Undifferentiated In Vitro Cultured Actinidia deliciosa as Cell Factory for the Production of Quercetin Glycosides.

Land plants produce a vast arsenal of specialized metabolites and many of them display interesting bioactivities in humans. Recently, flavonol quercetin gained great attention in the light of the COVID-19 pandemic because, in addition to the anti-inflammatory, antiviral and anti-cancer activity already described, it emerged as possible inhibitor of 3CLpro, the major protease of SARS-CoV-2 virus. Plant cell and tissue culture (PCTC) is an attractive platform for the biotechnological production of plant metabolites. This technology allows a large amount of water and agricultural land to be saved and, being free of contaminants in the process, it is suitable for scaling up the production in bioreactors. In a project aimed to generate and screen in vitro plant cells for the production of valuable specialized metabolites for commercial production, we generated various cell lines from Actinidia deliciosa (kiwi fruit tree) and Actinidia chinensis (gold kiwi fruit tree), that were able to produce relevant amounts of quercetin derivatives, mainly quercetin glycosides. Three cell lines from A. deliciosa were characterized by targeted and untargeted metabolomics. In standard growing conditions, they produce and accumulate up to 13.26 mg/100 g fresh weight (419.76 mg/100 g dry weight) of quercetin derivatives. To address future industrial applications, these cell lines should be entered into an acceleration program to further increase the amount of these metabolites by optimizing the culture conditions and elicitation.

Metabolomic Profiling and Antioxidant Activity of Fruits Representing Diverse Apple and Pear Cultivars.

The false fruits of apple (Malus domestica) and pear (Pyrus communis) are consumed all over the world, contributing to the dietary intake of health-promoting antioxidant phytochemicals. For example, polyphenols confer many beneficial effects (according to their chemical structure, bioavailability, and absorption efficiency in the gut) and the consumption of polyphenol-rich apple and pear fruits may therefore reduce the risk of some diseases. However, the content of such molecules is highly dependent on the specific fruit cultivar. To examine this metabolic diversity in detail, we used metabolomic analysis (NMR and HPLC-DAD/MS) to profile the metabolome of six apple and five pear cultivars. We also determined the antioxidant capacity of the extracts (FRAP assay) and correlated this with the metabolomic composition and abundance of specific metabolites. We observed the cultivar-specific accumulation of sugars, amino acids, malic acid, and various polyphenols, which was also related to the growing season for some cultivars. We found that the ancient Italian apple Pom Prussian was enriched for chlorogenic acid as well as more characteristic polyphenols (phloretin derivatives), the pear cultivar Abate Fetel was low in sucrose, and both cultivars displayed high in vitro antioxidant activity. These cultivars may, therefore, be particularly attractive to health-conscious consumers.

Untargeted and Targeted Metabolomics and Tryptophan Decarboxylase In Vivo Characterization Provide Novel Insight on the Development of Kiwifruits (Actinidia deliciosa).

Kiwifruit (Actinidia deliciosa cv. Hayward) is a commercially important crop with highly nutritional green fleshy fruits. The post-harvest maturation of the fruits is well characterized, but little is known about the metabolic changes that occur during fruit development. Here we used untargeted metabolomics to characterize the non-volatile metabolite profile of kiwifruits collected at different time points after anthesis, revealing profound metabolic changes before the onset of ripening including the depletion of many classes of phenolic compounds. In contrast, the phytohormone abscisic acid accumulated during development and ripening, along with two indolamines (serotonin and its precursor tryptamine), and these were monitored in greater detail by targeted metabolomics. The role of indolamines in kiwifruit development is completely unknown, so we also characterized the identity of genes encoding tryptophan decarboxylase in A. deliciosa and its close relative A. chinensis to provide insight into the corresponding biological processes. Our results indicate that abscisic acid and indolamines fulfill unrecognized functions in the development and ripening of kiwifruits.

Multi-approach metabolomics analysis and artificial simplified phytocomplexes reveal cultivar-dependent synergy between polyphenols and ascorbic acid in fruits of the sweet cherry (Prunus avium L.).

Fruits of the sweet cherry (Prunus avium L.) accumulate a range of antioxidants that can help to prevent cardiovascular disease, inflammation and cancer. We tested the in vitro antioxidant activity of 18 sweet cherry cultivars collected from 12 farms in the protected geographical indication region of Marostica (Vicenza, Italy) during two growing seasons. Multiple targeted and untargeted metabolomics approaches (NMR, LC-MS, HPLC-DAD, HPLC-UV) as well as artificial simplified phytocomplexes representing the cultivars Sandra Tardiva, Sandra and Grace Star were then used to determine whether the total antioxidant activity reflected the additive effects of each compound or resulted from synergistic interactions. We found that the composition of each cultivar depended more on genetic variability than environmental factors. Furthermore, phenolic compounds were the principal source of antioxidant activity and experiments with artificial simplified phytocomplexes indicated strong synergy between the anthocyanins and quercetins/ascorbic acid specifically in the cultivar Sandra Tardiva. Our data therefore indicate that the total antioxidant activity of sweet cherry fruits may originate from cultivar-dependent interactions among different classes of metabolite.

Impact of Phenylpropanoid Compounds on Heat Stress Tolerance in Carrot Cell Cultures.

The phenylpropanoid and flavonoid families include thousands of specialized metabolites that influence a wide range of processes in plants, including seed dispersal, auxin transport, photoprotection, mechanical support and protection against insect herbivory. Such metabolites play a key role in the protection of plants against abiotic stress, in many cases through their well-known ability to inhibit the formation of reactive oxygen species (ROS). However, the precise role of specific phenylpropanoid and flavonoid molecules is unclear. We therefore investigated the role of specific anthocyanins (ACs) and other phenylpropanoids that accumulate in carrot cells cultivated in vitro, focusing on their supposed ability to protect cells from heat stress. First we characterized the effects of heat stress to identify quantifiable morphological traits as markers of heat stress susceptibility. We then fed the cultures with precursors to induce the targeted accumulation of specific compounds, and compared the impact of heat stress in these cultures and unfed controls. Data modeling based on projection to latent structures (PLS) regression revealed that metabolites containing coumaric or caffeic acid, including ACs, correlate with less heat damage. Further experiments suggested that one of the cellular targets damaged by heat stress and protected by these metabolites is the actin microfilament cytoskeleton.

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics.

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to particular habitats and management practices. This article shows how certain terroir signatures can be detected in the berry metabolome and transcriptome of the grapevine cultivar Corvina using multivariate statistical analysis. The method first requires an appropriate sampling plan. In this case study, a specific clone of the Corvina cultivar was selected to minimize genetic differences, and samples were collected from seven vineyards representing three different macro-zones during three different growing seasons. An untargeted LC-MS metabolomics approach is recommended due to its high sensitivity, accompanied by efficient data processing using MZmine software and a metabolite identification strategy based on fragmentation tree analysis. Comprehensive transcriptome analysis can be achieved using microarrays containing probes covering ~99% of all predicted grapevine genes, allowing the simultaneous analysis of all differentially expressed genes in the context of different terroirs. Finally, multivariate data analysis based on projection methods can be used to overcome the strong vintage-specific effect, allowing the metabolomics and transcriptomics data to be integrated and analyzed in detail to identify informative correlations.

Disclosing the Molecular Basis of the Postharvest Life of Berry in Different Grapevine Genotypes.

The molecular events that characterize postripening grapevine berries have rarely been investigated and are poorly defined. In particular, a detailed definition of changes occurring during the postharvest dehydration, a process undertaken to make some particularly special wine styles, would be of great interest for both winemakers and plant biologists. We report an exhaustive survey of transcriptomic and metabolomic responses in berries representing six grapevine genotypes subjected to postharvest dehydration under identical controlled conditions. The modulation of phenylpropanoid metabolism clearly distinguished the behavior of genotypes, with stilbene accumulation as the major metabolic event, although the transient accumulation/depletion of anthocyanins and flavonols was the prevalent variation in genotypes that do not accumulate stilbenes. The modulation of genes related to phenylpropanoid/stilbene metabolism highlighted the distinct metabolomic plasticity of genotypes, allowing for the identification of candidate structural and regulatory genes. In addition to genotype-specific responses, a core set of genes was consistently modulated in all genotypes, representing the common features of berries undergoing dehydration and/or commencing senescence. This included genes controlling ethylene and auxin metabolism as well as genes involved in oxidative and osmotic stress, defense responses, anaerobic respiration, and cell wall and carbohydrate metabolism. Several transcription factors were identified that may control these shared processes in the postharvest berry. Changes representing both common and genotype-specific responses to postharvest conditions shed light on the cellular processes taking place in harvested berries stored under dehydrating conditions for several months.

Towards a scientific interpretation of the terroir concept: plasticity of the grape berry metabolome.

BACKGROUND: The definition of the terroir concept is one of the most debated issues in oenology and viticulture. The dynamic interaction among diverse factors including the environment, the grapevine plant and the imposed viticultural techniques means that the wine produced in a given terroir is unique. However, there is an increasing interest to define and quantify the contribution of individual factors to a specific terroir objectively. Here, we characterized the metabolome and transcriptome of berries from a single clone of the Corvina variety cultivated in seven different vineyards, located in three macrozones, over a 3-year trial period. RESULTS: To overcome the anticipated strong vintage effect, we developed statistical tools that allowed us to identify distinct terroir signatures in the metabolic composition of berries from each macrozone, and from different vineyards within each macrozone. We also identified non-volatile and volatile components of the metabolome which are more plastic and therefore respond differently to terroir diversity. We observed some relationships between the plasticity of the metabolome and transcriptome, allowing a multifaceted scientific interpretation of the terroir concept. CONCLUSIONS: Our experiments with a single Corvina clone in different vineyards have revealed the existence of a clear terroir-specific effect on the transcriptome and metabolome which persists over several vintages and allows each vineyard to be characterized by the unique profile of specific metabolites.

Carrot-specific features of the phenylpropanoid pathway identified by feeding cultured cells with defined intermediates.

Plants produce a vast array of secondary metabolites, many of which have important biological properties in animals when consumed as part of the diet. Interestingly, although the activities and benefits of plant secondary metabolites in animals are well established, comparatively little is known about the endogenous functions of these compounds in plants. One way to investigate the role of secondary products in plants is to modify the secondary metabolome and investigate the impact of such modifications on the phenotype. We have designed a novel feeding approach using different hydroxycinnamic acids (HCAs) and the cyanidin precursor dihydroquercetin (DHQ) to modify the metabolome of carrot R3M suspension cells. This strategy increased the accumulation of specific metabolites in a predictable way, and provided novel insights into the carrot phenylpropanoid pathway, suggesting that (a) cells use HCA hexose esters as substrates in the biosynthetic pathway leading to the accumulation of the various HCA derivatives and (b) p-coumaric acid derivative levels play a key roles in the regulation the flux of HCAs along the pathway. Moreover, this rapid strategy for metabolome modification does not depend on the availability of molecular tools or knowledge and can therefore be applied to any plant species.

Metabolomics of Daucus carota cultured cell lines under stressing conditions reveals interactions between phenolic compounds.

A metabolomic approach followed by principal components and partial least square analysis was used for investigating the effect of environmental factors on two Daucus carota L. cv. Flakkese cell lines (R3M and R4G), selected for their ability to produce anthocyanins in the light and the dark, respectively. A positive correlation between total anthocyanin, hydroxycinnamic and hydroxybenzoic acid accumulation was found in both lines. Furthermore, the experimental design and the combination of biochemical and statistical analyses allowed us to unravel complex relationships between environmental factors and phenylpropanoid composition. Among these, the induction by mechanical stress of overproduction of all anthocyanins, hydroxycinnamic and hydroxybenzoic acids except sinapic acid derivatives, whose accumulation was inhibited.

Image analysis and in vivo imaging as tools for investigation of productivity dynamics in anthocyanin-producing cell cultures of Daucus carota.

An anthocyanin-producing suspension culture of Daucus carota (L.) cv. Flakkese was used as model system to study secondary metabolite production in cell culture at the individual cell level. An approach was set up in which growth and production of anthocyanins were investigated using a combination of biochemical analysis, image (colour) analysis and in vivo imaging. This novel approach was used to segment the culture in different subpopulations and dissect the productive process in the cell culture grown under two different conditions, known to differ mainly for oxygen supply and mixing intensity (volume of 50 ml or 20 ml in 250 ml flasks). The 20 ml batch cultures gave a higher content and yield of anthocyanins, which depended on a complex balance between events that positively or negatively affected anthocyanin production. A model is proposed in which the different ability of cells to respond to environmental stimuli and stress depends on the different amount of anthocyanins accumulated within cells.

Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant.

The biochemical properties of PsbS protein, a nuclear-encoded Photosystem II subunit involved in the high energy quenching of chlorophyll fluorescence, have been studied using preparations purified from chloroplasts or obtained by overexpression in bacteria. Despite the homology with chlorophyll a/b/xanthophyll-binding proteins of the Lhc family, native PsbS protein does not show any detectable ability to bind chlorophylls or carotenoids in conditions in which Lhc proteins maintain full pigment binding. The recombinant protein, when refolded in vitro in the presence of purified pigments, neither binds chlorophylls nor xanthophylls, differently from the homologous proteins LHCII, CP26, and CP29 that refold into stable pigment-binding complexes. Thus, it is concluded that if PsbS is a pigment-binding protein in vivo, the binding mechanism must be different from that present in other Lhc proteins. Primary sequence analysis provides evidence for homology of PsbS helices I and III with the central 2-fold symmetric core of chlorophyll a/b-binding proteins. Moreover, a structural homology owed to the presence of acidic residues in each of the two lumen-exposed loops is found with the dicyclohexylcarbodiimide/Ca(2+)-binding domain of CP29. Consistently, both native and recombinant PsbS proteins showed [(14)C]dicyclohexylcarbodiimide binding, thus supporting a functional basis for its homology with CP29 on the lumen-exposed loops. This domain is suggested to be involved in sensing low luminal pH.