NMR-Based Approaches in the Study of Foods.

In this review, the three different NMR-based approaches usually used to study foodstuffs are described, reporting specific examples. The first approach starts with the food of interest that can be investigated using different complementary NMR methodologies to obtain a comprehensive picture of food composition and structure; another approach starts with the specific problem related to a given food (frauds, safety, traceability, geographical and botanical origin, farming methods, food processing, maturation and ageing, etc.) that can be addressed by choosing the most suitable NMR methodology; finally, it is possible to start from a single NMR methodology, developing a broad range of applications to tackle common food-related challenges and different aspects related to foods.

The Effect of Semen Cryopreservation Process on Metabolomic Profiles of Turkey Sperm as Assessed by NMR Analysis.

Semen cryopreservation represents the main tool for preservation of biodiversity; however, in avian species, the freezing−thawing process results in a sharp reduction in sperm quality and consequently fertility. Thus, to gain a first insight into the molecular basis of the cryopreservation of turkey sperm, the NMR-assessed metabolite profiles of fresh and frozen−thawed samples were herein investigated and compared with sperm qualitative parameters. Cryopreservation decreased the sperm viability, mobility, and osmotic tolerance of frozen−thawed samples. This decrease in sperm quality was associated with the variation in the levels of some metabolites in both aqueous and lipid sperm extracts, as investigated by NMR analysis. Higher amounts of the amino acids Ala, Ile, Leu, Phe, Tyr, and Val were found in fresh than in frozen−thawed sperm; on the contrary, Gly content increased after cryopreservation. A positive correlation (p < 0.01) between the amino acid levels and all qualitative parameters was found, except in the case of Gly, the levels of which were negatively correlated (p < 0.01) with sperm quality. Other water-soluble compounds, namely formate, lactate, AMP, creatine, and carnitine, turned out to be present at higher concentrations in fresh sperm, whereas cryopreserved samples showed increased levels of citrate and acetyl-carnitine. Frozen−thawed sperm also showed decreases in cholesterol and polyunsaturated fatty acids, whereas saturated fatty acids were found to be higher in cryopreserved than in fresh sperm. Interestingly, lactate, carnitine (p < 0.01), AMP, creatine, cholesterol, and phosphatidylcholine (p < 0.05) levels were positively correlated with all sperm quality parameters, whereas citrate (p < 0.01), fumarate, acetyl-carnitine, and saturated fatty acids (p < 0.05) showed negative correlations. A detailed discussion aimed at explaining these correlations in the sperm cell context is provided, returning a clearer scenario of metabolic changes occurring in turkey sperm cryopreservation.

Commercial Bio-Packaging to Preserve the Quality and Extend the Shelf-Life of Vegetables: The Case-Study of Pumpkin Samples Studied by a Multimethodological Approach.

A multidisciplinary protocol is proposed to monitor the preservation of fresh pumpkin samples (FP) using three commercial polymeric films: A made of biodegradable cellophane from regenerated cellulose pulp; B from corn starch, cassava and eucalyptus, C made of polylactic acid from corn starch, and a polyethylene film used as reference (REF). Chemical, mechanical and microbiological analyses were applied on packaging and fresh and packaged samples at different times. After an 11-day period, NMR spectroscopy results showed a sucrose increase and a malic acid decrease in all the biofilms with respect to FP; fructose, glucose, galactose levels remained quite constant in biofilms B and C; the most abundant amino acids remained quite constant in biofilm A and decreased significantly in biofilm B. From microbiological analyses total microbial count was below the threshold value up to 7 days for samples in all the films, and 11 days for biofilm C. The lactic acid bacteria, and yeasts and molds counts were below the acceptability limit during the 11 days for all packages. In the case of biofilm C, the most promising packaging for microbiological point of view, aroma analysis was also carried out. In this paper, you can find all the analysis performed and all the values found.

NMR, RP-HPLC-PDA-ESI-MSn, and RP-HPLC-FD Characterization of Green and Oolong Teas (Camellia sinensis L.).

Untargeted (NMR) and targeted (RP-HPLC-PDA-ESI-MSn, RP-HPLC-FD) analytical methodologies were used to determine the bioactive components of 19 tea samples, characterized by different production processes (common tea and GABA tea), degrees of fermentation (green and oolong teas), and harvesting season (autumn and spring). The combination of NMR data and a multivariate statistical approach led to a statistical model able to discriminate between GABA and non-GABA teas and green and oolong teas. Targeted analyses showed that green and GABA green teas had similar polyphenol and caffeine contents, but the GABA level was higher in GABA green teas than in regular green tea samples. GABA oolong teas showed lower contents of polyphenols, caffeine, and amino acids, and a higher content of GABA, in comparison with non-GABA oolong teas. In conclusion, the results of this study suggest that the healthy properties of teas, especially GABA teas, have to be evaluated via comprehensive metabolic profiling rather than only the GABA content.

Metabolomic Profiling of Fresh Goji (Lycium barbarum L.) Berries from Two Cultivars Grown in Central Italy: A Multi-Methodological Approach.

The metabolite profile of fresh Goji berries from two cultivars, namely Big Lifeberry (BL) and Sweet Lifeberry (SL), grown in the Lazio region (Central Italy) and harvested at two different periods, August and October, corresponding at the beginning and the end of the maturation, was characterized by means of nuclear magnetic resonance (NMR) and electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR MS) methodologies. Several classes of compounds such as sugars, amino acids, organic acids, fatty acids, polyphenols, and terpenes were identified and quantified in hydroalcoholic and organic Bligh-Dyer extracts. Sweet Lifeberry extracts were characterized by a higher content of sucrose with respect to the Big Lifeberry ones and high levels of amino acids (glycine, betaine, proline) were observed in SL berries harvested in October. Spectrophotometric analysis of chlorophylls and total carotenoids was also carried out, showing a decrease of carotenoids during the time. These results can be useful not only to valorize local products but also to suggest the best harvesting period to obtain a product with a chemical composition suitable for specific industrial use. Finally, preliminary studies regarding both the chemical characterization of Goji leaves generally considered a waste product, and the biological activity of Big Lifeberry berries extracts was also investigated. Goji leaves showed a chemical profile rich in healthy compounds (polyphenols, flavonoids, etc.) confirming their promising use in the supplements/nutraceutical/cosmetic field. MG63 cells treated with Big Lifeberry berries extracts showed a decrease of iNOS, COX-2, IL-6, and IL-8 expression indicating their significant biological activity.

A Multimethodological Characterization of Cannabis sativa L. Inflorescences from Seven Dioecious Cultivars Grown in Italy: The Effect of Different Harvesting Stages.

The chemical profile of the female inflorescence extracts from seven Cannabis sativa L. dioecious cultivars (Carmagnola, Fibranova, Eletta Campana, Antal, Tiborszallasi, Kompolti, and Tisza) was monitored at three harvesting stages (4, 14, and 30 September), reaching from the beginning of flowering to end of flowering/beginning of seed formation, using untargeted nuclear magnetic resonance (NMR) and targeted (ultra-high-performance liquid chromatography (UHPLC) and spectrophotometry) analyses. The tetrahydrocannabinol content was always below the legal limits (<0.6%) in all the analyzed samples. The NMR metabolite profile (sugars, organic acids, amino acids, and minor compounds) subjected to principal components analysis (PCA) showed a strong variability according to the harvesting stages: samples harvested in stage I were characterized by a high content of sucrose and myo-inositol, whereas the ones harvested in stage II showed high levels of succinic acid, alanine, valine, isoleucine, phenylalanine, and threonine. Samples harvested in stage III were characterized by high levels of glucose, fructose, choline, trigonelline, malic acid, formic acid, and some amino acids. The ratio between chlorophylls and carotenoids content indicated that all plants grew up exposed to the sun, the Eletta Campana cultivar having the highest pigment amount. Tiborszallasi cultivar showed the highest polyphenol content. The highest antioxidant activity was generally observed in stage II. All these results suggested that the Cannabis sativa L. inflorescences of each analyzed dioecious hemp cultivar presented a peculiar chemical profile affected by the harvesting stage. This information could be useful for producers and industries to harvest inflorescences in the appropriate stage to obtain samples with a peculiar chemical profile suitable for proper applications.

Synthesis and Evaluation of Thymol-Based Synthetic Derivatives as Dual-Action Inhibitors against Different Strains of H. pylori and AGS Cell Line.

Following a similar approach on carvacrol-based derivatives, we investigated the synthesis and the microbiological screening against eight strains of H. pylori, and the cytotoxic activity against human gastric adenocarcinoma (AGS) cells of a new series of ether compounds based on the structure of thymol. Structural analysis comprehended elemental analysis and 1H/13C/19F NMR spectra. The analysis of structure-activity relationships within this molecular library of 38 structurally-related compounds reported that some chemical modifications of the OH group of thymol led to broad-spectrum growth inhibition on all isolates. Preferred substitutions were benzyl groups compared to alkyl chains, and the specific presence of functional groups at para position of the benzyl moiety such as 4-CN and 4-Ph endowed the most anti-H. pylori activity toward all the strains with minimum inhibitory concentration (MIC) values up to 4 µg/mL. Poly-substitution on the benzyl ring was not essential. Moreover, several compounds characterized by the lowest minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) values against H. pylori were also tested in order to verify a cytotoxic effect against AGS cells with respect to 5-fluorouracil and carvacrol. Three derivatives can be considered as new lead compounds alternative to current therapy to manage H. pylori infection, preventing the occurrence of severe gastric diseases. The present work confirms the possibility to use natural compounds as templates for the medicinal semi-synthesis.

Impact of Dealcoholization by Osmotic Distillation on Metabolic Profile, Phenolic Content, and Antioxidant Capacity of Low Alcoholic Craft Beers with Different Malt Compositions.

Beer antioxidants originate mainly from malts, classified as colored, caramel, and roasted, according to the malting process. This study aimed to characterize, in terms of phenolic antioxidants, three types of Pale Ale craft beers brewed using increasing percentage of dark malt (0, 5, and 15% Caraamber malt, called PA100, PA95, PA85, respectively) and to evaluate the impact of dealcoholization by osmotic distillation (OD) on the same antioxidants. All the alcoholic (PA, 6.2-6.8 vol %) and low alcoholic (LA-PA, 1 vol %) beers were analyzed by HPLC-ESI-MS/MS, total phenolic content (TPC), and antioxidant activity (AA): similar phenolic profiles were evidenced and 43 compounds identified or tentatively identified. Some differences were found among PA100, PA95, and PA85: PA85 was richer in free phenolic compounds (10.55 mg/L) and had a higher TPC (463.7 GAE mg/L) and AA (852.1 TE mg/L). LA-PA beers showed the same phenolic profile and similar TPC and AA compared to PA beers; however, there were some differences regarding LA-PA85 (5.91 mg/L). Dealcoholization by OD seemed to weakly affect the phenolic fraction. ESI-MS/MS infusion experiments evidenced oligosaccharides, small organic acids, and amino acids, whose presence was confirmed and quantitated by NMR: besides ethanol and other alcohols, weak to strong loss of low-molecular-weight metabolites was evidenced in LA-PA beers.

NMR Characterization of Ten Apple Cultivars from the Piedmont Region.

The metabolite profile of ten traditional apple cultivars grown in the Piedmont region (Italy) was studied by means of nuclear magnetic resonance spectroscopy, identifying an overall number of 36 compounds. A more complete assignment of the proton nuclear magnetic resonance (1H NMR) resonances from hydroalcoholic and organic apple extracts with respect to literature data was reported, identifying fructose tautomeric forms, galacturonic acid, γ-aminobutyric acid (GABA), p-coumaroyl moiety, phosphatidylcholine, and digalactosyldiacylglycerol. The chemical profile of each apple cultivar was defined by thorough quantitative NMR analysis of four sugars (fructose, glucose, sucrose, and xylose), nine organic acids (acetic, citric, formic, citramalic, lactic, malic, quinic, and galacturonic acids), six amino acids (alanine, asparagine, aspartate, GABA, isoleucine, and valine), rhamnitol, p-coumaroyl derivative, phloretin/phloridzin and choline, as well as ß-sitosterol, fatty acid chains, phosphatidylcholine, and digalactosyldiacylglycerol. Finally, the application of PCA analysis allowed us to highlight possible differences/similarities. The Magnana cultivar showed the highest content of sugars, GABA, valine, isoleucine, and alanine. The Runsé cultivar was characterized by high amounts of organic acids, whereas the Gamba Fina cultivar showed a high content of chlorogenic acid. A significant amount of quinic acid was detected in the Carla cultivar. The knowledge of apple chemical profiles can be useful for industries interested in specific compounds for obtaining ingredients of food supplements and functional foods and for promoting apple valorization and preservation.

Synthesis and Biological Evaluation of Carvacrol-Based Derivatives as Dual Inhibitors of H. pylori Strains and AGS Cell Proliferation.

This study reports on the synthesis, structural assessment, microbiological screening against several strains of H. pylori and antiproliferative activity against human gastric adenocarcinoma (AGS) cells of a large series of carvacrol-based compounds. Structural analyses consisted of elemental analysis, 1H/13C/19F NMR spectra and crystallographic studies. The structure-activity relationships evidenced that among ether derivatives the substitution with specific electron-withdrawing groups (CF3 and NO2) especially in the para position of the benzyl ring led to an improvement of the antimicrobial activity, whereas electron-donating groups on the benzyl ring and ethereal alkyl chains were not tolerated with respect to the parent compound (MIC/MBC = 64/64 µg/mL). Ester derivatives (coumarin-carvacrol hybrids) displayed a slight enhancement of the inhibitory activity up to MIC values of 8-16 µg/mL. The most interesting compounds exhibiting the lowest MIC/MBC activity against H. pylori (among others, compounds 16 and 39 endowed with MIC/MBC values ranging between 2/2 to 32/32 µg/mL against all the evaluated strains) were also assayed for their ability to reduce AGS cell growth with respect to 5-Fluorouracil. Some derivatives can be regarded as new lead compounds able to reduce H. pylori growth and to counteract the proliferation of AGS cells, both contributing to the occurrence of gastric cancer.

Chemico-Biological Characterization of Torpedino Di Fondi® Tomato Fruits: A Comparison with San Marzano Cultivar at Two Ripeness Stages.

Torpedino di Fondi (TF) is a hybrid tomato landrace developed in Sicily and recently introduced in the south Lazio area along with the classical San Marzano (SM) cultivar. The present study aimed at characterizing TF tomatoes at both pink and red ripening stages, and at comparing them with traditional SM tomatoes. A multidisciplinary approach consisting of morphological, chemical (FT-ICR MS, NMR, HPLC, and spectrophotometric methods), and biological (antioxidant and antifungal in vitro activity) analyses was applied. Morphological analysis confirmed the mini-San Marzano nature and the peculiar crunchy and solid consistency of TF fruits. Pink TF tomatoes displayed the highest content of hydrophilic antioxidants, like total polyphenols (0.192 mg/g), tannins (0.013 mg/g), flavonoids (0.204 mg/g), and chlorophylls a (0.344 mg/g) and b (0.161 mg/g), whereas red TF fruits were characterized by the highest levels of fructose (3000 mg/100 g), glucose (2000 mg/100 g), tryptophan (2.7 mg/100 g), phenylalanine (13 mg/100 g), alanine (25 mg/100 g), and total tri-unsaturated fatty acids (13% mol). Red SM fruits revealed the greatest content of lipophilic antioxidants, with 1234 mg/g of total carotenoids. In agreement with phenolics content, TF cultivar showed the greatest antioxidant activity. Lastly, red TF inhibited Candida species (albicans, glabrata and krusei) growth.

Combined Microscopic and Metabolomic Approach to Characterize the Skeletal Muscle Fiber of the Ts65Dn Mouse, A Model of Down Syndrome.

Down syndrome (DS) is a genetically based disease caused by triplication of chromosome 21. DS is characterized by severe muscle weakness associated with motor deficits; however, understanding the DS-associated skeletal muscle condition is limited. In this study, we used a combined methodological approach involving light and electron microscopy, as well as nuclear magnetic resonance spectroscopy metabolomics, to investigate morphology and composition of the quadriceps muscles in the Ts65Dn mouse, a model of DS, to identify structural and/or functional trisomy-associated alterations. Morphometric analysis demonstrated a larger size of myofibers in trisomic versus euploid mice; however, myofibrils were thinner and contained higher amounts of mitochondria and lipid droplets. In trisomic mice, magnetic resonance spectroscopy showed a tendency to an overall increase in muscle metabolites involved in protein synthesis. These data strongly suggest that in DS, a sarcoplasmic hypertrophy associated with myofibril loss characterizes quadriceps myofibers. In addition, large-sized mitochondria suggestive of impaired fission/fusion events, as well as metabolites modifications suggestive of decreased mitochondrial function, were found in the trisomic muscle. Albeit preliminary, the results provided by this novel approach consistently indicate structural and compositional alterations of the DS skeletal muscle, which are typical of early aging.

Characterization of Local Products for Their Industrial Use: The Case of Italian Potato Cultivars Analyzed by Untargeted and Targeted Methodologies.

The chemical characterization of local Italian potato cultivars is reported to promote their preservation and use as high quality raw material in food industries. Twenty potato (Solanum tuberosum L.) cultivars from Piedmont and Liguria Italian regions were investigated using NMR (Nuclear Magnetic Resonance) and RP-HPLC-PDA-ESI-MSn (Reversed Phase High-Performance Liquid Chromatography with Photodiode Array Detector and Electrospray Ionization Mass Detector) methodologies. Water soluble and lipophilic metabolites were identified and quantified. With respect to literature data, a more complete 1H (protonic) spectral assignment of the aqueous potato extracts was reported, whereas the 1H NMR assignment of potato organic extracts was reported here for the first time. Phenolics resulted to be in high concentrations in the purple-blue colored Rouge des Flandres, Bergerac, Fleur Bleu, and Blue Star cultivars. Servane, Piatlina, and Malou showed the highest amount of galacturonic acid, a marker of pectin presence, whereas Jelly cultivar was characterized by high levels of monosaccharides. Roseval and Rubra Spes contained high levels of citric acid involved in the inhibition of the enzymatic browning in fresh-cut potato. High levels of the amino acids involved in the formation of pleasant-smell volatile compounds during potato cooking were detected in Rouge des Flandres, Blue Star, Bergerac, Roseval, and Ratte cultivars. These results suggest that each local cultivar is characterized by a proper chemical profile related to specific proprieties that can be useful to obtain high quality industrial products.

Synthesis and evaluation of a large library of nitroxoline derivatives as pancreatic cancer antiproliferative agents.

Pancreatic cancer (PC) is one of the deadliest carcinomas and in most cases, which are diagnosed with locally advanced or metastatic disease, current therapeutic options are highly unsatisfactory. Based on the anti-proliferative effects shown by nitroxoline, an old urinary antibacterial agent, we explored a large library of newly synthesised derivatives to unravel the importance of the OH moiety and pyridine ring of the parent compound. The new derivatives showed a valuable anti-proliferative effect and some displayed a greater effect as compared to nitroxoline against three pancreatic cancer cell lines with different genetic profiles. In particular, in silico pharmacokinetic data, clonogenicity assays and selectivity indexes of the most promising compounds showed several advantages for such derivatives, as compared to nitroxoline. Moreover, some of these novel compounds had stronger effects on cell viability and/or clonogenic capacity in PC cells as compared to erlotinib, a targeted agent approved for PC treatment.

Cannabis sativa L. Inflorescences from Monoecious Cultivars Grown in Central Italy: An Untargeted Chemical Characterization from Early Flowering to Ripening.

The chemical composition of the inflorescences from four Cannabis sativa L. monoecious cultivars (Ferimon, Uso-31, Felina 32 and Fedora 17), recently introduced in the Lazio Region, was monitored over the season from June to September giving indications on their sensorial, pharmaceutical/nutraceutical proprieties. Both untargeted (NMR) and targeted (GC/MS, UHPLC, HPLC-PDA/FD and spectrophotometry) analyses were carried out to identify and quantify compounds of different classes (sugars, organic acids, amino acids, cannabinoids, terpenoids, phenols, tannins, flavonoids and biogenic amines). All cultivars in each harvesting period showed a THC content below the Italian legal limit, although in general THC content increased over the season. Citric acid, malic acid and glucose showed the highest content in the late flowering period, whereas the content of proline drastically decreased after June in all cultivars. Neophytadiene, nerolidol and chlorogenic acid were quantified only in Felina 32 cultivar, characterized also by a very high content of flavonoids, whereas alloaromadendrene and trans-cinnamic acid were detected only in Uso-31 cultivar. Naringenin and naringin were present only in Fedora 17 and Ferimon cultivars, respectively. Moreover, Ferimon had the highest concentration of biogenic amines, especially in July and August. Cadaverine was present in all cultivars but only in September. These results suggest that the chemical composition of Cannabis sativa L. inflorescences depends on the cultivar and on the harvesting period. Producers can use this information as a guide to obtain inflorescences with peculiar chemical characteristics according to the specific use.

Phytochemical and biological characterization of Italian "sedano bianco di Sperlonga" Protected Geographical Indication celery ecotype: A multimethodological approach.

Celery is a widely used vegetable known for its peculiar sensorial and nutritional properties. Here, the white celery (Apium graveolens L.) "sedano bianco di Sperlonga" PGI ecotype was investigated to obtain the metabolic profile of its edible parts (blade leaves and petioles) also related to quality, freshness and biological properties. A multi-methodological approach, including NMR, MS, HPLC-PDA, GC-MS and spectrophotometric analyses, was proposed to analyse celery extracts. Sugars, polyalcohols, amino acids, organic acids, phenols, sterols, fatty acids, phthalides, chlorophylls, tannins and flavonoids were detected in different concentrations in blade leaf and petiole extracts, indicating celery parts as nutraceutical sources. The presence of some phenols in celery extracts was here reported for the first time. Low contents of biogenic amines and mycotoxins confirmed celery quality and freshness. Regarding the biological properties, ethanolic celery extracts inhibited the oxidative-mediated DNA damage induced by tert-butylhydroperoxide and scavenged DPPH and ABTS radicals.

Antidepressive effects of a chemically characterized maqui berry extract (Aristotelia chilensis (molina) stuntz) in a mouse model of Post-stroke depression.

Mood disorders occur in 30% of stroke patients, and of these post-stroke depression (PSD) is the most significant. This study aimed to evaluate the antidepressive-like effects and in vivo antioxidant activity of a chemically characterized maqui berry (Aristotelia chilensis (Molina) Stuntz) extract obtained from an optimized extraction method, on a murine PSD model. The extraction process was optimized to maximize anthocyanin content, and the phytochemical profile of the extract was evaluated using a multi-methodological approach including a liquid chromatographic method coupled with mass spectrometry and nuclear magnetic resonance spectroscopy. The antidepressive-like activity was investigated through despair swimming and tail suspension tests. The in vivo antioxidant activity was evaluated in mouse brain tissue by measuring the activity of antioxidant enzymes and lipid peroxidation products. A number of compounds have been first identified in maqui berry here, including malvidin-glucoside, GABA, choline and trigonelline. Moreover, the results showed that the antidepressive-like activity exerted by the extract, which was found to restore normal mouse behavior in both despair swimming and tail suspension tests, could be linked to its antioxidant activity, leading to the conclusion that maqui berries might be useful for supporting pharmacological therapy of PSD by modulating oxidative stress.

Extra-Virgin Olive Oils from Nine Italian Regions: An ¹H NMR-Chemometric Characterization.

Extra-virgin olive oil (383 samples; EVOOs) of three consecutive harvesting years from nine Italian regions were collected and submitted to an ¹H NMR-chemometric protocol to characterize the samples according to their origin (geographical area and variety). A more complete assignment of the olive oil ¹H spectrum in CDCl3 and DMSOd6 was reported identifying 24-methylencycolartanol. A single classification model provided the discrimination of EVOOs among the three geographical macro-areas (North, Islands, Center-South), whereas a hierarchical approach based on breaking the overall classification problem into a series of smaller linear discriminant analysis (LDA) sub-models was tested to differentiate olive oils according to their geographical regions. Specific compounds responsible for olive oil characterization were identified.

Dynamic changes in ABA content in water-stressed Populus nigra: effects on carbon fixation and soluble carbohydrates.

BACKGROUND AND AIMS: Hydraulic and chemical signals operate in tandem to regulate systemic plant responses to drought. Transport of abscisic acid (ABA) through the xylem and phloem from the root to shoot has been suggested to serve as the main signal of water deficit. There is evidence that ABA and its ABA-glycosyl-ester (ABA-GE) are also formed in leaves and stems through the chloroplastic 2-C-methylerythritol-5-phosphate (MEP) pathway. This study aimed to evaluate how hormonal and hydraulic signals contribute to optimize stomatal (gs), mesophyll (gm) and leaf hydraulic (Kleaf) conductance under well-watered and water-stressed conditions in Populus nigra (black poplar) plants. In addition, we assessed possible relationships between ABA and soluble carbohydrates within the leaf and stem. METHODS: Plants were subjected to three water treatments: well-watered (WW), moderate stress (WS1) and severe stress (WS2). This experimental set-up enabled a time-course analysis of the response to water deficit at the physiological [leaf gas exchange, plant water relations, (Kleaf)], biochemical (ABA and its metabolite/catabolite quantification in xylem sap, leaves, wood, bark and roots) and molecular (gene expression of ABA biosynthesis) levels. KEY RESULTS: Our results showed strong coordination between gs, gm and Kleaf under water stress, which reduced transpiration and increased intrinsic water use efficiency (WUEint). Analysis of gene expression of 9-cis-epoxycarotenoid dioxygenase (NCED) and ABA content in different tissues showed a general up-regulation of the biosynthesis of this hormone and its finely-tuned catabolism in response to water stress. Significant linear relationships were found between soluble carbohydrates and ABA contents in both leaves and stems, suggesting a putative function for this hormone in carbohydrate mobilization under severe water stress. CONCLUSIONS: This study demonstrates the tight regulation of the photosynthetic machinery by levels of ABA in different plants organs on a daily basis in both well-watered and water stress conditions to optimize WUEint and coordinate whole plant acclimation responses to drought.

Unveiling Kiwifruit Metabolite and Protein Changes in the Course of Postharvest Cold Storage.

Actinidia deliciosa cv. Hayward fruit is renowned for its micro- and macronutrients, which vary in their levels during berry physiological development and postharvest processing. In this context, we have recently described metabolic pathways/molecular effectors in fruit outer endocarp characterizing the different stages of berry physiological maturation. Here, we report on the kiwifruit postharvest phase through an integrated approach consisting of pomological analysis combined with NMR/LC-UV/ESI-IT-MSn- and 2D-DIGE/nanoLC-ESI-LIT-MS/MS-based proteometabolomic measurements. Kiwifruit samples stored under conventional, cold-based postharvest conditions not involving the use of dedicated chemicals were sampled at four stages (from fruit harvest to pre-commercialization) and analyzed in comparison for pomological features, and outer endocarp metabolite and protein content. About 42 metabolites were quantified, together with corresponding proteomic changes. Proteomics showed that proteins associated with disease/defense, energy, protein destination/storage, cell structure and metabolism functions were affected at precise fruit postharvest times, providing a justification to corresponding pomological/metabolite content characteristics. Bioinformatic analysis of variably represented proteins revealed a central network of interacting species, modulating metabolite level variations during postharvest fruit storage. Kiwifruit allergens were also quantified, demonstrating in some cases their highest levels at the fruit pre-commercialization stage. By lining up kiwifruit postharvest processing to a proteometabolomic depiction, this study integrates previous observations on metabolite and protein content in postharvest berries treated with specific chemical additives, and provides a reference framework for further studies on the optimization of fruit storage before its commercialization.