Multi-Omics Exploration of ABA Involvement in Identifying Unique Molecular Markers for Single and Combined Stresses in tomato plants.

Over the past decade, our research group has found that plant responses to combined abiotic stresses are unique and cannot be inferred from studying plants exposed to individual stresses. Understanding how adaptative plant mechanisms integrate from stress perception to biochemical and physiological adjustments is a major challenge in abiotic stress signaling studies. Considering abscisic acid (ABA) as a key regulator in plant abiotic stress responses, in our study, ABA-deficient plants (flc) exposed to single or combined salinity and heat stresses were evaluated and different -omics analyses were conducted. Significant changes in biomass, photosynthesis, ions, transcripts, and metabolites occurred in mutant plants under single or combined stresses. Exogenous ABA application in flc mutants did not fully recover plant phenotypes or metabolic levels but induced cellular reprogramming with changes in specific markers. Multi-omics analysis aimed to identify ABA-dependent, ABA-independent, or stress-dependent markers in plant responses to single or combined stresses. We demonstrated that studying different -omics as a whole led to the identification of specific markers for each stress condition that were not detectable when each -omic was studied individually. This resource article provides an important and novel reference for scientists working in the field of plant abiotic stress. Future exploration of the transcriptomic, ionomic and metabolomic data presented in this study could lead to the identification of new pathways and genes associated with ABA signaling processes. These findings may be utilized to enhance crop resilience to heat waves, salinity, and their combination, contributing to addressing food security challenges in a climate change scenario.

Exploring the Potential of Invasive Species Sargassum muticum: Microwave-Assisted Extraction Optimization and Bioactivity Profiling.

Sargassum muticum (SM) poses a serious environmental issue since it is a fast-expanding invasive species occupying key areas of the European shoreline, disrupting the autochthonous algae species, and disturbing the ecosystem. This problem has concerned the general population and the scientific community. Nevertheless, as macroalgae are recognized as a source of bioactive molecules, the abundance of SM presents an opportunity as a raw material. In this work, response surface methodology (RSM) was applied as a tool for the optimization of the extraction of bioactive compounds from SM by microwave-assisted extraction (MAE). Five different parameters were used as target functions: yield, total phenolic content (TPC); and the antioxidant measurements of 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and ß-carotene bleaching (BC). After the optimal extraction conditions were determined (time = 14.00 min; pressure = 11.03 bar; ethanol = 33.31%), the chemical composition and bioactivity of the optimum extract was evaluated to appraise its antioxidant capability to scavenge reactive species and as a potential antibacterial, antidiabetic, antiproliferation, and neuroprotective agent. The results lead to the conclusion that MAE crude extract has bioactive properties, being especially active as an antiproliferation agent and as a nitric oxide and superoxide radical scavenger.

Unraveling the Bioactive Potential of Camellia japonica Edible Flowers: Profiling Antioxidant Substances and In Vitro Bioactivity Assessment.

In recent years, the search for novel natural-based ingredients by food and related industries has sparked extensive research aimed at discovering new sources of functional molecules. Camellia japonica, traditionally known as an ornamental plant, has gained attention due to its diverse array of bioactive compounds with potential industrial applications. Although C. japonica flowers are edible, their phytochemical profile has not been thoroughly investigated. In this study, a phenolic profile screening through an HPLC-ESI-QQQ-MS/MS approach was applied to C. japonica flower extracts, revealing a total of 36 compounds, including anthocyanins, curcuminoids, dihydrochalcones, dihydroflavonols, flavonols, flavones, hydroxybenzoic acids, hydroxycinnamic acids, isoflavonoids, stilbenes, and tyrosols. Following extract profiling, their bioactivity was assessed by means of in vitro antioxidant, antimicrobial, cytotoxic, and neuroprotective activities. The results showed a multifaceted high correlation of phenolic compounds with all the tested bioactivities according to Pearson's correlation analysis, unraveling the potential of C. japonica flowers as promising sources of nutraceuticals. Overall, these findings provide insight into the valorization of C. japonica flowers from different unexplored cultivars thus diversifying their industrial outcome.

Integrated multi-omic approach reveals the effect of a Graminaceae-derived biostimulant and its lighter fraction on salt-stressed lettuce plants.

Plant biostimulants are widely applied in agriculture for their ability to improve plant fitness. In the present work, the impact of Graminaceae-derived protein hydrolysate (P) and its lighter molecular fraction F3 (< 1 kDa) on lettuce plants, subjected to either no salt or high salt conditions, was investigated through the combination of metabolomics and transcriptomics. The results showed that both treatments significantly modulated the transcriptome and metabolome of plants under salinity stress, highlighting an induction of the hormonal response. Nevertheless, P and F3 also displayed several peculiarities. F3 specifically modulated the response to ethylene and MAPK signaling pathway, whereas P treatment induced a down-accumulation of secondary metabolites, albeit genes controlling the biosynthesis of osmoprotectants and antioxidants were up-regulated. Moreover, according with the auxin response modulation, P promoted cell wall biogenesis and plasticity in salt-stressed plants. Notably, our data also outlined an epigenetic control of gene expression induced by P treatment. Contrarily, experimental data are just partially in agreement when not stressed plants, treated with P or F3, were considered. Indeed, the reduced accumulation of secondary metabolites and the analyses of hormone pathways modulation would suggest a preferential allocation of resources towards growth, that is not coherent with the down-regulation of the photosynthetic machinery, the CO2 assimilation rate and leaves biomass. In conclusion, our data demonstrate that, although they might activate different mechanisms, both the P and F3 can result in similar benefits, as far as the accumulation of protective osmolytes and the enhanced tolerance to oxidative stress are concerned. Notably, the F3 fraction exhibits slightly greater growth promotion effects under high salt conditions. Most importantly, this research further corroborates that biostimulants' mode of action is dependent on plants' physiological status and their composition, underscoring the importance of investigating the bioactivity of the different molecular components to design tailored applications for the agricultural practice.

Peculiarity of the early metabolomic response in tomato after urea, ammonium or nitrate supply.

Nitrogen (N) is the nutrient most applied in agriculture as fertilizer (as nitrate, Nit; ammonium, A; and/or urea, U, forms) and its availability strongly constrains the crop growth and yield. To investigate the early response (24 h) of N-deficient tomato plants to these three N forms, a physiological and molecular study was performed. In comparison to N-deficient plants, significant changes in the transcriptional, metabolomic and ionomic profiles were observed. As a probable consequence of N mobility in plants, a wide metabolic modulation occurred in old leaves rather than in young leaves. The metabolic profile of U and A-treated plants was more similar than Nit-treated plant profile, which in turn presented the lowest metabolic modulation with respect to N-deficient condition. Urea and A forms induced some changes at the biosynthesis of secondary metabolites, amino acids and phytohormones. Interestingly, a specific up-regulation by U and down-regulation by A of carbon synthesis occurred in roots. Along with the gene expression, data suggest that the specific N form influences the activation of metabolic pathways for its assimilation (cytosolic GS/AS and/or plastidial GS/GOGAT cycle). Urea induced an up-concentration of Cu and Mn in leaves and Zn in whole plant. This study highlights a metabolic reprogramming depending on the N form applied, and it also provide evidence of a direct relationship between urea nutrition and Zn concentration. The understanding of the metabolic pathways activated by the different N forms represents a milestone in improving the efficiency of urea fertilization in crops.

Exploring the bioaccessibility of polyphenols and glucosinolates from Brassicaceae microgreens by combining metabolomics profiling and computational chemometrics.

Microgreens constitute natural-based foods with health-promoting properties mediated by the accumulation of glucosinolates (GLs) and phenolic compounds (PCs), although their bioaccessibility may limit their nutritional potential. This work subjected eight Brassicaceae microgreens to in vitro gastrointestinal digestion and large intestine fermentation before the metabolomics profiling of PCs and GLs. The application of multivariate statistics effectively discriminated among species and their interaction with in vitro digestion phases. The flavonoids associated with arugula and the aliphatic GLs related to red cabbage and cauliflower were identified as discriminant markers among microgreen species. The multi-omics integration along in vitro digestion and fermentation predicted bioaccessible markers, featuring potential candidates that may eventually be responsible for these functional foods' nutritional properties. This combined analytical and computational framework provided a promising platform to predict the nutritional metabolome-wide outcome of functional food consumption, as in the case of microgreens.

Unravelling the biostimulant activity of a protein hydrolysate in lettuce plants under optimal and low N availability: a multi-omics approach.

The application of protein hydrolysates (PH) biostimulants is considered a promising approach to promote crop growth and resilience against abiotic stresses. Nevertheless, PHs bioactivity depends on both the raw material used for their preparation and the molecular fraction applied. The present research aimed at investigating the molecular mechanisms triggered by applying a PH and its fractions on plants subjected to nitrogen limitations. To this objective, an integrated transcriptomic-metabolomic approach was used to assess lettuce plants grown under different nitrogen levels and treated with either the commercial PH Vegamin® or its molecular fractions PH1(>10 kDa), PH2 (1-10 kDa) and PH3 (<1 kDa). Regardless of nitrogen provision, biostimulant application enhanced lettuce biomass, likely through a hormone-like activity. This was confirmed by the modulation of genes involved in auxin and cytokinin synthesis, mirrored by an increase in the metabolic levels of these hormones. Consistently, PH and PH3 upregulated genes involved in cell wall growth and plasticity. Furthermore, the accumulation of specific metabolites suggested the activation of a multifaceted antioxidant machinery. Notwithstanding, the modulation of stress-response transcription factors and genes involved in detoxification processes was observed. The coordinated action of these molecular entities might underpin the increased resilience of lettuce plants against nitrogen-limiting conditions. In conclusion, integrating omics techniques allowed the elucidation of mechanistic aspects underlying PH bioactivity in crops. Most importantly, the comparison of PH with its fraction PH3 showed that, except for a few peculiarities, the effects induced were equivalent, suggesting that the highest bioactivity was ascribable to the lightest molecular fraction.

A metabolomics perspective on the effect of environmental micro and nanoplastics on living organisms: A review.

The increasing trend regarding the use of plastics has arisen an exponential concern on the fate of their derived products to the environment. Among these derivatives, microplastics and nanoplastics (MNPs) have been featured for their associated environmental impact due to their low molecular size and high surface area, which has prompted their ubiquitous transference among all environmental interfaces. Due to the heterogenous chemical composition of MNPs, the study of these particles has focused a high number of studies, as a result of the myriad of associated physicochemical properties that contribute to the co-transference of a wide range of contaminants, thus becoming a major challenge for the scientific community. In this sense, both primary and secondary MNPs are well-known to be adscribed to industrial and urbanized areas, from which they are massively released to the environment through a multiscale level, involving the atmosphere, hydrosphere, and lithosphere. Consequently, much research has been conducted on the understanding of the interconnection between those interfaces, that motivate the spread of these contaminants to biological systems, being mostly represented by the biosphere, especially phytosphere and, finally, the anthroposphere. These findings have highlighted the potential hazardous risk for human health through different mechanisms from the environment, requiring a much deeper approach to define the real risk of MNPs exposure. As a result, there is a gap of knowledge regarding the environmental impact of MNPs from a high-throughput perspective. In this review, a metabolomics-based overview on the impact of MNPs to all environmental interfaces was proposed, considering this technology a highly valuable tool to decipher the real impact of MNPs on biological systems, thus opening a novel perspective on the study of these contaminants.

Untargeted metabolomics and machine learning unveil quality and authenticity interactions in grated Parmigiano Reggiano PDO cheese.

The chemical composition of Parmigiano Reggiano (PR) hard cheese can be significantly affected by different factors across the dairy supply chain, including ripening, altimetric zone, and rind inclusion levels in grated hard cheeses. The present study proposes an untargeted metabolomics approach combined with machine learning chemometrics to evaluate the combined effect of these three critical parameters. Specifically, ripening was found to exert a pivotal role in defining the signature of PR cheeses, with amino acids and lipid derivatives that exhibited their role as key discriminant compounds. In parallel, a random forest classifier was used to predict the rind inclusion levels (> 18%) in grated cheeses and to authenticate the specific effect of altimetry dairy production, achieving a high prediction ability in both model performances (i.e., ∼60% and > 90%, respectively). Overall, these results open a novel perspective to identifying quality and authenticity markers metabolites in cheese.

Phytochemical compounds with promising biological activities from Ascophyllum nodosum extracts using microwave-assisted extraction.

Phytochemical-rich antioxidant extracts were obtained from Ascophyllum nodosum (AN) using microwave-assisted extraction (MAE). Critical extraction factors such as time, pressure, and ethanol concentration were optimized by response surface methodology with a circumscribed central composite design. Under the optimal MAE conditions (3 min, 10.4 bar, 46.8 % ethanol), the maximum recovery of phytochemical compounds (polyphenols and fucoxanthin) with improved antioxidant activity from AN was obtained. In addition, the optimized AN extract showed significant biological activities as it was able to scavenge reactive oxygen and nitrogen species, inhibit central nervous system-related enzymes, and exhibit cytotoxic activity against different cancer cell lines. In addition, the optimized AN extract showed antimicrobial, and anti-quorum sensing activities, indicating that this extract could offer direct and indirect protection against infection by pathogenic microorganisms. This work demonstrated that the sustainably obtained AN extract could be an emerging, non-toxic, and natural ingredient with potential to be included in different applications.

The addition of polysaccharide gums to Aronia melanocarpa purees modulates the bioaccessibility of phenolic compounds and gut microbiota: A multiomics data fusion approach following in vitro digestion and fermentation.

This study aimed to determine how the addition of gellan, guar, locust bean, and xanthan gums affected the polyphenol profile of Aronia melanocarpa puree and the human gut microbiota after in vitro gastrointestinal digestion and large intestine fermentation. The different gums distinctively affected the content and bioaccessibility of phenolics in Aronia puree, as outlined by untargeted metabolomics. The addition of locust bean gum increased the levels of low-molecular-weight phenolics and phenolic acids after digestion. Gellan and guar gums enhanced phenolic acids' bioaccessibility after fermentation. Interactions between digestion products and fecal bacteria altered the composition of the microbiota, with the greatest impact of xanthan. Locust bean gum promoted the accumulation of different taxa with health-promoting properties. Our findings shed light on the added-value properties of commercial gums as food additives, promoting a distinctive increase of polyphenol bioaccessibility and shifting the gut microbiota distribution, depending on their composition and structural features.

Exploring the Interplay between Metabolic Pathways and Taxane Production in Elicited Taxus baccata Cell Suspensions.

Taxus cell cultures are a reliable biotechnological source of the anticancer drug paclitaxel. However, the interplay between taxane production and other metabolic pathways during elicitation remains poorly understood. In this study, we combined untargeted metabolomics and elicited Taxus baccata cell cultures to investigate variations in taxane-associated metabolism under the influence of 1 µM coronatine (COR) and 150 µM salicylic acid (SA). Our results demonstrated pleiotropic effects induced by both COR and SA elicitors, leading to differential changes in cell growth, taxane content, and secondary metabolism. Metabolite annotation revealed significant effects on N-containing compounds, phenylpropanoids, and terpenoids. Multivariate analysis showed that the metabolomic profiles of control and COR-treated samples are closer to each other than to SA-elicited samples at different time points (8, 16, and 24 days). The highest level of paclitaxel content was detected on day 8 under SA elicitation, exhibiting a negative correlation with the biomarkers kauralexin A2 and taxusin. Our study provides valuable insights into the intricate metabolic changes associated with paclitaxel production, aiding its potential optimization through untargeted metabolomics and an evaluation of COR/SA elicitor effects.

Impact of Elicitation on Plant Antioxidants Production in Taxus Cell Cultures.

Elicited cell cultures of Taxus spp. are successfully used as sustainable biotechnological production systems of the anticancer drug paclitaxel, but the effect of the induced metabolomic changes on the synthesis of other bioactive compounds by elicitation has been scarcely studied. In this work, a powerful combinatorial approach based on elicitation and untargeted metabolomics was applied to unravel and characterize the effects of the elicitors 1 µM of coronatine (COR) or 150 µM of salicylic acid (SA) on phenolic biosynthesis in Taxus baccata cell suspensions. Differential effects on cell growth and the phenylpropanoid biosynthetic pathway were observed. Untargeted metabolomics analysis revealed a total of 83 phenolic compounds, mainly flavonoids, phenolic acids, lignans, and stilbenes. The application of multivariate statistics identified the metabolite markers attributed to elicitation over time: up to 34 compounds at 8 days, 41 for 16 days, and 36 after 24 days of culture. The most notable metabolic changes in phenolic metabolism occurred after 8 days of COR and 16 days of SA elicitation. Besides demonstrating the significant and differential impact of elicitation treatments on the metabolic fingerprint of T. baccata cell suspensions, the results indicate that Taxus ssp. biofactories may potentially supply not only taxanes but also valuable phenolic antioxidants, in an efficient optimization of resources.

The impact of metallic nanoparticles on gut fermentation processes: An integrated metabolomics and metagenomics approach following an in vitro digestion and fecal fermentation model.

Metallic nanoparticles (MNPs) are becoming widespread environmental contaminants. They are currently added to several food preparations and cause a fast-growing concern for human health. The present work aims to assess the impact of zinc oxide (ZnO), titanium dioxide (TiO2), and silver (Ag) nanoparticles (NPs) on the human gut metabolome and microbiome. Water samples spiked with two different concentrations of each MNPs were subjected to in-vitro gastrointestinal digestion and in-vitro large intestine fermentation. The effects of the treatments were determined through 16 S amplicon sequencing and untargeted metabolomics. Multi-omics data integration was then applied to correlate the two datasets. MNPs treatments modulated the microbial genera Bifidobacterium, Sutterella, Escherichia and Bacteroides. The treatments, especially the lower concentrations of Ag and ZnO, caused modulation of indole derivatives, peptides, and metabolites related to protein metabolism in the large intestine. Notably, these metabolites are implicated in ulcerative colitis and inflammatory bowel disease. TiO2 NPs treatment in all concentrations increased E.coli relative abundance and decreased the abundance of B. longum. Moreover, for TiO2, an enrichment in proinflammatory lipid mediators of arachidonic acid metabolites, such as prostaglandin E2 and leukotrienes B4, was detected. For all metals except TiO2, low NP concentrations promoted differentiated profiles, thus suggesting that MNPs aggregation can limit adverse effects on living cells.

The functional implications of high-amylose wholegrain wheat flours: An in vitro digestion and fermentation approach combined with metabolomics.

Wheat flour is one of the most prevalent foodstuffs for human consumption, and novel strategies are underway to enhance its nutritional properties. This work evaluated wholegrain flours from bread wheat lines with different amylose/amylopectin ratios through in vitro starch digestion and large intestine fermentation. High-amylose flours presented a higher resistant starch content and lower starch hydrolysis index. Moreover, UHPLC-HRMS metabolomics was carried out to determine the profile of the resulting in vitro fermentates. The multivariate analysis highlighted distinctive profiles between the flours derived from the different lines compared to the wild type. Peptides, glycerophospholipids, polyphenols, and terpenoids were identified as the main markers of the discrimination. The high-amylose flour fermentates showed the richest bioactive profile, containing stilbenes, carotenoids, and saponins. Present findings pave the way toward applying high-amylose flours to design novel functional foods.

Algal nutraceuticals: A perspective on metabolic diversity, current food applications, and prospects in the field of metabolomics.

The current consumers' demand for food naturalness is urging the search for new functional foods of natural origin with enhanced health-promoting properties. In this sense, algae constitute an underexplored biological source of nutraceuticals that can be used to fortify food products. Both marine macroalgae (or seaweeds) and microalgae exhibit a myriad of chemical constituents with associated features as a result of their primary and secondary metabolism. Thus, primary metabolites, especially polysaccharides and phycobiliproteins, present interesting properties to improve the rheological and nutritional properties of food matrices, whereas secondary metabolites, such as polyphenols and xanthophylls, may provide interesting bioactivities, including antioxidant or cytotoxic effects. Due to the interest in algae as a source of nutraceuticals by the food and related industries, novel strategies should be undertaken to add value to their derived functional components. As a result, metabolomics is considered a high throughput technology to get insight into the full metabolic profile of biological samples, and it opens a wide perspective in the study of algae metabolism, whose knowledge is still little explored. This review focuses on algae metabolism and its applications in the food industry, paying attention to the promising metabolomic approaches to be developed aiming at the functional characterization of these organisms.

Phenolic acids, lignans, and low-molecular-weight phenolics exhibit the highest in vitro cellular bioavailability in different digested and faecal-fermented phenolics-rich plant extracts.

Polyphenols are multifaceted bioactive compounds, but little is known about their real impact on human health after consumption. In this work, the phenolic profiling of quebracho, yellow maize, and violet rice extracts was comprehensively investigated, together with the impact of in vitro digestion and colonic fermentation on the bioaccessibility and bioavailability of these phytochemicals. The different matrices showed distinct profiles, potentially influencing in vitro starch digestion under cooking conditions. Furthermore, after the extracts underwent in vitro gastrointestinal digestion and faecal fermentation, phenolics exhibited a differential bioaccessibility trend at every digestion level, with matrix-dependent behaviour. The bioavailability results suggest that polyphenols are metabolised during colonic fermentation, mainly into tyrosols, phenolic acids, and lignans, which are partially absorbed by Caco-2 cells. By combining metabolomics with in vitro cellular methods, this research provides new insights into the fate of these phytochemicals in the gut, yielding comprehensive data on their consumption in food matrices.

Bioactive compounds, health benefits, and industrial applications of Tartary buckwheat (Fagopyrum tataricum).

Tartary buckwheat belongs to the family Polygonaceae, which is a traditionally edible and medicinal plant. Due to its various bioactive compounds, the consumption of Tartary buckwheat is correlated to a wide range of health benefits, and increasing attention has been paid to its potential as a functional food. This review summarizes the main bioactive compounds and important bioactivities and health benefits of Tartary buckwheat, emphasizing its protective effects on metabolic diseases and relevant molecular mechanisms. Tartary buckwheat contains a wide range of bioactive compounds, such as flavonoids, phenolic acids, triterpenoids, phenylpropanoid glycosides, bioactive polysaccharides, and bioactive proteins and peptides, as well as D-chiro-inositol and its derivatives. Consumption of Tartary buckwheat and Tartary buckwheat-enriched products is linked to multiple health benefits, e.g., antioxidant, anti-inflammatory, antihyperlipidemic, anticancer, antidiabetic, antiobesity, antihypertensive, and hepatoprotective activities. Especially, clinical studies indicate that Tartary buckwheat exhibits remarkable antidiabetic activities. Various tartary buckwheat -based foods presenting major health benefits as fat and blood glucose-lowering agents have been commercialized. Additionally, to address the safety concerns, i.e., allergic reactions, heavy metal and mycotoxin contaminations, the quality control standards for Tartary buckwheat and its products should be drafted and completed in the future.

Different fractions from wheat flour provide distinctive phenolic profiles and different bioaccessibility of polyphenols following in vitro digestion.

Wheat is a prevalent food worldwide, although its production arises several tonnes of industrial by-products that merit valorisation, owing to sustainable demands. In this work, an untargeted metabolomics approach was applied to shed light on the profile of free and bound phenolic compounds in white and wholegrain flours, as well as the major wheat by-products, namely wheat bran, wheat shorts and wheat middlings. By-products and wholegrain flours were found to be rich sources of ferulic acid derivatives, lignans, and alkylresorcinols, mostly bound to wheat fibre components, whereas different flavonoids were found in their free forms. Afterwards, the in vitro gastrointestinal digestion of these matrices showed that flour phenolics, in particular lignans, were more bioaccessible than those from wheat by-products. These results support the carrier effect attributed to dietary fibre and open a wide perspective on the use of underexploited wheat by-products to formulate novel functional foods.

UHPLC-QTOF-HRMS metabolomics insight on the origin and processing authentication of thyme by comprehensive fingerprinting and chemometrics.

The metabolic composition of thyme, one of the most used aromatic herbs, is influenced by environmental and post-harvest processing factors, presenting the possibility of exploiting thyme fingerprint to assess its authenticity. In this study, a comprehensive UHPLC-QTOF-HRMS fingerprinting approach was applied with a dual objective: (1) tracing thyme from three regions of production (Spain, Morocco, and Poland) and (2) evaluating the metabolic differences in response to processing, considering sterilized thyme samples. Multivariate statistics reveal 37 and 33 key origin and processing differentiation compounds, respectively. The findings highlighted the remarkable "terroir" influence on thyme fingerprint, noticing flavonoids, amino acids, and peptides among the most discriminant chemical classes. Thyme sterilization led to an overall metabolite enrichment, most likely due to the facilitated compound accessibility as a result of processing. The findings provide a comprehensive metabolomics insight into the origin and processing effect on thyme composition for product traceability and quality assessment.