Overview of transcriptome changes and phenomic profile of sanitized artichoke vis-à-vis non-sanitized plants.

Plant tissue in vitro culture is increasingly used in agriculture to improve crop production, nutritional quality, and commercial value. In plant virology, the technique is used as sanitation protocol to produce virus-free plants. Sanitized (S) artichokes show increased vigour compared to their non-sanitized (NS) counterparts, because viral infections lead to a decline of growth and development. To investigate mechanisms that control the complex traits related to morphology, growth, and yield in S artichokes compared to NS plants, RNAseq analysis and phenotyping by imaging were used. The role of peroxidases (POD) was also investigated to understand their involvement in sanitized plant development. Results showed that virus infection affected regulation of cell cycle, gene expression and signal transduction modulating cellular response to stimulus/stress. Moreover, primary metabolism and photosynthesis were also influenced, contributing to explain the main morphological differences observed between S and NS artichokes. Sanitized artichokes are also characterized by higher POD activity, probably associated with increased plant growth, rather than strengthening of cell walls. Overall, results show that the differences in development of S artichokes may be derived from the in vitro culture stressor, as well as through pathogen elimination, which, in turn, improve qualitative and quantitative artichoke production.

The Dark Side of the pollen: BSA-seq identified genomic regions linked to male sterility in globe artichoke.

Globe artichoke (Cynara cardunculus var. scolymus; 2n = 2x = 34) is a food crop consumed for its immature flower heads. Traditionally, globe artichoke varietal types are vegetatively propagated. However, seed propagation makes it possible to treat the crop as annual, increasing field uniformity and reducing farmers costs, as well as pathogens diffusion. Despite globe artichoke's significant agricultural value and the critical role of heterosis in the development of superior varieties, the production of hybrids remains challenging without a reliable system for large-scale industrial seed production. Male sterility (MS) presents a promising avenue for overcoming these challenges by simplifying the hybridization process and enabling cost-effective seed production. However, within the Cynara genus, genic male sterility has been linked to three recessive loci in globe artichoke, with no definitive genetic mechanism elucidated to date. A 250 offsprings F2 population, derived from a cross between a MS globe artichoke and a male fertile (MF) cultivated cardoon (C. cardunculus var. altilis) and fitting a monogenic segregation model (3:1), was analyzed through BSA-seq, aiming at the identification of genomic regions/genes affecting male sterility. Four QTL regions were identified on chromosomes 4, 12, and 14. By analyzing the sequence around the highest pick on chromosome 14, a cytochrome P450 (CYP703A2) was identified, carrying a deleterious substitution (R/Q) fixed in the male sterile parent. A single dCAPS marker was developed around this SNP, allowing the discrimination between MS and MF genotypes within the population, suitable for applications in plant breeding programs. A 3D model of the protein was generated by homology modeling, revealing that the mutated amino acid is part of a highly conserved motif crucial for protein folding.

Identification, Sequencing, and Molecular Analysis of RNA2 of Artichoke Italian Latent Virus Isolates from Known Hosts and a New Host Plant Species.

Despite its first description in 1977 and numerous reports of its presence in various plant species in many countries, the molecular information available in GenBank for artichoke Italian latent virus (AILV) is still limited to a single complete genome sequence (RNA1 and 2) of a grapevine isolate (AILV-V) and a partial portion of the RNA2 sequence from an isolate of unknown origin and host. Here, we report the results of molecular analyses conducted on the RNA2 of some AILV isolates, sequenced for the first time in this study, together with the first-time identification of AILV in a new host plant species, namely chard (Beta vulgaris subsp. vulgaris), associated with vein clearing and mottling symptoms on leaves. The different AILV isolates sequenced were from artichoke (AILV-C), gladiolus (AILV-G), Sonchus (AILV-S), and chard (AILV-B). At the molecular level, the sequencing results of the RNA2 segments showed that AILV-C, AILV-G, AILV-S, and AILV-B had a length of 4629 nt (excluding the 3' terminal polyA tail), which is one nt shorter than that of the AILV-V reported in GenBank. A comparison of the RNA2 coding region sequences of all the isolates showed that AILV-V was the most divergent isolate, with the lowest sequence identities of 83.2% at the nucleotide level and 84.7% at the amino acid level. Putative intra-species sequence recombination sites were predicted among the AILV isolates, mainly involving the genomes of AILV-V, AILV-C, and AILV-B. This study adds insights into the variability of AILV and the occurrence of recombination that may condition plant infection.

Streptomyces cynarae sp. nov., a novel actinomycete isolated from the leaves of Cynara scolymus L.

Strain HUAS 13-4T, a novel endophytic actinobacterium, was isolated from the leaves of Cynara scolymus L. collected from Changde City in China and characterized using a polyphasic approach. Based on 16S rRNA gene sequence analysis, strain HUAS 13-4T shared the highest sequence similarities to Streptomyces leeuwenhoekii C34T (98.90%), Streptomyces harenosi PRKS01-65T (98.83%) and Streptomyces glomeratus LMG 19903T (98.76%). Phylogenetic analysis of 16S rRNA gene sequence indicated that strain HUAS 13-4T was clustered together with Streptomyces bluensis ISP 5564T and Streptomyces cavernae SYSU K10008T. Phylogenomic analysis revealed that strain HUAS 13-4T was most closely related to S. glomeratus JCM 9091T. However, the average nucleotide identity and the digital DNA-DNA hybridization values between them were less than 96.7% and 70% cut-off points recommended for delineating species. Based on a comprehensive comparison of the genome sequences and phenotypic characteristics between strain HUAS 13-4T and its relative, strain HUAS 13-4T (= MCCC 1K08364T = JCM 35919T) should evidently represent a novel Streptomyces species, and the name Streptomyces cynarae sp. nov. is proposed.

Structural and expression analysis of polyphenol oxidases potentially involved in globe artichoke (C. cardunculus var. scolymus L.) tissue browning.

Globe artichoke capitula are susceptible to browning due to oxidation of phenols caused by the activity of polyphenol oxidases (PPOs), this reduces their suitability for fresh or processed uses. A genome-wide analysis of the globe artichoke PPO gene family was performed. Bioinformatics analyses identified eleven PPOs and their genomic and amino acidic features were annotated. Cis-acting element analysis identified a gene regulatory and functional profile associated to plant growth and development as well as stress response. For some PPOs, phylogenetic analyses revealed a structural and functional conservation with different Asteraceae PPOs, while the allelic variants of the eleven PPOs investigated across four globe artichoke varietal types identified several SNP/Indel variants, some of which having impact on gene translation. By RTqPCR were assessed the expression patterns of PPOs in plant tissues and in vitro calli characterized by different morphologies. Heterogeneous PPO expression profiles were observed and three of them (PPO6, 7 and 11) showed a significant increase of transcripts in capitula tissues after cutting. Analogously, the same three PPOs were significantly up-regulated in calli showing a brown phenotype due to oxidation of phenols. Our results lay the foundations for a future application of gene editing aimed at disabling the three PPOs putatively involved in capitula browning.

Botanical authentication of globe artichoke-containing foods: Differentiation of Cynara scolymus by a novel HRM approach.

Cynara scolymus L., known as globe artichoke, is a medicinal plant widely used in plant food supplements (PFS) and herbal infusions due to its beneficial health properties. The high demand for artichoke-containing products can lead to adulteration practices. In this work, a real-time polymerase chain reaction (PCR) system coupled to high-resolution melting (HRM) analysis was proposed to differentiate C. scolymus from other Cynara species. Hence, a Cynara-specific real-time PCR assay was successfully developed with high analytical performance, achieving a sensitivity of 0.4 pg of globe artichoke DNA. HRM analysis enabled the discrimination of C. scolymus, with a high level of confidence (>98%), corroborating sequencing data. Application results to artichoke-containing PFS and mixed herbal infusions allowed confirming the presence of C. scolymus in 38% of the samples, suggesting the substitution/mislabelling of globe artichoke in 2 samples and the need for further efforts to increase DNA amplifiability of PFS.

(-)-α-Bisabolol Production in Engineered Escherichia coli Expressing a Novel (-)-α-Bisabolol Synthase from the Globe Artichoke Cynara cardunculus var. Scolymus.

(-)-α-Bisabolol is a functional ingredient in various health and cosmetic products and has antibacterial, anti-inflammatory, and wound healing properties. (-)-α-Bisabolol is chemically synthesized and produced by steam distillation of essential oils extracted from Brazilian Candeia (Eremanthus erythropappus). To sustainably produce pure (-)-α-bisabolol, we previously engineered Escherichia coli to produce 9.1 g/L (-)-α-bisabolol via heterologous mevalonate pathways and (-)-α-bisabolol synthase (BOS) from German chamomile, Matricaria recutita (MrBOS). BOS has only been reported in MrBOS and Brazilian Candeia (EeBOS). The limited availability of BOS has made it difficult to achieve high titer and yield and large-scale (-)-α-bisabolol production. We identified a novel BOS in globe artichoke (CcBOS) and examined its functionality in vitro and in vivo. CcBOS showed higher catalytic efficiency and (-)-α-bisabolol production rates than those from MrBOS or EeBOS. In fed-batch fermentation, CcBOS generated the highest reported (-)-α-bisabolol titer to date (23.4 g/L). These results may facilitate economically viable industrial (-)-α-bisabolol production.

Hop and artichoke extracts inhibit expression of extracellular matrix components in uterine leiomyoma cells.

OBJECTIVE: To screen 14 different plant extracts for their antifibrotic effect on human primary leiomyoma and healthy myometrial cells. DESIGN: Preclinical study. SETTING: University research laboratory. PATIENT(S): Human uterine leiomyoma and matched myometrial tissues were obtained from Caucasian premenopausal women with symptomatic uterine fibroids at the time of hysterectomy. INTERVENTION(S): Primary human leiomyoma and myometrial cells were cultured in the absence or presence of the plant extracts. MAIN OUTCOME MEASURE(S): Quantification of the expression of extracellular matrix components, such as fibronectin 1 (FN1), collagen type I alpha 1 (COL1A1), and versican (VCAN), and the profibrotic growth factor activin A or inhibin beta A subunit (INHBA). RESULT(S): The cells were treated with the 14 extracts for 48 hours, and we measured FN1 messenger RNA (mRNA) expression. Of the 14 extracts, about (ABO) ABO-2 (hop) and ABO-9 (artichoke) significantly reduced FN1 expression in both the cell types. Next, we evaluated the effect of fractions of these 2 extracts on the mRNA expression of FN1 and other extracellular matrix components, such as COL1A1, VCAN, and INHBA, in leiomyoma and myometrial cells. We found that ABO-2 (hop) and ABO-9 (artichoke) as well as their fractions, ABO-AR-2016-015 (fraction of ABO-2) and ABO-AR-2014-168 (fraction of ABO-9), reduced the mRNA expression of FN1, COL1A1, VCAN, and INHBA in primary leiomyoma cells. In primary myometrial cells, the mRNA expression of FN1, COL1A1, VCAN, and INHBA was not greatly affected. CONCLUSION(S): These results suggest that the hop and artichoke extracts possess antifibrotic properties and support additional evaluation using in vivo models.

"Mind the Gap": Hi-C Technology Boosts Contiguity of the Globe Artichoke Genome in Low-Recombination Regions.

Globe artichoke (Cynara cardunculus var. scolymus; 2n2x=34) is cropped largely in the Mediterranean region, being Italy the leading world producer; however, over time, its cultivation has spread to the Americas and China. In 2016, we released the first (v1.0) globe artichoke genome sequence (http://www.artichokegenome.unito.it/). Its assembly was generated using ∼133-fold Illumina sequencing data, covering 725 of the 1,084 Mb genome, of which 526 Mb (73%) were anchored to 17 chromosomal pseudomolecules. Based on v1.0 sequencing data, we generated a new genome assembly (v2.0), obtained from a Hi-C (Dovetail) genomic library, and which improves the scaffold N50 from 126 kb to 44.8 Mb (∼356-fold increase) and N90 from 29 kb to 17.8 Mb (∼685-fold increase). While the L90 of the v1.0 sequence included 6,123 scaffolds, the new v2.0 just 15 super-scaffolds, a number close to the haploid chromosome number of the species. The newly generated super-scaffolds were assigned to pseudomolecules using reciprocal blast procedures. The cumulative size of unplaced scaffolds in v2.0 was reduced of 165 Mb, increasing to 94% the anchored genome sequence. The marked improvement is mainly attributable to the ability of the proximity ligation-based approach to deal with both heterochromatic (e.g.: peri-centromeric) and euchromatic regions during the assembly procedure, which allowed to physically locate low recombination regions. The new high-quality reference genome enhances the taxonomic breadth of the data available for comparative plant genomics and led to a new accurate gene prediction (28,632 genes), thus promoting the map-based cloning of economically important genes.

Analysis of DNA Methylation Patterns Associated with In Vitro Propagated Globe Artichoke Plants Using an EpiRADseq-Based Approach.

Globe artichoke represents one of the main horticultural species of the Mediterranean basin, and 'Spinoso sardo' is the most widespread and economically relevant varietal type in Sardinia, Italy. In the last decades, in vitro culture of meristematic apices has increased the frequency of aberrant plants in open-field production. These off-type phenotypes showed highly pinnate-parted leaves and late inflorescence budding, and emerged from some branches of the true-to-type 'Spinoso sardo' plants. This phenomenon cannot be foreseen and is reversible through generations, suggesting the occurrence of epigenetic alterations. Here, we report an exploratory study on DNA methylation patterns in off-type/true-to-type globe artichoke plants, using a modified EpiRADseq technology, which allowed the identification of 2,897 differentially methylated loci (DML): 1,998 in CG, 458 in CHH, and 441 in CHG methylation contexts of which 720, 88, and 152, respectively, were in coding regions. Most of them appeared involved in primary metabolic processes, mostly linked to photosynthesis, regulation of flower development, and regulation of reproductive processes, coherently with the observed phenotype. Differences in the methylation status of some candidate genes were integrated with transcriptional analysis to test whether these two regulation levels might interplay in the emergence and spread of the 'Spinoso sardo' non-conventional phenotype.

Genotyping-by-sequencing highlights patterns of genetic structure and domestication in artichoke and cardoon.

Exploiting the biodiversity of crops and their wild relatives is fundamental for maintaining and increasing food security. The species Cynara cardunculus includes three taxa: the globe artichoke, one of the most important Mediterranean vegetables, the leafy cardoon, and the wild cardoon. In this study, genotyping by sequencing (GBS) was successfully applied to reveal thousands of polymorphisms in a C. cardunculus germplasm collection, including 65 globe artichoke, 9 leafy cardoon, and 21 wild cardoon samples. The collection showed a strong population structure at K = 2, separating the globe artichoke from the leafy and wild cardoon. At higher K values, further substructures were observed, in which the wild cardoon was separated from the leafy cardoon, and the latter included the Spanish wild cardoons, while the wild sample from Portugal was admixed. Moreover, subpopulations within the globe artichoke set were highlighted. Structure analysis restricted to the globe artichoke dataset pointed out genetic differentiation between the ˝Catanesi˝ typology and all the other samples (K = 2). At higher values of K, the separation of the ˝Catanesi˝ group still held true, and green headed landraces from Apulia region, Italy (˝Green Apulian˝) formed a distinct subpopulation. ˝Romaneschi˝ artichoke types fell in a variable group with admixed samples, indicating that they should not be considered as a genetically uniform typology. The results of principal component analysis and Neighbor-Joining hierarchical clustering were consistent with structure results, and in addition provided a measure of genetic relationships among individual genotypes. Both analyses attributed the wild material from Spain and Portugal to the cultivated cardoon group, supporting the idea that this might be indeed a feral form of the leafy cardoon. Different reproductive habit and possibly selective pressure led to a slower LD decay in artichoke compared to cardoon. Genotyping by sequencing has proven a reliable methodology to obtain valuable SNPs and assess population genetics in C. cardunculus.

Nutritional value and chemical composition of Greek artichoke genotypes.

The nutritional value and chemical composition of various artichoke genotypes cultivated in Greece were evaluated. Significant differences were detected in water content, as well as in fat, ash, protein and carbohydrate content. Proteins and carbohydrates were detected in high amounts in all the genotypes. Antioxidant activity was also varied between the studied samples. Palmitic and linoleic acids were the most abundant fatty acids in artichoke heads, while stearic, oleic, alpha-linolenic, arachidic, behenic and lignoceric acids were detected in lesser amounts. 3,5-O-Dicaffeoylquinic acid and 5-O-caffeoylquinic acid were the main phenolic compounds. In conclusion, heads of artichoke genotypes cultivated in Greece showed a high nutritional value and antioxidant activity which signifies the importance of this vegetable for the Mediterranean diet, while the diversity in chemical composition between the studied samples should be further exploited for the selection of elite cultivars with specific end-uses of the final product.

The genome-wide identification and transcriptional levels of DNA methyltransferases and demethylases in globe artichoke.

Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases) and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1), CMT (chromomethyltransferases) and DRM (domains rearranged methyltransferases). Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus) is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs) and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

Leaf parts from Greek artichoke genotypes as a good source of bioactive compounds and antioxidants.

Globe artichoke is an important vegetable in the Mediterranean diet. However, the marketable part of the plant constitutes a small portion of its total crop biomass and large amounts of waste are produced during its cultivation. In this study, the phenolic compound content and composition and the antioxidant activity of leaf parts [blades (SLB), petioles and midribs (SPM)] from various Greek artichoke genotypes were evaluated. The results showed significant variation in antioxidant activities and bioactive compound contents between the studied genotypes, with the leaf blades of most of the genotypes having a high content of flavonoids (mainly luteolin glycoside derivatives), which ranged between 49 and 78% of total phenolic compounds (TPC). In contrast, in petioles and midribs phenolic acids (mainly hydroxycinnamic acid derivatives) were the main phenolic compounds, ranging between 64 and 76% of TPC. The antioxidant activity of blades was significantly higher than that of petiole and midribs for all the genotypes and the tested assays. In conclusion, artichoke leaves (blades, petioles and midribs) of the studied genotypes showed different bioactive compound profiles and significant antioxidant properties, and could be further used in the food and nutraceuticals industries as a cheap source of phenolic compounds and antioxidants.

A Genome-Wide Survey of the Microsatellite Content of the Globe Artichoke Genome and the Development of a Web-Based Database.

The recently acquired genome sequence of globe artichoke (Cynara cardunculus var. scolymus) has been used to catalog the genome's content of simple sequence repeat (SSR) markers. More than 177,000 perfect SSRs were revealed, equivalent to an overall density across the genome of 244.5 SSRs/Mbp, but some 224,000 imperfect SSRs were also identified. About 21% of these SSRs were complex (two stretches of repeats separated by <100 nt). Some 73% of the SSRs were composed of dinucleotide motifs. The SSRs were categorized for the numbers of repeats present, their overall length and were allocated to their linkage group. A total of 4,761 perfect and 6,583 imperfect SSRs were present in 3,781 genes (14.11% of the total), corresponding to an overall density across the gene space of 32,5 and 44,9 SSRs/Mbp for perfect and imperfect motifs, respectively. A putative function has been assigned, using the gene ontology approach, to the set of genes harboring at least one SSR. The same search parameters were applied to reveal the SSR content of 14 other plant species for which genome sequence is available. Certain species-specific SSR motifs were identified, along with a hexa-nucleotide motif shared only with the other two Compositae species (sunflower (Helianthus annuus) and horseweed (Conyza canadensis)) included in the study. Finally, a database, called "Cynara cardunculus MicroSatellite DataBase" (CyMSatDB) was developed to provide a searchable interface to the SSR data. CyMSatDB facilitates the retrieval of SSR markers, as well as suggested forward and reverse primers, on the basis of genomic location, genomic vs genic context, perfect vs imperfect repeat, motif type, motif sequence and repeat number. The SSR markers were validated via an in silico based PCR analysis adopting two available assembled transcriptomes, derived from contrasting globe artichoke accessions, as templates.

The genome sequence of the outbreeding globe artichoke constructed de novo incorporating a phase-aware low-pass sequencing strategy of F1 progeny.

Globe artichoke (Cynara cardunculus var. scolymus) is an out-crossing, perennial, multi-use crop species that is grown worldwide and belongs to the Compositae, one of the most successful Angiosperm families. We describe the first genome sequence of globe artichoke. The assembly, comprising of 13,588 scaffolds covering 725 of the 1,084 Mb genome, was generated using ~133-fold Illumina sequencing data and encodes 26,889 predicted genes. Re-sequencing (30×) of globe artichoke and cultivated cardoon (C. cardunculus var. altilis) parental genotypes and low-coverage (0.5 to 1×) genotyping-by-sequencing of 163 F1 individuals resulted in 73% of the assembled genome being anchored in 2,178 genetic bins ordered along 17 chromosomal pseudomolecules. This was achieved using a novel pipeline, SOILoCo (Scaffold Ordering by Imputation with Low Coverage), to detect heterozygous regions and assign parental haplotypes with low sequencing read depth and of unknown phase. SOILoCo provides a powerful tool for de novo genome analysis of outcrossing species. Our data will enable genome-scale analyses of evolutionary processes among crops, weeds, and wild species within and beyond the Compositae, and will facilitate the identification of economically important genes from related species.

Accumulation of cynaropicrin in globe artichoke and localization of enzymes involved in its biosynthesis.

Globe artichoke (Cynara cardunculus var. scolymus) belongs to the Asteraceae family, in which one of the most biologically significant class of secondary metabolites are sesquiterpene lactones (STLs). In globe artichoke the principal STL is the cynaropicrin, which contributes to approximately 80% of its characteristic bitter taste. Cynaropicrin content was assessed in globe artichoke tissues and was observed to accumulate in leaves of different developmental stages. In the receptacle, a progressive decrease was observed during inflorescence development, while the STL could not be detected in the inflorescence bracts. Almost undetectable amounts were found in the roots and inflorescence stems at the commercial stage. Cynaropicrin content was found to correlate with expression of genes encoding CcGAS, CcGAO and CcCOS, which are involved in the STL biosynthesis. A more detailed study of leaf material revealed that cynaropicrin predominantly accumulates in the trichomes, and not in the apoplastic cavity fluids. Analysis of the promoter regions of CcGAO and CcCOS revealed the presence of L1-box motifs, which confers trichome-specific expression in Arabidopsis, suggesting that cynaropicrin is not only stored but also synthesized in trichomes. A transient expression of GFP fusion proteins was performed in Nicotiana benthamiana plants: the CcGAS fluorescence signal was located in the cytoplasm while the CcGAO and CcCOS localized to the endoplasmatic reticulum.

Complete chloroplast genome of the multifunctional crop globe artichoke and comparison with other Asteraceae.

With over 20,000 species, Asteraceae is the second largest plant family. High-throughput sequencing of nuclear and chloroplast genomes has allowed for a better understanding of the evolutionary relationships within large plant families. Here, the globe artichoke chloroplast (cp) genome was obtained by a combination of whole-genome and BAC clone high-throughput sequencing. The artichoke cp genome is 152,529 bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 25,155 bp, representing the longest IRs found in the Asteraceae family so far. The large (LSC) and the small (SSC) single-copy regions span 83,578 bp and 18,641 bp, respectively. The artichoke cp sequence was compared to the other eight Asteraceae complete cp genomes available, revealing an IR expansion at the SSC/IR boundary. This expansion consists of 17 bp of the ndhF gene generating an overlap between the ndhF and ycf1 genes. A total of 127 cp simple sequence repeats (cpSSRs) were identified in the artichoke cp genome, potentially suitable for future population studies in the Cynara genus. Parsimony-informative regions were evaluated and allowed to place a Cynara species within the Asteraceae family tree. The eight most informative coding regions were also considered and tested for "specific barcode" purpose in the Asteraceae family. Our results highlight the usefulness of cp genome sequencing in exploring plant genome diversity and retrieving reliable molecular resources for phylogenetic and evolutionary studies, as well as for specific barcodes in plants.

Evaluation of the bioactive properties of avenanthramide analogs produced in recombinant yeast.

Saccharomyces cerevisiae has been proven to be a valuable tool for the expression of plant metabolic pathways. By engineering a S. cerevisiae strain with two plant genes (4cl-2 from tobacco and hct from globe artichoke) we previously set up a system for the production of two novel phenolic compounds, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid (Yeast avenanthramide I, Yav I) and N-(E)-caffeoyl-3-hydroxyanthranilic acid (Yeast avenanthramide II, Yav II). These compounds have a structural similarity with a class of bioactive oat compounds called avenanthramides. By developing a fermentation process for the engineered S. cerevisiae strain, we obtained a high-yield production of Yav I and Yav II. To examine the biological relevance of these compounds, we tested their potential antioxidant and antiproliferative properties upon treatment of widely used cell models, including immortalized mouse embryonic fibroblast cell lines and HeLa cancer cells. The outcomes of our experiments showed that both Yav I and Yav II enter the cell and trigger a significant up-regulation of master regulators of cell antioxidant responses, including the major antioxidant protein SOD2 and its transcriptional regulator FoxO1 as well as the down-regulation of Cyclin D1. Intriguingly, these effects were also demonstrated in cellular models of the human genetic disease Cerebral Cavernous Malformation, suggesting that the novel phenolic compounds Yav I and Yav II are endowed with bioactive properties relevant to biomedical applications. Taken together, our data demonstrate the feasibility of biotechnological production of yeast avenanthramides and underline a biologically relevant antioxidant activity of these molecules.

Genetic relatedness of artichoke (Cynara scolymus L.) hybrids using random amplified polymorphic DNA (RAPD) fingerprinting.

The artichoke (Cynara scolymus L.) is an important food and medicinal crop that is cultivated in Mediterranean countries. Morphological characteristics, such as head shape and diameter, leaf shape, and bract shape, are mainly affected by environmental conditions. A molecular marker approach was used to analyze the degree of polymorphism between artichoke hybrid lines. The degree of genetic difference among three artichoke hybrids was evaluated using random amplified polymorphic DNA-PCR (RAPD-PCR). In this study, the DNA fingerprints of three artichoke lines (A13-010, A11-018, and A12-179) were generated, and a total of 10 decamer primers were applied for RAPD-PCR analyses. Polymorphism  (16.66 to 62.50%) was identified using eight arbitrary decamers and total genomic DNA extracted from the hybrids. Of the 59 loci detected, there were 25 polymorphic and 34 monomorphic loci. Jaccard's similarity index (JSI) ranged between 1.0 and 0.84. Based on the unweighted pair group method with arithmetic mean (UPGMA) similarity matrix and dendrogram, the results indicated that two hybrids (A13-010 and A11-018) were closely related to each other, and the A12-179 line showed more divergence. When identifying correct accessions, consideration of the genetic variation and genetic relationships among the genotypes are required. The RAPD-PCR fingerprinting of artichoke lines clearly showed that it is possible to analyze the RAPD patterns for correlation between genetic means and differences or resemblance between close accessions (A13-010 and A11- 018) at the genomic level.