in pea (susceptible plant) and faba bean (tolerant plant) at three days after inoculation. Pea and faba bean were inoculated with A. euteiches zoospores. Root colonization was examined. Root exudates from pea and faba bean were harvested and their impact on A. euteiches development were assessed by using in vitro assays. A. euteiches root colonization and the influence of the oomycete inoculation on specialized metabolites patterns and arabinogalactan protein (AGP) concentration of root exudates were also determined. In faba bean root, A. euteiches colonization was very low as compared with that of pea. Whereas infected pea root exudates have a positive chemotaxis index (CI) on zoospores, faba bean exudate CI was negative suggesting a repellent effect. While furanoacetylenic compounds were only detected in faba bean exudates, AGP concentration was specifically increased in pea.This work showed that early in the course of infection, host susceptibility to A. euteiches is involved via a plant-species specific root exudation opening new perspectives in pea root rot disease management.

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC50 values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.

Grape canes are viticulture byproducts representing a sustainable source of valuable bioactive polyphenols. However, varietal origin and postharvest treatment greatly influence the effective concentration of biomolecules, thus limiting industrial development. With the aim to develop grape cane extracts with high polyphenol contents, the selection of a specific grape variety combined with optimized postharvest treatment is the major determinant. A previously described postharvest treatment comprising cutting grapevine stalks of 0.5 cm length and storage at 15-20 degrees C over 2 weeks was applied on a selection of 44 grape varieties representative of the genetic diversity of the whole European collection and performing the screening of polyphenol contents. Varietal rankings according to major polyphenols (catechin, epicatechin, E-resveratrol, E-piceatannol, E-epsilon-viniferin, E-miyabenol C, ampelopsin A, E-vitisin B, hopeaphenol, and isophopeaphenol) were performed with and without the postharvest treatment. We observed that postharvest treatment greatly influenced the total polyphenol composition but also the ranking of polyphenol-rich varieties. This polyphenol screening of grape canes from a large collection of European varieties revealed the importance of postharvest treatment together with the selection of varieties to develop natural extracts based on grapevine wood biomass enriched with molecules with health benefits.

Grape downy mildew is a devastating disease worldwide and new molecular phenotyping tools are required to detect metabolic changes associated to plant disease symptoms. In this purpose, we used UPLC-DAD-MS-based semi-targeted metabolomics to screen downy mildew symptomatic leaves that expressed oil spots (6 dpi, days post-infection) and necrotic lesions (15 dpi) under natural infections in the field. Leaf extract analyses enabled the identification of 47 metabolites belonging to the primary metabolism including 6 amino acids and 1 organic acid, as well as an important diversity of specialized metabolites including 9 flavonols, 11 flavan-3-ols, 3 phenolic acids, and stilbenoids with various degree of polymerization (DP) including 4 stilbenoids DP1, 8 stilbenoids DP2, and 4 stilbenoids DP3. Principal component analysis (PCA) was applied as unsupervised multivariate statistical analysis method to reveal metabolic variables that were affected by the infection status. Univariate and multivariate statistics revealed 33 and 27 metabolites as relevant infection biomarkers at 6 and 15 dpi, respectively. Correlation-based networks highlighted a general decrease of flavonoid-related metabolites, whereas stilbenoid DP1 and DP2 concentrations increased upon downy mildew infection. Stilbenoids DP3 were identified only in necrotic lesions representing late biomarkers of downy mildew infection.

Grape canes are waste biomass of viticulture containing bioactive polyphenols valuable in cosmetics. Whereas several studies reported the cosmetic activities of E-resveratrol, only few described the potential of E-ε-viniferin, the second major constituent of grape cane extracts (GCE), and none of them investigated GCE as a natural blend of polyphenols for cosmetic applications. In this study, we considered the potential of GCE from polyphenol-rich grape varieties as multifunctional cosmetic ingredients. HPLC analysis was performed to quantify major polyphenols in GCE i.e., catechin, epicatechin, E-resveratrol, E-piceatannol, ampelopsin A, E-ε-viniferin, hopeaphenol, isohopeaphenol, E-miyabenol C and E-vitisin B from selected cultivars. Skin whitening potential through tyrosinase inhibition assay and the activation capacity of cell longevity protein (SIRT1) of GCE were compared to pure E-resveratrol and E-ε-viniferin. Drug-likeness of GCE polyphenols were calculated, allowing the prediction of skin permeability and bioavailability. Finally, the present data enabled the consideration of GCE from polyphenol-rich varieties as multifunctional cosmetic ingredients in accordance with green chemistry practices.

Ommochromes are widespread pigments that mediate multiple functions in invertebrates. The two main families of ommochromes are ommatins and ommins, which both originate from the kynurenine pathway but differ in their backbone, thereby in their coloration and function. Despite its broad significance, how the structural diversity of ommochromes arises in vivo has remained an open question since their first description. In this study, we combined organic synthesis, analytical chemistry and organelle purification to address this issue. From a set of synthesized ommatins, we derived a fragmentation pattern that helped elucidating the structure of new ommochromes. We identified uncyclized xanthommatin as the elusive biological intermediate that links the kynurenine pathway to the ommatin pathway within ommochromasomes, the ommochrome-producing organelles. Due to its unique structure, we propose that uncyclized xanthommatin functions as a key branching metabolite in the biosynthesis and structural diversification of ommatins and ommins, from insects to cephalopods.

Over the last few decades, methods relating to plant tissue culture have become prevalent within the cosmetic industry. Forecasts predict the cosmetic industry to grow to an annual turnover of around a few hundred billion US dollars. Here we focused on Linum usitatissimum L., a plant that is well-known for its potent cosmetic properties. Following the a) establishment of cell cultures from three distinct initial explant origins (root, hypocotyl, and cotyledon) and b) selection of optimal hormonal concentrations, two in vitro systems (callus vs cell suspensions) were subjected to different light conditions. Phytochemical analysis by UPLC-HRMS not only confirmed high (neo)lignan accumulation capacity of this species with high concentrations of seven newly described (neo)lignans. Evaluation over 30 days revealed strong variations between the two different in vitro systems cultivated under light or dark, in terms of their growth kinetics and phytochemical composition. Additionally, antioxidant (i.e. four different in vitro assays based on hydrogen-atom transfer or electron transfer mechanism) and anti-aging (i.e. four in vitro inhibition potential of the skin remodeling enzymes: elastase, hyaluronidase, collagenase and tyrosinase) properties were evaluated for the two different in vitro systems cultivated under light or dark. A prominent hydrogen-atom transfer antioxidant mechanism was illustrated by the DPPH and ABTS assays. Potent tyrosinase and elastase inhibitory activities were also observed, which was strongly influenced by the in vitro system and light conditions. Statistical treatments of the data showed relationship of some (neo)lignans with these biological activities. These results confirmed the accumulation of flax (neo)lignans in different in vitro systems that were subjected to distinct light conditions. Furthermore, we showed the importance of optimizing these parameters for specific applications within the cosmetic industry.

Grape canes are under-valued byproducts of viticulture that accumulate large amounts of stilbenoids. These specialized metabolites are phytoalexins of Vitis vinifera that play a keyrole in defence of Vitis vinifera against pathogens. Since in vitro assays demonstrated the antifungal activity of several pure stilbenoids, grape cane extracts (GCE) have been proposed as eco-friendly alternative to classical fungicides in a context of sustainable viticulture. Following the grape pruning during winter, a storage period associated to an efficient extraction procedure, are determinant to obtain GCE with optimal stilbenoid titration. Stilbenoid-enriched extracts have shown promising protection in vitro against various grape pathogens including downy mildew, powdery mildew, gray mold and wood diseases. Additionally, large-scale studies in vineyards reported a significant partial protection against downy mildew. Protection level is relative to the concentration of stilbenoids contained in the grape cane extract. Therefore, the huge varietal diversity of grape could be explored to select high-producing varieties for future use as biocontrol agent.

Monoterpene indole alkaloids (MIAs) are specialized metabolites synthesized in many plants of the Apocynaceae family including Catharanthus roseus and Rauvolfia sp. MIAs are part of the chemical arsenal that plants evolved to face pet and herbivore attacks, and their high biological activities also confer pharmaceutical properties exploited in human pharmacopeia. Developing robust and straightforward tools to elucidate each step of MIA biosynthetic pathways thus constitutes a prerequisite to the understanding of Apocynaceae defense mechanisms and to the exploitation of MIA cytotoxicity through their production by metabolic engineering. While protocols of virus-induced gene silencing (VIGS) based on Agrobacterium-based transformation have emerged, the recalcitrance of Apocynaceae to this type of transformation prompted us to develop an universal procedure of VIGS vector inoculation. Such procedure relies on the delivery of the transforming plasmids through a particle bombardment performed using a biolistic device and offers the possibility to overcome host specificity to silence genes in any plant species. Using silencing of geissoschizine oxidase as an example, we described the main steps of this biolistic mediated VIGS in C. roseus and R. tetraphylla.

Two cytochrome P450s with high substrate specificity catalyze the synthesis of the alkaloid lochnericine in Catharanthus roseus, representing valuable tools for metabolic engineering in yeast. Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (Catharanthus roseus). Lochnericine is derived from the stereoselective C6,C7-epoxidation of tabersonine and can be metabolized further to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2,3-epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to hörhammericine production. TEX1 and TEX2 displayed complementary expression profiles, with TEX1 expressed mainly in roots and TEX2 in aerial organs. Our results suggest that TEX1 and TEX2 originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through the sequential expression of TEX1 and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhörhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for the metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.

Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (Catharanthus roseus). Lochnericine is derived from the stereoselective C6,C7-epoxidation of tabersonine and can be metabolized further to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2,3-epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to hörhammericine production. TEX1 and TEX2 displayed complementary expression profiles, with TEX1 expressed mainly in roots and TEX2 in aerial organs. Our results suggest that TEX1 and TEX2 originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through the sequential expression of TEX1 and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhörhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for the metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.