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In recent decades, the food system has been faced with the significant problem of increasing food waste. Therefore, the feed industry, supported by scientific research, is attempting to valorise the use of discarded biomass as co-products for the livestock sector, in line with EU objectives. In parallel, the search for functional products that can ensure animal health and performances is a common fundamental goal for both animal husbandry and feeding. In this context, camelina cake (CAMC), cardoon cake (CC) and cardoon meal (CM), due valuable nutritional profile, represent prospective alternatives. Therefore, the aim of this work was to investigate the antioxidant activity of CAMC, CC and CM following in vitro digestion using 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Total phenolic content (TPC) and angiotensin converting enzyme (ACE) inhibitory activity, actively involved in modulating antioxidant properties, were also studied. Further, a peptidomic analysis was adopted to substantiate the presence of bioactive peptides after in vitro digestion. The results obtained confirmed an interesting nutritional profile of CAMC, CC and CM and relevant antioxidant and ACE inhibitory activities. In particular, considering antioxidant profile, CM and CC revealed a significantly higher (10969.80 ± 18.93 mg TE/100 g and 10451.40 ± 149.17 mg TE/100 g, respectively; p < 0.05) ABTS value than CAMC (9511.18 ± 315.29 mg TE/100 g); a trend also confirmed with the FRAP assay (306.74 ± 5.68 mg FeSO4/100 g; 272.84 ± 11.02 mg FeSO4/100 g; 103.84 ± 3.27 mg FeSO4/100 g, for CC, CM and CAMC, respectively). Similar results were obtained for TPC, demonstrating the involvement of phenols in modulating antioxidant activity. Finally, CAMC was found to have a higher ACE inhibitory activity (40.34 ± 10.11%) than the other matrices. Furthermore, potentially bioactive peptides associated with ACE inhibitory, anti-hypertensive, anti-cancer, antimicrobial, antiviral, antithrombotic, DPP-IV inhibitory and PEP-inhibitory activities were identified in CAMC. This profile was broader than that of CC and CM. The presence of such peptides corroborates the antioxidant and ACE profile of the sample. Although the data obtained report the important antioxidant profile of CAMC, CC, and CM and support their possible use, future investigations, particularly in vivo trials will be critical to evaluate and further investigate their effects on the health and performance of farm animals.

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Serra da Estrela is the oldest and most recognizable traditional protected designation of origin (PDO) cheese from Portugal. It has been extensively studied over the years, but the latest microbial characterization is 20 years old. Hence, this work aimed to perform an updated characterization of Serra da Estrela PDO cheeses and raw materials. Our analysis showed that lactic acid bacteria content on Serra da Estrela cheeses exceeded 8.8 log CFUsg-1, in all analyzed samples, with lactococci, lactobacilli and Leuconostoc spp. predominating over enterococci strains. Moreover, lactococci and lactobacilli abundance increased across the production season, while enterococci dropped considerably in late manufactures. Lastly, Leuconostoc spp. content remained unchanged in all analyzed periods. A correspondence analysis showed that L. paracasei, L. lactis, E. durans, E. faecium and L. mesenteroides are transversal in Serra da Estrela cheese manufacturing and were closely associated with milk, curd and cheese matrices. Additionally, L. casei, L. plantarum and L. curvatus were specifically associated with cheese matrices, possibly active during ripening and contributing for the development of these cheeses' organoleptic characteristics.

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The objective of the present study was to develop tofu with soybean-water soluble extract coagulated with cardoon flower (F1) and magnesium chloride (MgCl2, F2). The produced tofu was characterized in terms of physical, chemical, microbiological, and sensory properties during 14 days of storage. The yield of F1 was higher (p < 0.05) (195 g/100 soybean seeds) than F2 (162 g/100 soybean seeds). F1 presented higher moisture, protein, acidity, syneresis, and lipids when compared with F2, and a reduction of these contents during the storage. F1 presented lower hardness, stickiness, springiness, and cohesiveness compared with F2. The acceptability of F1 showed a score of 6.00 and F2 of 4.68, and the purchase intention was 3.22 for F1 and 2.23 for F2. This study recommended the use of cardoon flower at 35% level as it has great potential as a coagulant for the elaboration of tofu with higher yield, and acceptability and reasonable purchasing intention.

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The food enzyme phytepsin (EC 3.4.23.40) is extracted from the pistils of the cardoon (Cynara cardunculus L.) by different manufacturers represented by the Dirección General de Salud Pública, Gobierno de Canarias, España. It is intended to be used in milk processing for cheese production. As no concerns arose from the source of the food enzyme, from its manufacture, and based on a history of safe use and consumption, the Panel considered that toxicological data and the estimation of dietary exposure were not required. A search for the similarity of the amino acid sequences of the food enzyme to known allergens was made and no matches were found. The Panel considered that allergic reactions to this phytepsin cannot be excluded in individuals allergic to cardoon. However, the likelihood of allergic reactions to the phytepsin from C. cardunculus L. is expected not to exceed the likelihood of allergic reactions to cardoon. As the prevalence of allergic reactions to cardoon is low, also the likelihood of such reactions to occur to the food enzyme is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

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The study reports the enzymatic synthesis of bio-based oligoesters and chemo-enzymatic processes for obtaining epoxidized bioplasticizers and biolubricants starting from cardoon seed oil. All of the molecules had MW below 1000 g mol-1 and were analyzed in terms of marine biodegradation. The data shed light on the effects of the chemical structure, chemical bond lability, thermal behavior, and water solubility on biodegradation. Moreover, the analysis of the biodegradation of the building blocks that constituted the different bio-based products allowed us to distinguish between different chemical and physicochemical factors. These hints are of major importance for the rational eco-design of new benign bio-based products. Overall, the high lability of ester bonds was confirmed, along with the negligible effect of the presence of epoxy rings on triglyceride structures. The biodegradation data clearly indicated that the monomers/building blocks undergo a much slower process of abiotic or biotic transformations, potentially leading to accumulation. Therefore, the simple analysis of the erosion, hydrolysis, or visual/chemical disappearance of the chemical products or plastic is not sufficient, but ecotoxicity studies on the effects of such small molecules are of major importance. The use of natural feedstocks, such as vegetable seed oils and their derivatives, allows the minimization of these risks, because microorganisms have evolved enzymes and metabolic pathways for processing such natural molecules.

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The main interest in the valorization of vegetable wastes is due to the peculiarity of their chemical composition in substances that present important properties. Among these substances, antioxidants could replace those industrially manufactured. In the present study, three solvents of different polarities (hexane, ethanol, and water) were applied for the extraction of phenolic compounds from Cynara cardunculus L. waste using two extraction methods: Soxhlet Extraction (SE) and Ultrasonic-Assisted Extraction (UAE). The obtained extracts were then characterized by Fourier-Transform Infrared (FTIR) spectroscopy and spectrophotometric determination of Total Phenolics (TPC), Total Flavonoids (TFC), and Condensed Tannins (CT). Total Antioxidant Capacity (TAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity of ethanol and water extracts of leaves and stems were also evaluated. High extraction yields were obtained by UAE. Water extracts had high yield regardless of the technique used for leaves and stems, and these extracts showed high TAC of 534.72 ± 3.83 mg AAE/g FM for leaves and 215.70 ± 8.87 mg AAE/g FM (mg of ascorbic acid equivalent per g of FM) for stems, and IC50 of 2077.491 µg/mL for leaves and 1248.185 µg/mL for stems. We explain the latter by the high total phenolic contents (TPCs), which reach 579.375 ± 3.662 mg GAE/g FM (mg of gallic acid equivalents per g of fresh matter) for leaves and 264.906 ± 3.500 mg GAE/g FM for stems. These results confirmed that the leaves and stems of the studied cardoon waste were, indeed, interesting sources of natural antioxidants.

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The search for innovative and sustainable solutions to improve the energy efficiency of the construction industry has been a hot topic for researchers due to the tremendous impact of insulator materials in the thermal comfort of buildings. In the present work, an innovative lightweight composite material with thermal insulation properties was developed, for the first time, by using cardoon particles and polyurethane. The formulation of the composite material was optimized in terms of cardoon fraction and the polyol/isocyanate ratio, to achieve the best compromise between internal bond (IB) strength and thickness swelling (TS). The best performing composite was PU75-CP45, with 45 wt% of cardoon particles and 75% of isocyanate, achieving an IB of 0.41 MPa and a TS of 5.3%. Regarding insulation properties, the PU75-CP45 composite material exhibits a promising performance when compared to conventional construction industry materials by tuning its thickness. Additionally, the composite material presented very low emissions of volatile organic compounds and formaldehyde (bellow to legislation levels) and high resistance to biological degradation.

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The food enzyme phytepsin (EC 3.4.23.40) is extracted from the pistils of the cardoon (Cynara cardunculus L.) by seven manufacturers represented by the Regulation Council of Protected Designation of Origin Torta del Casar. It is intended to be used in milk processing for cheese production. As no concerns arose from the source of the food enzyme, from its manufacture, and based on the history of safe use and consumption, the Panel considered that toxicological data and the estimation of dietary exposure were not required. A search for similarity of the amino acid sequences of the food enzyme to known allergens was made and no matches were found. The Panel considered that allergic reactions to this phytepsin cannot be excluded in individuals allergic to this plant. However, the likelihood of allergic reactions to the phytepsin from C. cardunculus L. is expected not to exceed the likelihood of allergic reactions to cardoon. As the prevalence of allergic reactions to cardoon is low, also the likelihood of such reaction to occur to the food enzyme is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

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The food enzyme phytepsin (EC 3.4.23.40) is extracted from the pistils of the cardoon Cynara cardunculus L. by ABIASA. It is intended to be used in milk processing for cheese production. As no concerns arose from the source of the food enzyme, from its manufacture, and based on the history of safe use and consumption, the Panel considered that toxicological data and the estimation of dietary exposure were not required. The Panel considered that allergic reactions to this phytepsin cannot be excluded in individuals allergic to this plant. However, the likelihood of allergic reactions to the phytepsin from C. cardunculus L. is expected not to exceed the likelihood of allergic reactions to cardoon. As the prevalence of allergic reactions to cardoon is low, also the likelihood of such reaction to occur to the food enzyme is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

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The food enzyme phytepsin (EC 3.4.23.40) is extracted from the pistils of cardoon (Cynara cardunculus L.) by QUALIFICA/oriGIn PORTUGAL. It is intended to be used in milk processing for cheese production. As no concerns arose from the source of the food enzyme, from its manufacture, and based on the history of safe use and consumption, the Panel considered that toxicological data and the estimation of dietary exposure were not required. The Panel considered that allergic reactions to this phytepsin cannot be excluded in individuals allergic to this plant. However, the likelihood of allergic reactions to the phytepsin from C. cardunculus L. is expected not to exceed the likelihood of allergic reactions to cardoon. As the prevalence of allergic reactions to cardoon is low, also the likelihood of such reaction to occur to the food enzyme is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

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This study investigates the bioactive properties of different extracts of cardoon leaves in rescuing neuronal development arrest in an in vitro model of Rett syndrome (RTT). Samples were obtained from plants harvested at different maturity stages and extracted with two different methodologies, namely Naviglio® and supercritical carbon dioxide (scCO2). While scCO2 extracts more hydrophobic fractions, the Naviglio® method extracts phenolic compounds and less hydrophobic components. Only the scCO2 cardoon leaves extract obtained from plants harvested in spring induced a significant rescue of neuronal atrophy in RTT neurons, while the scCO2 extract from the autumn harvest stimulated dendrite outgrowth in Wild-Type (WT) neurons. The scCO2 extracts were the richest in squalene, 3ß-taraxerol and lupeol, with concentrations in autumn harvest doubling those in spring harvest. The Naviglio® extract was rich in cynaropicrin and exerted a toxic effect at 20 µM on both WT and RTT neurons. When cynaropicrin, squalene, lupeol and 3ß-taraxerol were tested individually, no positive effect was observed, whereas a significant neurotoxicity of cynaropicrin and lupeol was evident. In conclusion, cardoon leaves extracts with high content of hydrophobic bioactive molecules and low cynaropicrin and lupeol concentrations have pharmacological potential to stimulate neuronal development in RTT and WT neurons in vitro.

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In the present research study, Cynara cardunculus (wild cardoon) flowers were blended and extracted using different types of buffers (phosphate buffer; citrate buffer and distilled water) for different maceration times. The most reliable, quick and efficient buffer was found to be phosphate (pH = 6.5) with a 6-h maceration time, which was used throughout this study. C. cardunculus extract (CE) was found to have high clotting and proteolytic activities. The extracted enzyme was found to be very stable against a wide range of pH values as well as of temperature. The formation of milk gels prepared in the presence of CE with different types of milk was evaluated using dynamic rheology and Turbiscan. The evolution of both elastic (G') and viscous (G″) moduli was monitored with time. The values of the whole milk enriched with milk powder gels were higher. Coagulum stability was evaluated using Turbiscan. The textural properties and the curd-firming rate of coagulum were also determined. In conclusion, the prepared CE could be an efficient milk-clotting agent in the production of dairy products.

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The development of effective tools for the sustainable supply of phyto-ingredients and natural substances with reduced environmental footprints can help mitigate the dramatic scenario of climate change. Plant cell cultures-based biorefineries can be a technological advancement to face this challenge and offer a potentially unlimited availability of natural substances, in a standardized composition and devoid of the seasonal variability of cultivated plants. Monounsaturated (MUFA) fatty acids are attracting considerable attention as supplements for biodegradable plastics, bio-additives for the cosmetic industry, and bio-lubricants. Cardoon (Cynara cardunculus L. var. altilis) callus cultures accumulate fatty acids and polyphenols and are therefore suitable for large-scale production of biochemicals and valuable compounds, as well as biofuel precursors. With the aim of boosting their potential uses, we designed a biotechnological approach to increase oleic acid content through Agrobacterium tumefaciens-mediated metabolic engineering. Bioinformatic data mining in the C. cardunculus transcriptome allowed the selection and molecular characterization of SAD (stearic acid desaturase) and FAD2.2 (fatty acid desaturase) genes, coding for key enzymes in oleic and linoleic acid formation, as targets for metabolic engineering. A total of 22 and 27 fast-growing independent CcSAD overexpressing (OE) and CcFAD2.2 RNAi knocked out (KO) transgenic lines were obtained. Further characterization of five independent transgenic lines for each construct demonstrated that, successfully, SAD overexpression increased linoleic acid content, e.g., to 42.5%, of the relative fatty acid content, in the CcSADOE6 line compared with 30.4% in the wild type (WT), whereas FAD2.2 silencing reduced linoleic acid in favor of the accumulation of its precursor, oleic acid, e.g., to almost 57% of the relative fatty acid content in the CcFAD2.2KO2 line with respect to 17.7% in the WT. Moreover, CcSADOE6 and CcFAD2.2KO2 were also characterized by a significant increase in total polyphenolic content up to about 4.7 and 4.1 mg/g DW as compared with 2.7 mg/g DW in the WT, mainly due to the accumulation of dicaffeoyl quinic and feruloyl quinic acids. These results pose the basis for the effective creation of an engineered cardoon cells-based biorefinery accumulating high levels of valuable compounds from primary and specialized metabolism to meet the industrial demand for renewable and sustainable sources of innovative bioproducts.

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Cardoon (Cynara cardunculus var. altilis) blades were collected at sixteen sampling dates (B1-B16) to study the influence of the phenological growth stage on the phenolic composition and biological properties. Twenty phenolic compounds were identified, among which trans 3,4-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, and luteolin-O-hexoside (39.6, 42.6, and 101.0 mg/g extract, respectively) were the main compounds. Immature blades (B3) had a higher content of phenolic compounds (178 mg/g extract) and a greater ability to inhibit the formation of thiobarbituric acid reactive substances (IC50 of 1.61 µg/mL). Samples at more advanced growth stages revealed a greater capacity to inhibit oxidative hemolysis (B8, IC50 of 25 and 47.4 µg/mL for Δt of 60 and 120 min, respectively) and higher cytotoxic (B8-B13, GI50 between 7.1 and 17 µg/mL), anti-inflammatory (B13, IC50 of 10 µg/mL), and antibacterial activities. In turn, the antifungal activity varied depending on the tested fungi. All these results suggest that maturity influences the phenolic composition and bioactive properties of cardoon blades, which reveal great potential for the development of bioactive ingredients for food and pharmaceutical applications, among others.

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Cynara cardunculus (Asteraceae family) is a perennial plant native to Mediterranean regions. This plant represents a source of high-value compounds, such as polyphenols and fatty acids that have several industrial applications. However, in vitro plant cell cultures can represent a valid alternative to in-field cultivation and facilitate the extraction of metabolites of commercial interest. Generally, sucrose is the main sugar used for plant cell cultures, but other carbon sources can be considered. Here, we investigated the potential use of alternative organic carbon sources, such as galactose, maltose, glucose, glycerol, fructose, lactose, and starch, for the cultivation of C. cardunculus cells. Moreover, cardoon cells were collected, and an extraction of polyphenols and oils was performed to study the effects of different carbon sources on the production of bioactive molecules. This study provided evidence that cardoon cell growth can be supported by carbon sources other than sucrose. However, the carbon source inducing optimum growth, did not necessarily induce the highest production of high-value compounds.

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Cardoon seeds collected in Greece at four different maturity stages (samples S1 to S4) were analysed in terms of chemical composition and in vitro bioactivities. The content of phenolic compounds (six compounds in total) increased with increasing maturity, and 3,5-O-dicaffeyolquinic (14.8-33.8 mg/g extract) acid was the compound detected in higher abundance. Mature seeds (sample S4) also revealed the highest content in lipids (23 g/100 g extract) and tocopherols (29.62 mg/100 g dw) and demonstrated the highest cytotoxic (GI50 of 97-216 µg/mL) and anti-inflammatory (IC50 = 148 µg/mL) activities, and capacity to inhibit the formation of thiobarbituric acid reactive substances (TBARS) (IC50 = 5 µg/mL). Cardoon seed hydroethanolic extracts also revealed high antibacterial and antifungal potential, particularly samples S3 and S1, respectively. This study proved the multifaceted potential associated with valorisation of cardoon seeds, while their biological and chemical composition can be influenced by the maturity stage.

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Hydroethanolic extracts of cardoon petioles collected at sixteen growth stages (P1-P16) were characterized in terms of their phenolic composition and bioactive potential (antioxidant, cytotoxic, anti-inflammatory, and antimicrobial activities). Fifteen phenolic compounds were tentatively identified (i.e., ten phenolic acids and five flavonoid glycosides); the main compounds were 5-O-caffeoylquinic and 1,5-di-O-caffeoylquinic acids. Samples collected at early maturity (P1-P4) presented a weak positive correlation between the higher content in polyphenols (P3: 101-mg/g extract) and better inhibition capacity against thiobarbituric acid reactive substance formation (TBARS; P3: IC50 = 5.0 µg/mL). Samples at intermediate maturation stages (P9) presented higher cytotoxic and anti-inflammatory potential. Moreover, immature petioles showed greater antihemolytic (OxHLIA; P4: IC50 = 65 and 180 µg/mL for Δt of 60 and 120 min, respectively) and antibacterial activity. The antifungal activity varied depending on the maturation stage and the fungi strain. In conclusion, the maturation stage may greatly affect the polyphenols composition and content and the bioactive potential of cardoon petioles.

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The production of lignocellulosic ethanol calls for a robust fermentative yeast able to tolerate a wide range of toxic molecules that occur in the pre-treated lignocellulose. The concentration of inhibitors varies according to the composition of the lignocellulosic material and the harshness of the pre-treatment used. It follows that the versatility of the yeast should be considered when selecting a robust strain. This work aimed at the validation of seven natural Saccharomyces cerevisiae strains, previously selected for their industrial fitness, for their application in the production of lignocellulosic bioethanol. Their inhibitor resistance and fermentative performances were compared to those of the benchmark industrial yeast S. cerevisiae Ethanol Red, currently utilized in the second-generation ethanol plants. The yeast strains were characterized for their tolerance using a synthetic inhibitor mixture formulated with increasing concentrations of weak acids and furans, as well as steam-exploded lignocellulosic pre-hydrolysates, generally containing the same inhibitors. The eight non-diluted liquors have been adopted to assess yeast ability to withstand bioethanol industrial conditions. The most tolerant S. cerevisiae Fm17 strain, together with the reference Ethanol Red, was evaluated for fermentative performances in two pre-hydrolysates obtained from cardoon and common reed, chosen for their large inhibitor concentrations. S. cerevisiae Fm17 outperformed the industrial strain Ethanol Red, producing up to 18 and 39 g/L ethanol from cardoon and common reed, respectively, with ethanol yields always higher than those of the benchmark strain. This natural strain exhibits great potential to be used as superior yeast in the lignocellulosic ethanol plants.

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Serra da Estrela PDO cheese is the oldest traditional cheese manufactured in Portugal. In this work, its microbiome as well as the main raw materials used in cheese production, raw ewes' milk and thistle flowers (Cynara cardunculus L.), were characterized using next generation sequencing. Samples were accordingly retrieved from a local producer over two consecutive production campaigns and at different time periods within each campaign. The bacterial and fungi communities associated with each matrix were accessed through sequencing of V3-V4 and Internal Transcribed Spacer 2 regions of rRNA gene amplicons, respectively. A high microbial diversity was found associated to each matrix, differing significantly (p < 0.05) from each other. Over 500 taxa were identified in each analyzed matrix, ranging from dominant (relative abundance > 1%), sub-dominant (0.01-1%) and rare taxa (<0.01%). Specifically, in cheese, 30 taxa were present in all analyzed samples (core taxa), including species of Leuconostoc spp. and Lactococcus spp. for bacteria and Candida spp., Debaryomyces spp. and Yarrowia spp. for fungi, that were cumulatively the most prevalent genera in Serra da Estrela PDO cheese (average relative abundance ≥10%). Ultimately, this characterization study may contribute to a better understanding of the microbial dynamics of this traditional PDO product, namely the influence of raw materials on cheese microbiome, and could assist producers interested in preserving the identity, quality and safety of Serra da Estrela PDO cheese.

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The contamination of environments by heavy metals has become an urgent issue causing undesirable accumulations and severe damages to agricultural crops, especially cadmium and lead which are among the most widespread and dangerous metal pollutants worldwide. The selection of proper species is a crucial step in many plant-based restoration approaches; therefore, the aim of the present work was to check for early morphophysiological responsive traits in three cultivars of Cynara cardunculus (Sardo, Siciliano, and Spagnolo), helping to select the best performing cultivar for phytoremediation. For all three tested cultivars, our results indicate that cardoon displays some morphophysiological traits to face Cd and Pb pollution, particularly at the root morphology level, element uptake ability, and photosynthetic pigment content. Other traits show instead a cultivar-specific behavior; in fact, stomata plasticity, photosynthetic pattern, and antioxidant power provide different responses, but only Spagnolo cv. achieves a successful strategy attaining a real resilience to metal stress. The capacity of Spagnolo plants to modify leaf structural and physiological traits under heavy metal contamination to maintain high photosynthetic efficiency should be considered an elective trait for its use in contaminated environments.

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