Cynara Cardunculus Research Articles

Long-term cardoon (Cynara cardunculus) productivity under reduced irrigation and weed control inputs

ABSTRACT The long-term productivity data for the energy crops under reduced inputs are decisive for farmers’ decisions. An 11-year field study was conducted in central Greece to evaluate the productivity of two cardoon varieties under reduced irrigation and weed control inputs. During the initial six plant stage ages, the cardoon total dry biomass, seed, oil and potential energy yields, averaged 21.65, 1.92 and 0.53 t ha−1, as well as 364.719 GJ ha−1, respectively, with the var. C12, in most cases, the most productive. Both conditions of non-irrigation and weed competition caused a slight reduction in cardoon yield components. However, during the next five plant stage ages, the corresponding yields were dramatically reduced and averaged 11.10, 0.52 and 0.15 t ha−1, as well as 185.153 GJ ha−1, mainly due to reduced regrowth and the shorter height of cardoon stems. In this period, irrigation and weed management did not affect in most cases cardoon productivity. So, the under-reduced inputs cultivation of cardoon after the sixth year may not be economically acceptable for farmers. Conclusively, cardoon can be cultivated under reduced inputs, at least for 6 years, constituting one of the eligible energy crops for farmers in the hot and semi-arid Mediterranean fields.

Bioguided isolation of potential antitumor agents from the aerial parts of cultivated cardoon (Cynara cardunculus var. altilis)

Hepatocellular carcinoma (HCC) is one of the leading causes of mortality worldwide; therefore, searching for an effective treatment for this illness is of great importance. In the present work,in vitrocytotoxic activity of the ethanol extract of the aerial parts ofCynara cardunculusL. against human liver carcinoma cells (Hep G2) was tested. Additionally, the antitumor activity of the extract was confirmed using chemically induced rat liver carcinogenesis with diethylnitrosamine (DEN). Moreover, bioguided fractionation and column chromatographic separation of the active compounds were carried out. The extract of C. cardunculus showed a promising cytotoxic activity according to the protocols of the National Cancer Institute. Bioguided chromatographic separation of the ethanol extract of C. cardunculus led to the isolation of seven secondary metabolites including two sesquiterpene lactones as the principal active components of the methylene chloride soluble fraction, grosheimin (IC50 = 7.49 µg/mL) and cynaropicrin (IC50 = 13.9 µg/mL). The compounds were characterized by different spectroscopic techniques such as EI-MS, IR and NMR. Additionally, in silico analysis of the two active compounds revealed their ability to bind with caspase-3 via hydrogen bonds interactions to initiate apoptosis of cancer cells. The results shed the light on the significance of C. cardunculus as a potential source of antitumor agents.

Trends in scientific literature on the environmental sustainability of the artichoke (Cynara cardunculus L. spp.) supply chain

PurposeThe research provides a systematic literature review on the environmental sustainability of the globe artichoke [Cynara cardunculus L. var. scolymus (L.) Fiori], in the cultivated cardoon [Cynara cardunculus L. var. altilis DC.] and in the wild cardoon [Cynara cardunculus L. var. sylvestris (Lamk)] supply chains, to fill in the literature data gaps and to identify new research directions.Design/methodology/approachIn the light of the PRISMA standard guidelines, the research provides a systematic literature review according to six research strings on Scopus and Web of Science. 45 scientific articles have been selected out of 407 contributions. Data have been synthesized according to a co-word analysis through the VOSviewer software, to provide insights into the structure of the research network, to offer a multidimensional scaling and clustering into research groups and to discuss the results.FindingsThe research identifies five main research trends: (1) biomass-to-bioenergy or biomass-to-biocomposite materials; (2) waste-to-bioenergy or waste-to-bioproducts; (3) crop management to increase yield productivity; (4) environmental impacts assessment; (5) irrigation water management. Besides, the research highlights momentous challenges and adopted strategies to tackle climate change and to reduce natural resources consumption, as well as the nexus between circular economy and environmental impact assessment.Originality/valueThe novelty of this study relies on the fact that it analyses the environmental sustainability of the Cynara cardunculus L. spp. Supply chain in a systematic way, giving the opportunity to identify future research directions regarding the environmental impacts associated with agricultural production and industrial transformation.

Gibberellin treatments increase the global performance of globe artichoke hybrid propagated by seeds and suckers

• GA 3 applications increase the global performance of an artichoke hybrid propagated from both seeds and suckers. • The year affects more the productivity, commercial quality and caffeoylquinic acids content followed by the type of propagation. • GA 3 treatments have strong impact on productive and chemical variables. • An adequate combination of propagation type and GA 3 treatments for the artichoke cultivation enhance its production. In the globe artichoke ( Cynara cardunculus var . scolymus ), the application of gibberellins substitutes, partially or totally, the cold required for the entry into production. The needed amount of gibberellic acid depends on the genotype and consequently it would affect the product quality. So, the effect of gibberellic acid (GA 3 ) application in the globe artichoke hybrid Madrigal F 1 multiplied by seeds was evaluated in the course of two years. Seed and sucker propagations were compared for precocity, productivity performance, commercial quality and caffeoylquinic acids content in the second year. One application of GA 3 (50 ppm, 30 mL) was enough to produce an increase of active compounds in leaves of plants obtained from suckers. With two applications, the content of active principles increased in the edible part of plants propagated by seeds. With three applications, fibre content and browning decreased in the edible part of all plants, while yield increased and precocity was maintained. Both univariate and multivariate analyses demonstrated that the year affected most of the variables (78%) followed by the type of multiplication (61%). GA 3 treatments and the interactions affected 46% of the variables. These results would be useful for producers, manufacturers and consumers to achieve better globe artichoke products, since GA 3 applications increase the global performance of hybrid Madrigal F 1 propagated from both seeds and suckers.

Life Cycle Assessment of Cynara cardunculus L. -Based Polygeneration and Biodiesel Chains

Given the current scenario of increasing environmental problems associated with the need for rapid energy transition, this article aimed to investigate the implementation of Cynara cardunculus L. (cardoon), a plant with high environmental performance, as a source of energy resources. This study presented thLife Cycle Assessment of two energy production chains—for the polygeneration of power, heat, and cooling; and to produce biodiesel—fed with cardoon seeds, in addition to comparing these chains with the use of the traditional Italian grids (power and gas) and with the production of diesel based on palm, soybean, and rapeseed. Approximately 49 t of seeds were cultivated and processed, yielding 8.5 t of oil. The system boundaries encompass three main macro-phases, cardoon production, oil extraction, and, depending on the application, energy generation (polygeneration) or transesterification (biodiesel). The models were developed using the software SimaPro V9.3.0.2, and the inventory was based on the database ecoinvent V3.8. The Life Cycle Impact Assessment was performed using the ReCiPe V1.06 method at the midpoint (H) and endpoint (H/A) levels. Crude cardoon oil showed a global warming of 20–55% lower than other vegetable oils (palm, soybean, and rapeseed). In the case of biodiesel production, cardoon-based biodiesel presented a reduction in the impact burden by 12–57% compared to biodiesel based on palm, soybean, and rapeseed. With the use of oil in Polygeneration, a subtle increase in the impact burden was observed, with 13% more impact than the use of Italian power and gas grids.

Valorisation, Green Extraction Development, and Metabolomic Analysis of Wild Artichoke By-Product Using Pressurised Liquid Extraction UPLC-HRMS and Multivariate Data Analysis.

Valorisation of food by-products has recently attracted considerable attention due to the opportunities to improve the economic and environmental sustainability of the food production chain. Large quantities of non-edible parts of the artichoke plant (Cynara cardunculus L.) comprising leaves, stems, roots, bracts, and seeds are discarded annually during industrial processing. These by-products contain many phytochemicals such as dietary fibres, phenolic acids, and flavonoids, whereby the most challenging issue concerns about the recovery of high-added value components from these by-products. The aim of this work is to develop a novel valorisation strategy for the sustainable utilisation of artichoke leaves' waste, combining green pressurised-liquid extraction (PLE), spectrophotometric assays and UPLC-HRMS phytochemical characterization, to obtain bioactive-rich extract with high antioxidant capacity. Multivariate analysis of the major selected metabolites was used to compare different solvent extraction used in PLE.

New Insights into the Role of Alternating Temperatures and Cyanide in the ROS-Mediated Cardoon Seed Dormancy Termination

Physiological dormancy in wild cardoon (Cynara cardunculus var. sylvestris) can be terminated by achenes exposure to alternating temperatures, likely with the participation of reactive oxygen species (ROS). Cyanide is a natural compound that mediates seed dormancy removal in some plant species in association with oxidative signalling exerted by ROS. To date, no study has been conducted on the cyanide effect on ROS homeostasis during the germination of cardoon. Here, we showed that the addition of cyanide at low concentrations in dormant cardoon achenes promotes dormancy breakage at a constant temperature, speeds up germination to alternating temperatures and promotes ROS accumulation in embryonic axes of dormant achenes. The in-silico transcriptome analysis showed that the expression levels of transcripts of genes associated with ROS signalling and production, calcium signalling, gibberellins biosynthesis and cell wall loosening were significantly up-regulated at the alternating temperatures imbibition condition. In contrast, the expression of gene transcripts associated with the inhibition of germination, ABA biosynthesis and signalling were up-regulated at the constant temperature imbibition. However, no significant difference in lipid peroxidation or protein carbonylation levels was observed when achenes were imbibed at constant or alternating temperature conditions. These results suggest that dormancy termination triggered by alternating temperatures or cyanide could be mediated by ROS production and signalling in the cardoon embryonic axis, but this does not determine extensive protein carbonylation.

Microbiological, morpho-textural, and volatile characterization of Portuguese Queijo de Nisa PDO cheese

Queijo de Nisa PDO (Protected Designation of Origin) is a semi-hard cheese obtained from raw ewe's milk clotted with cardoon (Cynara cardunculus, L.) rennet. The aim of the present study was to characterize the bacterial communities naturally occurring in Queijo de Nisa PDO cheese samples through viable counting and metataxonomic analysis. Moreover, physico-chemical and morpho-textural analyses were also performed, together with the analysis of volatile organic compounds (VOCs) through solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC–MS). In the analyzed samples, pH values were comprised between 4.84 and 5.74. As for lactic acid, the concentration varied between 0.83 and 2.10 g 100 g-1. Water activity varied between 0.942 and 0.960. Lightness of the samples ranged from 107.82 to 119.16, whereas hardness ranged between 34.45 N and 126.05 N. As for microbiological analyses, lactococci and lactobacilli counts were up to 9.01 Log CFU g-1. Coagulase-negative cocci and enterococci counts were up to 7 Log CFU g-1. Metataxonomic analysis revealed that Lactococcus lactis and Leuconostoc mesenteroides occurred at high frequency in all the analyzed samples. Moreover, Lactiplantibacillus plantarum, Lactococcus piscium, and Lacticaseibacillus zeae were also detected. The most represented VOCs were carboxylic acids, carbonyl compounds, alcohols, and esters. In the analyzed cheese samples, significant relationships between bacteria and VOCs were also evidenced. Of note, research on the microbiological and chemical features, as well as on morpho-textural characteristics, of Queijo de Nisa PDO cheese can increase the knowledge on the interactions between raw materials, environment, and autochthonous microorganisms, thus enabling protection of biodiversity and tradition.

Extraction, Identification and Quantification of Bioactive Compounds from Globe Artichoke (Cynara cardunculus var. scolymus)

This study looked at the best conditions for extracting bioactive compounds from globe artichoke (Cynara cardunculus var. scolymus), such as total phenolics, flavonoids, ascorbic acid, and inulin. The obtained results showed that the optimum conditions for extraction of total phenolics and flavonoids from receptacle and bracts of artichoke, when applying maceration at room temperature, with 70% methanol for 4 h. Genstin was the major phenolic compound (57.86 and 25.6 mg/100 g DM) in artichoke receptacle and bracts, respectively. The highest content of ascorbic acid extracted from artichoke parts was obtained using 1% citric acid solution at 25 °C. In addition, the optimum conditions for extraction of inulin were autoclave at 120 °C for 15 min. The most abundant essential amino acids were aromatic amino acids (phenylalanine and tyrosine), followed by valine, lysine, and leucine but sulfur amino acids were the limited amino acids found in the artichoke parts. Our results also suggested that bioactive compounds from artichoke extracts might have a promising future in the management of oxidative stress on the gastrointestinal tract and recommended as an attractive ingredient for developing functional food.

Upgrading cardoon biomass into Polyhydroxybutyrate based blends: A holistic approach for the synthesis of biopolymers and additives

Cardoon, Cynara cardunculus L. represents a biorefinery crop with a great potential in the bioplastic field. This work investigates the valorization of different cardoon components into high added value products, finally recombined into novel upgraded bioplastics. Bioprocesses for Polyhydroxybutyrate (PHB) and medium-chain-length Polyhydroxyalkanoates (mcl-PHA) production were set up starting from root inulin and seed oil respectively, highlighting the effect of process conditions on polymer properties. The ternary blend, in which the PHB polymer matrix was added with mcl-PHA and epoxidized cardoon oil, evidenced a synergic effect of both additives in modulating PHB structural and thermal properties, promoted by the physical interaction occurring among the components. This proof-of concept frames the paper in the holistic approach of circular economy applied to bioplastic production.

Antimicrobial evaluation of the Cynara cardunculus extract in Helicobacter pylori cells using mid-infrared spectroscopy and chemometric methods

A preliminary assay, using both therapeutic-based antibiotics (clarithromycin and metronidazole), was conducted to optimize experimental conditions in terms of the sensibility of the Fourier-transform mid-infrared (MIR-FTIR) spectroscopy associated with chemometric methods. Principal component analysis was applied to understand how the Cynara extract concentration acts differentially against H. pylori bacteria. The partial least squares model, characterized by R2 =0.98, and root mean square error cross-validation, 0.011, was developed for the spectral regions (3600-2500 cm-1 and 2000-698 cm-1 ). MIR-FTIR spectroscopy associated with chemometric methods can be considered a suitable approach to discover and analyse the promissory antimicrobial agents based on the biomolecular changes observed according to the Cynara extract. MIR-FTIR spectroscopy and chemometric methods allowed to register the biomolecular changes due to the potential antimicrobial drugs at reduced concentrations comparatively to the conventional assay based on an agar-dilution method, being considered a useful approach to develop a platform to discover new bioactive molecules, allowing to reduce time and costs related to the exploratory step.

Antiproliferative Effects of Cynara Cardunculus in Colorectal Cancer Cells Are Modulated by the Circadian Clock.

The circadian clock generates 24 h rhythms in behavioural, cellular and molecular processes. Malfunctions of the clock are associated with enhanced susceptibility to cancer, worse treatment response and poor prognosis. Clock-controlled genes are involved in cellular processes associated with tumour development and progression including metabolism of drugs and the cell cycle. Cynara cardunculus, a plant of the Asteraceae family, has been reported to have antiproliferative effects on breast cancer cells. Here, we used the human colorectal cancer (CRC) cell line HCT116 and its knockout variants for different core-clock genes (BMAL1, PER2, NR1D1), to investigate the treatment effect of C. cardunculus lipophilic leaf extract under different clock scenarios. Our results show a direct effect of C. cardunculus on the circadian phenotype of the cells, as indicated by alterations in the phase, amplitude, and period length of core-clock gene oscillations. Furthermore, our data indicate a role for the circadian clock in sensitivity to C. cardunculus treatment. In particular, the treatment inhibited proliferation and induced cytotoxicity and apoptosis in a clock knockout-specific manner, in CRC cells. These results point to a potential effect of C. cardunculus lipophilic leaf extracts as a modulator of the circadian clock, in addition to its anti-proliferative properties.

Preparation and characterization of activated carbons from cardoon “Cynara Cardunculus” waste: Application to the adsorption of synthetic organic dyes

There is a growing interest in the adsorption of pollutants on biosorbents, especially those of plant origin. This study deals with the development of two activated carbons based on Cardoon leaf waste and their application as adsorbents of synthetic organic dyes. The activated carbons obtained were characterized by physicochemical methods and complementary analyses. The different adsorption experiments were followed by varying the mass of adsorbent, the contact time, the pH of the medium, the initial dye concentration and the salinity of the medium. The obtained results indicate that the adsorption equilibrium is reached after a contact time of 75 min for Crystal Violet and 105 min for Congo Red. The adsorption rates remain proportional to the mass of each adsorbent. The influence of the ionic strength is negligible for both dyes. Cardoon leaf waste could be a potential precursor for the preparation of ecological carbon for the reduction or elimination of organic pollutants.

Caffeoylquinic acids and flavones profile in Cynara cardunculus L. seedlings under controlled conditions as affected by light and water-supply treatments

• A standard, cheap and valuable production system of health-promoting compounds was proposed. • Water-supply and light role on Cynara cardunculus seedling polyphenols was studied. • Full water-supply increases the polyphenol compounds amount than water-stressed ones. • The trend 0 < 12< 24 h was found for all phytochemical compounds. • The polyphenol profile was genotype-dependent. Cynara cardunculus L. is an Asteraceae member widely diffused in the Mediterranean Basin, rich in phenolic acids (caffeoylquinic acids and derivatives), flavones (luteolin, apigenin and their conjugates), anthocyanins, inulin and vitamin C. Thanks to their numerous biological activities, these compounds are in high demand for food and no-food applications. To match such request, in this research we evaluated the effect of three water-supply (100%, 75% and 50% of plant requirement) and light (24 h, 12 h and 0 h) treatments on the polyphenols profile of C. cardunculus seedlings, with the aim of developing a production system under controlled conditions. Overall, the 100% of plant water requirement increased the amount of caffeoylquinic acids (+28%), luteolines (+27%) and total measured polyphenols (+26%) respect to water-stressed plants (75% and 50% of plant requirement), with cultivated cardoon showing a higher concentration than the globe artichoke. Concerning the light treatment, the trend 0 < 12< 24 h was found for all phytochemical compounds. In particular, 24 h of light strongly induced the biosynthesis of caffeoylquinic acids (+119%), luteolines (+273%) and total measured polyphenols (+129%) compared to 0 h of light. In both experiments, the most abundant compounds were 5- O -caffeoylquinic acid and 1,5- O -dicaffeoylquinic acid. Regardless of experiment, the genetic background showed a significant role, since the responses were genotype-dependent. From these results clearly emerged the possibility of producing polyphenols-enriched C. cardunculus seedlings in controlled conditions.

Hydromulching Enhances the Growth of Artichoke (Cynara cardunculus var. scolymus) Plants Subjected to Drought Stress through Hormonal Regulation of Source–Sink Relationships

Mulching the soil with organic-based formulations (hydromulching) is a sustainable alternative to plastic mulching that is here hypothesized to maintain crop production under drought stress by hormonal and metabolic regulation of source–sink relationships. To test this hypothesis, artichoke plants were grown on non-mulched soil and on soil mulched with polyethylene and three different organic mixtures, and subjected to optimal and reduced irrigation regimes. Under drought stress, the growth parameters were higher in plants grown with the different mulching treatments compared to non-mulched plants, which was related to a higher photosynthetic rate and water-use efficiency. Importantly, mulching-associated growth improvement under stress was explained by higher sucrolytic activity in the leaves that was accompanied by a decline in the active cytokinins. Besides this, salicylic acid decreased in the leaves, and abscisic acid and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid were impaired in the artichoke heads, which is associated with better regulation of photoassimilate partitioning. Taken together, these results help to explain the hydromulching-associated growth improvement of artichokes under water stress through the hormonal regulation of sucrose metabolism, which could be very useful in future breeding programs for drought tolerance.

Cynara cardunculus as a potential source of milk coagulating protease: Effects on physical properties of cow's milk.

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.

Effects of Callus Cell Suspension Cultures and Elicitor Applications on Bioactive Components in Globe Artichoke [Cynara cardunculus var. scolymus (L.) Fiori

Globe artichoke [Cynara cardunculus var. scolymus (L.) Fiori] has many health-protecting properties due to its valuable bioactive components which are not stable and produced in high amounts in the raw plant material in nature. These bioactive components that gaining interest can be increased thanks to the contribution of valuable in vitro techniques, such as callus and cell suspension cultures, and various new applications such as elicitor treatments. Present study aimed to determined bioactive components in three globe artichoke cultivars by using callus cell suspension cultures in various media combinations and by applying two elicitor treatments, namely methyl jasmonate and chitosan, at 3 different concentrations (methyl jasmonate 50 µM, 100 µM, and 200 µM; chitosan 200 mg L-1, 400 mg L-1, and 800 mg L-1) with 3 different application durations (24h, 48h, and 72h). The bioactive compounds profile of cultivars was determined by HPLC-DAD. Obtained results clearly revealed that using well-balanced concentrations of auxin: cytokinin (1:1 or 10:1) in a media composition is a must for triggering the callus formation process for globe artichoke. Results also showed that accumulated bioactive components and their amounts varied based on cultivars. Experiment results revealed that different types of elicitors other than methyl jasmonate and chitosan, or different doses of elicitors and application durations should be used/tested to get desired levels of bioactive components. The findings of the present study may play a supportively and complementarily mission in several important fields such as agriculture, and pharmaceutical engineering.

Organoleptic Chemical Markers of Serpa PDO Cheese Specificity.

Serpa is a protected designation of origin cheese produced with a vegetable coagulant (Cynara cardunculus L.) and raw ovine milk. Despite the unique sensory profile of raw milk cheeses, numerous parameters influence their sensory properties and safety. To protect the Serpa cheese quality and contribute to unifying their distinctive features, some rheologic and physicochemical parameters of cheeses from four PDO producers, in distinct seasons and with different sensory scores, were monitored. The results suggested a high chemical diversity and variation according to the dairy, month and season, which corroborates the significant heterogeneity. However, a higher incidence of some compounds was found: a group of free amino acids (Glu, Ala, Leu, Val and Phe), lactic and acetic acids, some volatile fatty acids (e.g., iC4, iC5, C6 and C12) and esters (e.g., ethyl butanoate, decanoate and dodecanoate). Through the successive statistical analysis, 13 variables were selected as chemical markers of Serpa cheese specificity: C3, C4, iC5, C12, Tyr, Trp, Ile, 2-undecanone, ethyl isovalerate, moisture content on a fat-free basis, the nitrogen-fractions (maturation index and non-protein and total nitrogen ratio) and G’ 1 Hz. These sensory markers’ identification will be essential to guide the selection and development of an autochthonous starter culture to improve cheese quality and safety issues and maintain some of the Serpa authenticity.

Membrane fractionation of Cynara cardunculus swine blood hydrolysate: Ingredients of high nutritional and nutraceutical value

This work proposes an innovative approach to valorise swine blood based on enzymatic hydrolysis and membrane fractionations. Hydrolysis with Cynara cardunculus enzymes, followed by microfiltration and double nanofiltration generated three high protein fractions, retentate of microfiltration (RMF; >0.5 µm) and retentate of nanofiltration (RNF; >3 kDa) with approximately 90% of protein on a dry basis and filtrate of nanofiltrate (FNF; <3 kDa) with 65%. FNF, rich in low molecular weight peptides, showed excellent antioxidants (ABTS and ORAC of 911.81 and 532.82 µmol TE g−1 DB, respectively) and antihypertensive (IC50 of 28.51 µg mL−1) potential. By peptidomics and in silico analysis, 43 unique sequences of interest were found, among which LVV-Hemorphin-7 was identified. This hemorphin was demonstrated as the main responsible for the observed bioactivity. Complementary results showed a prebiotic effect mainly for the growth of Bifidobacterium animalis Bo, as well as interesting free amino acids (mainly glutamic acid, leucine, alanine, phenylalanine and aspartic acid) and mineral (e.g., Ca, Mg, P, K and Na) profiles. No antibacterial effect was verified for the seven pathogenic bacteria tested. This study allowed obtaining new ingredient of high nutritional and nutraceutical value for human consumption, with a perspective of sustainability and industrial viability.

Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing.

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.