Beyond the Visible and Below the Peel: How UV-B Radiation Influences the Phenolic Profile in the Pulp of Peach Fruit. A Biochemical and Molecular Study

In the last decades, UV-B radiation has attracted attention due to its potential to increase nutraceutical values of fruit and vegetables, especially by inducing the accumulation of phenolics in a structure-dependent way. However, most current studies have investigated the UV-B-driven changes only in the peel or focusing on individual phenolic classes. Adopting an \u201c-omics\u201d approach, this work aimed to deepen the knowledge about the effects of UV-B radiation on the phenolic profile in the pulp of peach fruit. Based on these considerations, melting flesh yellow peaches (Prunus persica L., cv. Fairtime) were subjected to either a 10- or 60-min UV-B treatment (1.39 and 8.33 kJ m\u20132, respectively), and sampled at different time points from the exposure. A UHPLC-ESI/QTOF-MS analysis coupled with a phenolics-specific database for the annotation of compounds and a multivariate discriminant analysis revealed a marked effect of UV-B radiation on the phenolic profiles of peach pulp. Particularly, a general, transient increase was observed after 24 h from the irradiation, especially for flavanols, flavonols, and flavones. Such behavior diverges from what was observed in the peel, where an overall increase of phenolics was observed after 36 h from the irradiation. Concerning the flavonols in the pulp, UV-B exposure stimulated a specific accumulation of isorhamnetin and kaempferol derivatives, with variations imposed by the different sugar moiety bound. Anthocyanins, which were the second most abundant flavonoid group after flavonols, displayed a general decrease after 36 h that was not attributable to specific molecules. The UV-B treatments also increased the glycoside/aglycone ratio of flavonols and anthocyanins after 24 h, by increasing the glycoside concentration of both, flavonols and anthocyanins, and decreasing the aglycone concentration of anthocyanins. In support of the biochemical results, targeted gene expression analysis by RT-qPCR revealed an UV-B-induced activation of many genes involved in the flavonoid pathway, e.g., CHS, F3H, F3\u2032H, DFR, as well as some MYB transcription factors and few genes involved in the UV-B perception. Generally, all the flavonoid-related and MYB genes showed a transient UV-B dose-dependent activation after 6 h from the irradiation, similarly to what was observed in the peel

A Microbial-Based Biostimulant Enhances Sweet Pepper Performance by Metabolic Reprogramming of Phytohormone Profile and Secondary Metabolism

Microbial-based biostimulants can improve crop productivity by modulating cell metabolic pathways including hormonal balance. However, little is known about the microbial-mediated molecular changes causing yield increase. The present study elucidates the metabolomic modulation occurring in pepper (Capsicum annuum L.) leaves at the vegetative and reproductive phenological stages, in response to microbial-based biostimulants. The arbuscular mycorrhizal fungi Rhizoglomus irregularis and Funneliformis mosseae, as well as Trichoderma koningii, were used in this work. The application of endophytic fungi significantly increased total fruit yield by 23.7% compared to that of untreated plants. Multivariate statistics indicated that the biostimulant treatment substantially altered the shape of the metabolic profile of pepper. Compared to the untreated control, the plants treated with microbial biostimulants presented with modified gibberellin, auxin, and cytokinin patterns. The biostimulant treatment also induced secondary metabolism and caused carotenoids, saponins, and phenolic compounds to accumulate in the plants. Differential metabolomic signatures indicated diverse and concerted biochemical responses in the plants following the colonization of their roots by beneficial microorganisms. The above findings demonstrated a clear link between microbial-mediated yield increase and a strong up-regulation of hormonal and secondary metabolic pathways associated with growth stimulation and crop defense to environmental stresses

Foliar application of different vegetal-derived protein hydrolysates distinctively modulates tomato root development and metabolism

Despite the scientific evidence supporting their biostimulant activity, the molecular mechanism(s) underlying the activity of protein hydrolysates (PHs) and the specificity among different products are still poorly explored. This work tested five different protein hydrolysates, produced from different plant sources using the same enzymatic approach, for their ability to promote rooting in tomato cuttings following quick dipping. Provided that all the different PHs increased root length (45\u201393%) and some of them increased root number (37\u201356%), untargeted metabolomics followed by multivariate statistics and pathway analysis were used to unravel the molecular processes at the basis of the biostimulant activity. Distinct metabolomic signatures could be found in roots following the PHs treatments. In general, PHs shaped the phytohormone profile, modulating the complex interaction between cytokinins and auxins, an interplay playing a pivotal role in root development, and triggered a down accumulation of brassinosteroids. Concerning secondary metabolism, PHs induced the accumulation of aliphatic glucosinolates, alkaloids, and phenylpropanoids, potentially eliciting crop resilience to stress conditions. Here, we confirm that PHs may have a hormone-like activity, and that their application can modulate plant growth, likely interfering with signaling processes. Noteworthy, the heterogenicity of the botanical origin supported the distinctive and peculiar metabolomic responses we observed across the products tested. While supporting their biostimulant activity, these findings suggest that a generalized crop response to PHs cannot be defined and that specific effects are rather to be investigated

Foliar and root comparative metabolomics and phenolic profiling of Micro-Tom tomato (Solanum lycopersicum L.) plants associated with a gene expression analysis in response to short daily UV treatments

Tomato (Solanum lycopersicum L.) is globally recognised as a high-value crop both for commercial profit and nutritional benefits. In contrast to the extensive data regarding the changes in the metabolism of tomato fruit exposed to UV radiation, less is known about the foliar and root metabolome. Using an untargeted metabolomic approach through UHPLC-ESI-QTOF-MS analysis, we detected thousands of metabolites in the leaves (3000) and roots (2800) of Micro-Tom tomato plants exposed to 11 days of short daily UV radiation, applied only on the aboveground organs. Multivariate statistical analysis, such as OPLS-DA and volcano, were performed to allow a better understanding of the modifications caused by the treatment. Based on the unexpected modulation to the secondary metabolism, especially the phenylpropanoid pathway, of which compounds were down and up accumulated respectively in leaves and roots of treated plants, a phenolic profiling was carried out for both organs. The phenolic profile was associated with a gene expression analysis to check the transcription trend of genes involved in the UVR8 signalling pathway and the early steps of the phenolic biosynthesis. The retention of the modifications at metabolic and phenolic levels was also investigated 3 days after the UV treatment, showing a prolonged effect on the modulation once the UV treatment had ceased

Nutraceutical Profiles of Two Hydroponically Grown Sweet Basil Cultivars as Affected by the Composition of the Nutrient Solution and the Inoculation With Azospirillum brasilense

Sweet basil (Ocimum basilicum L.) is one of the most produced aromatic herbs in the world, exploiting hydroponic systems. It has been widely assessed that macronutrients, like nitrogen (N) and sulfur (S), can strongly affect the organoleptic qualities of agricultural products, thus influencing their nutraceutical value. In addition, plant-growth-promoting rhizobacteria (PGPR) have been shown to affect plant growth and quality. Azospirillum brasilense is a PGPR able to colonize the root system of different crops, promoting their growth and development and influencing the acquisition of mineral nutrients. On the bases of these observations, we aimed at investigating the impact of both mineral nutrients supply and rhizobacteria inoculation on the nutraceutical value on two different sweet basil varieties, i.e., Genovese and Red Rubin. To these objectives, basil plants have been grown in hydroponics, with nutrient solutions fortified for the concentration of either S or N, supplied as SO42\u2013 or NO3\u2013, respectively. In addition, plants were either non-inoculated or inoculated with A. brasilense. At harvest, basil plants were assessed for the yield and the nutraceutical properties of the edible parts. The cultivation of basil plants in the fortified nutrient solutions showed a general increasing trend in the accumulation of the fresh biomass, albeit the inoculation with A. brasilense did not further promote the growth. The metabolomic analyses disclosed a strong effect of treatments on the differential accumulation of metabolites in basil leaves, producing the modulation of more than 400 compounds belonging to the secondary metabolism, as phenylpropanoids, isoprenoids, alkaloids, several flavonoids, and terpenoids. The primary metabolism that resulted was also influenced by the treatments showing changes in the fatty acid, carbohydrates, and amino acids metabolism. The amino acid analysis revealed that the treatments induced an increase in arginine (Arg) content in the leaves, which has been shown to have beneficial effects on human health. In conclusion, between the two cultivars studied, Red Rubin displayed the most positive effect in terms of nutritional value, which was further enhanced following A. brasilense inoculation

A combined phenotypic and metabolomic approach for elucidating the biostimulant action of a plant-derived protein hydrolysate on tomato grown under limited water availability

Plant-derived protein hydrolysates (PHs) are an important category of biostimulants able to increase plant growth and crop yield especially under environmental stress conditions. PHs can be applied as foliar spray or soil drench. Foliar spray is generally applied to achieve a relatively short-term response, whereas soil drench is used when a long-term effect is desired. The aim of the study was to elucidate the biostimulant action of PH application method (foliar spray or substrate drench) on morpho-physiological traits and metabolic profile of tomato grown under limited water availability. An untreated control was also included. A high-throughput image-based phenotyping (HTP) approach was used to non-destructively monitor the crop response under limited water availability (40% of container capacity) in a controlled environment. Moreover, metabolic profile of leaves was determined at the end of the trial. Dry biomass of shoots at the end of the trial was significantly correlated with number of green pixels (R2 = 0.90) and projected shoot area, respectively. Both drench and foliar treatments had a positive impact on the digital biomass compared to control while the photosynthetic performance of the plants was slightly influenced by treatments. Overall drench application under limited water availability more positively influenced biomass accumulation and metabolic profile than foliar application. Significantly higher transpiration use efficiency was observed with PH-drench applications indicating better stomatal conductance. The mass-spectrometry based metabolomic analysis allowed the identification of distinct biochemical signatures in PH-treated plants. Metabolomic changes involved a wide and organized range of biochemical processes that included, among others, phytohormones (notably a decrease in cytokinins and an accumulation of salicylates) and lipids (including membrane lipids, sterols, and terpenes). From a general perspective, treated tomato plants exhibited an improved tolerance to reactive oxygen species (ROS)-mediated oxidative imbalance. Such capability to cope with oxidative stress might have resulted from a coordinated action of signaling compounds (salicylic acid and hydroxycinnamic amides), radical scavengers such as carotenoids and prenyl quinones, as well as a reduced biosynthesis of tetrapyrrole coproporphyrins

Metabolomic, proteomic and physiological insights into the potential mode of action of thymol, a phytotoxic natural monoterpenoid phenol: The phytotoxic effect of thymol on adult plants of A. thaliana

Thymol is a natural phenolic monoterpene widely produced by different species belonging to the Labiateae family. Although the thymol phytotoxicity is well known, the knowledge of its potential toxic mechanism is still limited. In this regard, the model species Arabidopsis thaliana was treated for 16 days by sub-irrigation with 300 μM of thymol. The results confirmed the high phytotoxic potential of this phenolic compound, which caused a reduction in plant growth and development. Thymol induced a water status alteration accompanied by an increase in ABA content and stomatal closure. Furthermore, leaves appeared necrotic in the margins and their temperature rinsed. The increase in H2O2 content suggested an oxidative stress experienced by treated plants. Both metabolomic and proteomic analysis confirmed this hypothesis showing a strong increase in osmoprotectants content, such as galactinol and proline, and a significant up-accumulation of proteins involved in ROS detoxification. Furthermore, the down-accumulation of proteins and pigments involved in the photosynthetic machinery, the increase in light sensitivity and the lower PSII efficiency well indicated a reduction in photosynthetic activity. Overall, we can postulate that thymol-induced phytotoxicity could be related to a combined osmotic and oxidative stress that resulted in reduced plant development

Metabolomic Responses of Maize Shoots and Roots Elicited by Combinatorial Seed Treatments With Microbial and Non-microbial Biostimulants

Microbial and non-microbial plant biostimulants have been successfully used to improve agriculture productivity in a more sustainable manner. Since the mode of action of biostimulants is still largely unknown, the present work aimed at elucidating the morpho-physiological and metabolomic changes occurring in maize (Zea mays L.) leaves and roots following seed treatment with (i) a consortium of two beneficial fungi [arbuscular mycorrhizal fungi (AMF) and Trichoderma koningii TK7] and rhizobacteria, (ii) a protein hydrolyzate-based biostimulant (PH) alone, or (iii) in combination with a consortium of T. koningii TK7 and rhizobacteria. The application of PH alone or in combination with Trichoderma elicited significant increases (+16.6%) in the shoot biomass compared to untreated maize plants, whereas inoculation with AMF + Trichoderma elicited significant increases in root dry biomass (+48.0%) compared to untreated plants. Distinctive metabolomic signatures were achieved from the different treatments, hence suggesting that different molecular processes were involved in the plants response to the biostimulants. The metabolic reprogramming triggered by the treatments including the protein hydrolyzate was hierarchically more pronounced than the application of microorganisms alone. Most of the differential metabolites could be ascribed to the secondary metabolism, with phenylpropanoids and terpenes being the most represented compounds. The application of PH triggered an accumulation of secondary metabolites, whereas the opposite trend of accumulation was seen in the case of microorganisms alone. The increase in biomass could be related to two processes, namely the modulation of the multilayer phytohormone interaction network and a possible increase in nitrogen use efficiency via the GS-GOGAT system

Bioactivity and bioavailability of phytoene and strategies to improve its production

Phytoene is a valuable colourless carotenoid, which is used as an ingredient in nutraceuticals as well as in cosmetic products. Its use in formulations has several advantages over other carotenoids due to its stability, photo-insensitivity and long shelf life. In addition, phytoene has beneficial effects on human health because it is an antioxidant which inhibits lipoprotein oxidation and protects against UVB light, while some studies have suggested that it could also have anticancer activity and could decrease cholesterol levels. For these reasons, the demand for phytoene has increased, and new strategies that will allow the production of this bioactive compound in high amounts are needed. The use of in vitro cultures of plants, algae and microorganisms has been suggested as a biotechnological strategy to obtain phytoene. In addition, many tools and strategies are available for metabolic engineering that will allow increasing phytoene to be increased using a variety of in vivo systems. The source of the phytoene biosynthetic pathway genes used, the design of the construction to express the same and the host strains used, among other factors, can modify the efficiency of the process to obtain phytoene in high amounts. This review focuses on the strategies used to enhance the production of phytoene, using in vitro cultures of plants, algae and microorganisms. Special attention is paid to increasing the production of phytoene using metabolic engineering strategies

Effect of terbinafine on the biosynthetic pathway of isoprenoid compounds in carrot suspension cultured cells

Key message: Terbinafine induced a significant increase of squalene production. Terbinafine increased the expression levels of squalene synthase. Cyclodextrins did not work as elicitors due to the gene expression levels obtained. Abstract: Plant sterols are essential components of membrane lipids, which contributing to their fluidity and permeability. Besides their cholesterol-lowering properties, they also have anti-inflammatory, antidiabetic and anticancer activities. Squalene, which is phytosterol precursor, is widely used in medicine, foods and cosmetics due to its anti-tumor, antioxidant and anti-aging activities. Nowadays, vegetable oils constitute the main sources of phytosterols and squalene, but their isolation and purification involve complex extraction protocols and high costs. In this work, Daucus carota cell cultures were used to evaluate the effect of cyclodextrins and terbinafine on the production and accumulation of squalene and phytosterols as well as the expression levels of squalene synthase and cycloartenol synthase genes. D. carota cell cultures were able to produce high levels of extracellular being phytosterols in the presence of cyclodextrins (12 mg/L), these compounds able to increase both the secretion and accumulation of phytosterols in the culture medium. Moreover, terbinafine induced a significant increase in intracellular squalene production, as seen after 168 h of treatment (497.0 ± 23.5 µg g dry weight−1) while its extracellular production only increased in the presence of cyclodextrins.The analysis of sqs and cas gene expression revealed that cyclodextrins did not induce genes encoding enzymes involved in the phytosterol biosynthetic pathway since the expression levels of sqs and cas genes in cyclodextrin-treated cells were lower than in control cells. The results, therefore, suggest that cyclodextrins were only able to release phytosterols from the cells to the extracellular medium, thus contributing to their acumulation. To sum up, D. carota cell cultures treated with cyclodextrins or terbinafine were able to produce high levels of phytosterols and squalene, respectively, and, therefore, these suspension-cultured cells of carrot constitute an alternative biotechnological system, which is at the same time more sustainable, economic and ecological for the production of these bioactive compounds