La biodiversità rappresenta la variabilità di tutti gli esseri viventi e può essere riferita agli ecosistemi, alle specie, o a livellidi rango tassonomico infraspecifico, quali le varietà. In particolare, la biodiversità vegetale di interesse agrario è riferita allespecie coltivate e spontanee utilizzate dall"uomo a scopi alimentari o per altri usi e ai relativi progenitori selvatici.Nell"ambito dei Progetti Integrati per la Biodiversità, PSR Puglia 2007-2013 (Misura 214/4 sub-azione a), la Regione Pugliaha finanziato il progetto BiodiverSO - Biodiversità delle specie orticole della Puglia - con l"obiettivo primario disalvaguardare le risorse genetiche orticole della regione. Infatti, a tutt"oggi, sul territorio pugliese non è stata effettuataun"indagine capillare e sistematica delle risorse genetiche ortive presenti. Le azioni di conservazione del germoplasma sonospesso affidate alle iniziative di singoli agricoltori che tramandano semi o altre parti di pianta di generazione in generazione(conservazione on farm), e alla conservazione ex situ che viene normalmente praticata da enti pubblici, quali CNR edUniversità. La rivalutazione di genotipi di varietà locali e/o il loro risanamento possono ampliare la base genetica delcomparto orticolo consentendo migliore tolleranza agli stress biotici e abiotici, in un periodo in cui si assiste a notevolicambiamenti climatici, per salvaguardare la salute del consumatore e l"ambiente, nonché per valorizzare alcune produzionitipiche pugliesi. L"Italia meridionale rappresenta il centro di domesticazione e/o diversificazione di alcune colture, quali adesempio alcune Brassicacee, il carciofo, il melone, e questo significa che le nostre regioni sono estremamente ricche dibiodiversità di interesse agrario. La Puglia produce numerose varietà coltivate di ortaggi come la carota di Polignano e diSant"Ippazio; la cipolla di Acquaviva e di Margherita; il cavolfiore, cavolo broccolo (cima nera, cavolo riccio, mugnoli) ecima di rapa; il melone immaturo (carosello e barattiere) e d"inverno; la cicoria (di Molfetta, di Galatina, di Otranto); ilcarciofo (di Mola, Centofoglie, Bianco tarantino, nero di Ostuni, ecc.); il pomodoro Regina ed il pomodoro di Manduria.Alcune di queste varietà locali sono a rischio di estinzione e potrebbero scomparire per sempre per essere soppiantate davarietà moderne. La principale finalità del progetto integrato BiodiverSO è quella di contribuire a raggiungere unasignificativa riduzione del tasso attuale di erosione della biodiversità delle specie orticole pugliesi. Dopo aver reperito sulterritorio pugliese le risorse genetiche orticole a rischio, esse saranno catalogate mediante strumenti informatizzati ecaratterizzate sia dal punto di vista morfo-agronomico che nutrizionale, che genetico-molecolare. Il materiale geneticoraccolto verrà conservato presso le banche del germoplasma del CNR-IBBR di Bari e dell"Università di Bari. Laconservazione ex si

The infection of Arabidopsis thaliana plants with avirulent pathogens causes the accumulation of cGMP with a biphasic profile downstream of nitric oxide signalling. However, plant enzymes that modulate cGMP levels have yet to be identified, so we generated transgenic A. thaliana plants expressing the rat soluble guanylate cyclase (GC) to increase genetically the level of cGMP and to study the function of cGMP in plant defence responses. Once confirmed that cGMP levels were higher in the GC transgenic lines than in wild-type controls, the GC transgenic plants were then challenged with bacterial pathogens and their defence responses were characterized. Although local resistance was similar in the GC transgenic and wild-type lines, differences in the redox state suggested potential cross-talk between cGMP and the glutathione redox system. Furthermore, large-scale transcriptomic and proteomic analysis highlighted the significant modulation of both gene expression and protein abundance at the infection site, inhibiting the establishment of systemic acquired resistance. Our data indicate that cGMP plays a key role in local responses controlling the induction of systemic acquired resistance in plants challenged with avirulent pathogens.

Cyclic AMP is a recognized second messenger; however, knowledge of cAMP involvement in plant physiological processes originates primarily from pharmacological studies. To obtain direct evidence for cAMP function in plants, tobacco Bright Yellow-2 (BY-2) cells were transformed with the cAMP sponge, which is a genetically encoded tool that reduces cAMP availability. BY-2 cells expressing the cAMP sponge (cAS cells), showed low levels of free cAMP and exhibited growth inhibition that was proportional to the cAMP sponge transcript level. Growth inhibition in cAS cells was closely related to the precocious inhibition of mitosis due to a delay in cell cycle progression. The cAMP deficiency also enhanced antioxidant systems. Remarkable changes occurred in the cAS proteomic profile compared with that of wild-type (WT) cells. Proteins involved in translation, cytoskeletal organization, and cell proliferation were down-regulated, whereas stress-related proteins were up-regulated in cAS cells. These results support the hypothesis that BY-2 cells sense cAMP deficiency as a stress condition. Finally, many proteasome subunits were differentially expressed in cAS cells compared with WT cells, indicating that cAMP signaling broadly affects protein degradation via the ubiquitin/proteasome pathway.

Plant cyclic nucleotides (cGMP and cAMP) have been reported to be involved in several physiological processes, but the mechanisms of action and the following signal transduction events, are still poorly characterized. The main object of the 2010 FIRB project is the study of cyclic nucleotide roles in signalling events following biotic stress in Arabidopsis thaliana.For this aim transgenic plants with altered levels of both cGMP and cAMP, have been generated. Specifically, plants over-expressing a mammalian guanylate cyclase (GC) or a phosphodiesterase (PDE) to alter the cGMP levels, and plants over-expressing a chimeric protein namely "cAMP-sponge" (cAS), to buffer the cAMP, were produced. Moreover, in order to measure both in vivo and in vitro the cNMP levels and dynamics, different approaches have been followed: i) production of transgenic lines expressing genetically encoded FRET-based probes (EPACs and Red-cGES) and ii) set up of alpha screen technology for plant tissue. To gain insight into the inter-relationships of cNMPs and other second messengers in the plant signalling events, we then crossed the plants with altered levels of cNMPs with Arabidopsis transgenic lines expressing the genetically encoded probes Cameleon (for in vivo Ca2+ detection) and roGFP2 (for in vivo detection of redox potential). In Arabidopsis, it has been reported that Ca2+, redox status and cNMPs play important signalling roles in response to the avirulent attack of Pseudomonas syringae. Our preliminary results, using Cameleon and roGFP2 wild type plants, showed a transient increase of both Ca2+ and oxidation status upon avirulent attack of Pseudomonas syringae. Biochemical analyses confirmed that both Glutathione and Ascorbate pools were more oxidized upon such attack. In order to study if and how the cNMPs play a role in the regulation of Ca2+ and redox status similar experiments will be performed with the different generated lines with altered levels of cNMPs. Finally, to understand if the altered levels of cNMPs can affect gene expression in response to the considered pathogen attack, a transcriptomic and proteomic characterization of the transgenic lines will be performed. Following this multidisciplinary approach, the understanding of the specific biological responses regulated by cAMP and cGMP and the downstream signalling events actually represents a big challenge in plants

Cyclic AMP plays important roles in different physiological processes, including plant defence responses. However, since little is known on plant enzyme responsible for cAMP production/degradation, the study of cAMP functions relied, until now, on non-specific pharmacological approaches. We thus developed a more reliable approach, producing transgenic Arabidopsis thaliana lines overexpressing the "cAMP-sponge" (cAS), a genetic tool that specifically buffers cAMP levels. In response to an avirulent strain of Pseudomonas syringae pv. tomato (PstAvrB), cAS plants showed a higher bacterial growth and a reduced hypersensitive cell death in comparison with WT plants. The low cAMP availability after pathogen infection delayed cytosolic calcium elevation, as well as hydrogen peroxide increase and induction of redox systems. The proteomic analysis, performed 24 hours post-infection, indicated that a core of 49 proteins was modulated in both genotypes, while 16 and 42 proteins were uniquely modulated in WT and cAS lines, respectively. The involvement of these proteins in the impairment of defence response in cAS plants has been discussed. Moreover, an in-silico analysis revealed that the promoter regions of the genes coding for proteins uniquely accumulating in WT plants share the CGCG motif, target of the calcium-calmodulin binding transcription factor AtSR1 (Arabidopsis thaliana signal responsive1). Thus, following pathogen perception, the low free cAMP content, altering timing and levels of defence signals, likely in part through the mis-regulation of AtSR1 activity, affects the speed and strength of the immune response.

Ten accessions of wild Borago officinaliswere collected during missions in the course of aresearch activity carried out on plants producingnutraceutical compounds. The aim was to identifypopulations that produce a higher content ofc-linolenic acid, as this compound has importanttherapeutic properties and has been used in medicaltests to treat rheumatoid arthritis, eczema, and psoriasiswith good therapeutical results. The present studyreports on the characterization of both the polyunsaturatedfatty acid composition and on genetic diversityestimated by means of AFLP markers in severalB. officinalis accessions collected in Southern Italyand grown in isolation by means of screen housesspecifically designed for each single population.Polyunsaturated fatty acid were found in differentamounts in all investigated accessions. The populationswere very variable at the molecular level, edthat similarity based on Jaccard's index rangedbetween 0.330 and 0.742. Our investigations allowedus to identify a population that may be considered as agood source of GL

An integrated consensus linkage map is proposed for globe artichoke. Maternal and paternal genetic maps were constructed on the basis of an F(1) progeny derived from crossing an artichoke genotype (Mola) with its progenitor, the wild cardoon (Tolfa), using EST-derived SSRs, genomic SSRs, AFLPs, ten genes, and two morphological traits. For most genes, mainly belonging to the chlorogenic acid pathway, new markers were developed. Five of these were SNP markers analyzed through high-resolution melt technology. From the maternal (Mola) and paternal (Tolfa) maps, an integrated map was obtained, containing 337 molecular and one morphological markers ordered in 17 linkage groups (LGs), linked between Mola and Tolfa. The integrated map covers 1,488.8 cM, with an average distance of 4.4 cM between markers. The map was aligned with already existing maps for artichoke, and 12 LGs were linked via 31 bridge markers. LG numbering has been proposed. A total of 124 EST-SSRs and two genes were mapped here for the first time, providing a framework for the construction of a functional map in artichoke. The establishment of a consensus map represents a necessary condition to plan a complete sequencing of the globe artichoke genome.

An integrated consensus linkage map is proposed for globe artichoke. Maternal and paternal genetic maps were constructed on the basis of an F(1) progeny derived from crossing an artichoke genotype (Mola) with its progenitor, the wild cardoon (Tolfa), using EST-derived SSRs, genomic SSRs, AFLPs, ten genes, and two morphological traits. For most genes, mainly belonging to the chlorogenic acid pathway, new markers were developed. Five of these were SNP markers analyzed through high-resolution melt technology. From the maternal (Mola) and paternal (Tolfa) maps, an integrated map was obtained, containing 337 molecular and one morphological markers ordered in 17 linkage groups (LGs), linked between Mola and Tolfa. The integrated map covers 1,488.8 cM, with an average distance of 4.4 cM between markers. The map was aligned with already existing maps for artichoke, and 12 LGs were linked via 31 bridge markers. LG numbering has been proposed. A total of 124 EST-SSRs and two genes were mapped here for the first time, providing a framework for the construction of a functional map in artichoke. The establishment of a consensus map represents a necessary condition to plan a complete sequencing of the globe artichoke genome.

Phenolic acids are major components of cell walls in wheat and have important implications on human health as antioxidants with anti-tumor activity. Our objectives were to identify phenolic acid genes in wheat by single nucleotide polymorphisms (SNPs) detected within the coding sequences of candidate genes, and to identify chromosomal regions associated with single phenolic acids and total soluble phenolic compounds. A set of candidate genes involved in the biosynthesis of hydroxycinnamic acid derivatives were identified by comparative genomics. SNPs found in the coding sequences of six genes (PAL1, PAL2, C4H, C3H, COMT1 and COMT2) were used to determine their chromosomal location and accurate map position on two reference consensus linkage maps. The genome-wide association study (GWAS), based on genotyping a tetraploid wheat collection with 81,587 gene-associated SNPs, detected 22 quantitative trait loci (QTL) distributed on almost all durum wheat chromosomes. Two QTL for p-coumaric acid were coincident with the phenylalanine ammonia-lyase (PAL2) and p-coumarate 3-hydroxylase (C3H) genes on chromosome arms 2AL and 1AL, respectively. The availability of candidate gene-based markers can allow elucidating the mechanism of phenolic acids accumulation in wheat kernels and exploiting the genetic variability of phenolic acids content for the nutritional improvementof wheat end-products.

Exploiting the biodiversity of crops and their wild relatives is fundamental for maintaining andincreasing food security. The species Cynara cardunculus includes three taxa: the globe artichoke,one of the most important Mediterranean vegetables, the leafy cardoon, and the wildcardoon. In this study, genotyping by sequencing (GBS) was successfully applied to revealthousands of polymorphisms in a C. cardunculus germplasm collection, including 65 globeartichoke, 9 leafy cardoon, and 21 wild cardoon samples. The collection showed a strong populationstructure at K = 2, separating the globe artichoke from the leafy and wild cardoon. Athigher K values, further substructures were observed, in which the wild cardoon was separatedfrom the leafy cardoon, and the latter included the Spanish wild cardoons, while the wildsample from Portugal was admixed. Moreover, subpopulations within the globe artichoke setwere highlighted. Structure analysis restricted to the globe artichoke dataset pointed outgenetic differentiation between the ?Catanesi? typology and all the other samples (K = 2). Athigher values of K, the separation of the ?Catanesi? group still held true, and green headedlandraces from Apulia region, Italy (?Green Apulian?) formed a distinct subpopulation. ?Romaneschi?artichoke types fell in a variable group with admixed samples, indicating that theyshould not be considered as a genetically uniform typology. The results of principal componentanalysis and Neighbor-Joining hierarchical clustering were consistent with structureresults, 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 cardoongroup, 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 artichokecompared to cardoon. Genotyping by sequencing has proven a reliable methodology toobtain valuable SNPs and assess population genetics in C. cardunculus

Polyphenols are important constituents of food products of plant origin and represent major antioxidants in our diet, since endogenous defense mechanisms are inadequate for the complete prevention of oxidative damage. The most abundant types of polyphenols found in the human diet are flavonoids, most often conjugated as glycosides.Among different sources of dietary antioxidants, artichoke extracts are particularly rich in bioavailable poliphenolic compounds as flavonoids and are important for their marked antioxidative potential and cancer chemopreventive properties. The understanding of flavonoids biosynthesis and its regulation in artichoke is essential to enhance the levels of these bioactive molecules in plants used as food.The biosynthetic pathway leading to the accumulation of flavonoids has been elucidated using genetic and biochemical information from many plant species (parsley, maize, petunia, Antirrhinum species, Arabidopsis, apple and grapevine) and has been recently studied in artichoke, where several enzymes directly involved in the early steps of the biosynthesis of flavonoids have been isolated and characterized, but little is still known on the regulation of these genes.The fine regulation of flavonoids accumulation is achieved by combinatorial actions of transcription factors (TF) belonging to various classes, among which MYB TF. Two putative artichoke MYB TF genes were isolated from a BAC library and were sequenced using Illumina technology. Sequencing allowed the structural characterization of coding and promoter regions of both genes, which showed a high similarity to MYB TF of other plant species (e.g. Arabidopsis, tomato). A phylogenetic analysis of putative MYB factors showed that the two artichoke sequences cluster together in a group including MYB factors from other species involved in the pathway of flavonols and proanthocyanidin biosynthesis. Heterologous expression in bacteria to study protein/DNA interactions and quantitative real-time PCR have been performed to gain insights into the molecular mechanism of polyphenols regulation in this edible plant, contributing to future progress in the study of polyphenols accumulation.

Cyclic AMP is a well known second messenger involved in different cellular responses in all living organisms. In higher plants, its role as second messenger has been widely debated, due to its low content and to the difficulty of measuring it. However, its natural occurrence and the existence of adenylyl-cyclases and cAMP phosphodiesterases, that constitute the on-off switches needed for its action as second messenger, have been demonstrated. Data accumulated in the last three decades show the involvement of cAMP in several processes of higher plants, including cell cycle regulation, growth and reorientation of the pollen tube, seed germination and defense responses. However little is known on the mechanisms involved in the cAMP-dependent signal transduction in plants. To shed light on cAMP role in plant signaling pathways, Arabidopsis plants, whose genome has been completely sequenced, and tobacco Bright Yellow-2 (BY-2) cells, that are highly synchronizable, have been chosen as model systems. For this purpose, both Arabidopsis plants and tobacco BY-2 cells were transformed with the "cAMP-sponge", a non invasive tool able to selectively reduce cAMP concentration (Lefkimmiatis et al, 2009). The cAMP-sponge is composed of two high-affinity cAMP binding domains of the regulatory subunits I beta of human protein kinase A (PKARIbeta) that specifically bind cAMP and not cGMP. The construct containing the cAMP-sponge in frame with the reporter gene mCherry were mobilized into TBY-2 cells and Arabidopsis via A. tumefaciens-mediated transformation. Transgenic TBY-2 calli and Arabidopsis plants were selected in the presence of appropriate antibiotics, and several independent transgenic lines were obtained. Trans-gene integration and its expression in Arabidospsis and tobacco transformed lines were verified by PCR, RT-PCR and immunoblotting analyses. The low levels of cAMP negatively affected the growth of TBY-2 cells whereas no distinctive phenotype was observed in Arabidobsis plants. However, a stress condition was evidenced for both tobacco BY-2 cells and Arabidopsis plants, as shown by the alteration of their cellular redox state, analysed by ascorbate and glutathione measurements.

It is well known that artichoke possesses a number of beneficial properties, mainly attributable to polyphenols, a class of compounds with beneficial effects on human health. These are secondary metabolites including hydroxycinnamates, particularly caffeic acid, chlorogenic acid (CGA), di-caffeoylquinic acids, and flavonoids (e.g. luteolin). Chlorogenic acid, in particular, is a potent antioxidant very abundant in some vegetables, included artichoke. In artichoke the synthesis of CGA has not been completely elucidated yet. There are three proposed routes for the synthesis of CGA in plants, where the first common enzymatic step is the deamination of phenylalanine by means of the enzyme phenylalanine ammonia-liase (PAL), while the enzyme hydroxycynnamoyl-quinate transferase (HQT), belonging to the BAHD family is reported to be involved in the last step of the biosinthesys of CGA in some plant species. Recently, four members of the artichoke pal gene family have been isolated (De Paolis et al., 2008); in the present contribution, we report on the relative expression of three PAL isoforms in different plant parts, at different developmental stages and under stress simulation by mechanical wounding, and also on the isolation and characterization of two artichoke full-length hqt and hqt2 cDNA sequences. The HQT sequences showed a high level of similarity to homologous genes from other plant species, particularly tobacco and tomato. The transcripts of the two artichoke genes were heterologously expressed and biochemical assays were performed, in order to assess specificity of the enzymes for various substrates.

A pool of twelve cDNA sequences coding for Bowman Birk inhibitors (BBIs) was identified in the legume grass pea (Lathyrus sativus L.). The corresponding amino acid sequences showed a canonical first antitrypsin domain, predicted according to the identity of the determinant residue P-1. A more variable second binding loop was observed allowing to identify three groups based on the identity of residue P-1: two groups (Ls_BBI_1 and Ls_BBI_2) carried a second reactive site specific for chymotrypsin, while a third group (Ls_BB1_3) was predicted to inhibit elastase. A fourth variant carrying an Asp in the P-1 position of the second reactive site was identified only from genomic DNA. A phylogenetic tree constructed using grass pea BBIs with their homologs from other legume species revealed grouping based on taxonomy and on specificity of the reactive sites.

Flavonoids are a well-studied group of secondary metabolites, belonging to the phenylpropanoid pathway. Flavonoids are known to exhibit health promoting effects such as antioxidant capacities, anti-cancer and anti-inflammatory activity. Globe artichoke is an important source of bioactive phenolic compounds, including flavonoids. To study the regulation of their biosynthesis, a R2R3-MYB transcription factor, CcMYB12, was isolated from artichoke leaves. Phylogenetic analysis showed that this protein belongs to the MYB subgroup 7 (flavonol-specific MYB), which includes Arabidopsis AtMYB12, grapevine VvMYBF1, and tomato SlMYB12. CcMYB12 transcripts were detected specifically in artichoke immature inflorescence and young leaves and overlapped with the profiles of flavonol biosynthetic genes. Electrophoretic mobility shift assays (EMSAs) revealed that recombinant CcMYB12 protein is able to bind to ACII element, a DNA binding site ubiquitously present in the promoters of genes encoding flavonol biosynthetic enzymes. In transgenic Arabidopsis plants, the overexpression of CcMYB12 activated the expression of endogenous flavonol biosynthesis genes, leading to an increase of flavonol accumulation and a decrease of anthocyanins in leaves. Likewise, in transgenic tobacco petals and leaves, the overexpression of CcMYB12 decreased anthocyanin levels and increased flavonols.

The known nutraceutical properties ascribed to artichoke are mainly attributable to the presence of phenylpropanoid compounds, in particular of flavonoids, caffeic acid and its derivatives (caffeoylquinic acids). These metabolites play central roles in plant biology, as they are involved in the protection against various biotic and abiotic stresses, in the regulation of plant reproduction and development, and they act as signalling molecules. Phenylpropanoids have also significant beneficial effects on human health if consumed as part of the diet, as they act as antioxidants, reducing the incidence of cardiovascular disease, certain cancers and age-related degenerative diseases. Hence, there is considerable interest in improving our understanding of phenylpropanoid biosynthesis and its regulation, to enhance the levels of these bioactive molecules in plants used as food. The biosynthetic pathway leading to the accumulation of phenylpropanoids has been elucidated using genetic and biochemical information from many plant species. The fine regulation of this pathway is achieved by combinatorial actions of transcription factors (TF) belonging to various classes, among which MYB TF.The metabolic route for the biosynthesis of caffeoylquinic acids, particularly chlorogenic acid (CGA) and its derivatives has been recently studied in artichoke, where several enzymes directly involved in this biosynthetic pathway have been isolated and characterized. However, little is known on the regulation of these genes. We have previously studied the genes coding for phenylalanine ammonio lyase (PAL) and two hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferases (HQT) enzymes in artichoke, where we detected different binding sites recognized by MYB TF in their promoter regions, suggesting that, as in other plant species, this class of transcription factors might be involved in the regulation of these artichoke genes.In order to isolate one or more MYB TF from artichoke, sequences of the ortholog MYB12 from other species (e.g. Arabidopsis, tomato) were used to screen artichoke EST database. Primers were designed to amplify a fragment of putative MYB TF in artichoke cDNA. Sequence completion was performed by means of 5'-RACE and 3'-RACE technology. This strategy allowed us to isolate two putative transcription factor genes belonging to the MYB family. Further analyses are being performed to assess their role in phenylpropanoid accumulation in artichoke. Heterologous expression in bacteria, transient expression assays, quantitative real-time PCR may help elucidate their function and interaction with phenylpropanoid biosynthetic genes.

Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes.

Artichoke has beneficial effects on human health, since it possesses anticarcinogenic, anti-HIV, antioxidative, cholesterol-lowering, bile expelling, hepatoprotective, and diuretic properties. These nutraceutical qualities are mainly due to polyphenolic compounds, particularly mono- and dicaffeoylquinic acids (e.g. chlorogenic acid, cynarin), caffeic acid and flavonoids (e.g. luteolin-7-O-glucoside, naringerin), with chlorogenic acid (CGA) being the most abundant. The content of polyphenol compounds, especially chlorogenic acid and cynarin, was measured in various tissues (leaves, bracts and receptacles) and physiological stages of artichoke plants, and in different artichoke genotypes belonging to the CNR-IGV Cynara world collection. A variation among tissues and genotypes was observed, and these variations were quite reproducible among years.On the same tissues, transcript levels of key genes for the synthesis of chlorogenic acid were measured by means of real time PCR. In particular, hqt genes, coding for HQT enzymes (acyltransferases of the BAHD family), have been shown to play a fundamental role in the synthesis of CGA in some plant species. Recently, hqt1 and hqt2 genes, possessing two exons and one intron each, have been isolated from artichoke, starting from Asteraceae EST sequences. Coding sequences were heterologously expressed in E. coli and the crude extract was used for enzyme chatracterization and substrate specificity. Both HQT1 and HQT2 were able to synthesize CGA in vitro and showed a remarkable preference towards quinate over shikimate, which distinguishes HQT enzyme class from HCT class (preferring shikimate). Based on the available crystallized structures of two BAHD enzymes, modeling and docking analyses were used to assume structural models for our HQT enzymes and to predict their potential binding sites.The content of CGA in the various tissues and genotypes analysed was more directly correlated with hqt1 expression levels. Moreover, transient and stable expression of HQT1 in Nicotiana benthamiana and in N. tabacum respectively, produced an increase in the content of CGA and cynarin, a derivative of CGA. Our findings indicate that both hqt1 and hqt2 are involved in the synthesis of CGA, but possibly at different steps of the metabolic pathway, and according to the plant exigencies. Moreover, the synthesis of cynarin (which is still unclear) might take place starting from CGA.

Polyphenols are widespread constituents of plants that have been used in the treatment ofdiseases for centuries. The current growing interest in dietary plants has led to renewed attention onartichoke, because of its high polyphenolic content. Polyphenolic artichoke extracts have surprisingpharmacological and biochemical effects, such as a marked antioxidative potential and cancerchemopreventive properties. Hence, there is considerable interest in improving our understanding ofpolyphenol biosynthesis and its regulation in artichoke, to enhance the levels of these bioactivemolecules in plants used as food, starting from the evaluation of phenolic content of differentgenotypes. A combination of genetic and biochemical analyses was used to help identifying genesinvolved in the accumulation of polyphenols and characterizing different artichoke varietal typesdiffused in the Mediterranean region by polyphenolic content.Six traditionally cultivated varieties of artichoke (Mola, Tondo di Paestum, Sant'Erasmo,Bianco di Ostuni, Blanca de Tudela and Violet de Provence) were selected, and qualitative andquantitative evaluations of phenolic profile were carried out on different artichoke tissues (threebract orders and receptacle of flower heads and leaves) in several developmental stages, by HPLCand mass spectrometry analyses. Our results show that polyphenols content mainly depend on thegenotype and part of the plants. Total polyphenols were more abundant in leaves than in heads,while cynarin and chlorogenic acid (CGA) were the most abundant hydroxycinnammates (mainly incapitula).The fine regulation of biosynthetic pathway leading to the accumulation of phenylpropanoidsis achieved by combinatorial actions of transcription factors (TF) belonging to various classes,among which MYB TF. Two putative MYB TF genes were isolated from artichoke (Tondo diPaestum and Locale di Mola), which showed a high similarity to MYB12 TF of other plant species(e.g. Arabidopsis, tomato). These genes were over-expressed under UV-light stress conditions.Total polyphenols content will be measured in artichoke tissues to possibly confirm correlation ofthe artichoke MYB factors with accumulation of these compounds. Heterologous expression inbacteria and quantitative real-time PCR are being performed to gain insights into the molecularmechanism of polyphenols regulation in this edible plant, contributing to future progress in thestudy of polyphenols accumulation.

L"Italia possiede il più ampio pool genico di carciofo coltivato, includendo così una ricca biodiversità cinaricola. Le regionicentro-meridionali conservano una moltitudine di tipi varietali ed ecotipi che si differenziano sulla base di tratti morfologicie per composizione chimica, e di conseguenza presentano diverse proprietà nutrizionali e attività farmacologiche. Il carciofoè una rilevante fonte di polifenoli, composti bioattivi dall"elevata capacità antiossidante e dunque molto salutari per l"uomo.Acquisire informazioni sul contenuto e sulla biosintesi di polifenoli in differenti varietà di carciofo, risulta quindi difondamentale importanza per la promozione e l"utilizzo di risorse genetiche autoctone di questa specie. A tale scopo sonostate effettuate dettagliate analisi biochimiche per caratterizzare sei diverse varietà di carciofo (Mola, Tondo di Paestum,Sant"Erasmo, Bianco di Ostuni, Blanca de Tudela e Violetto di Provenza) diffuse non solo in Italia ma anche in altri Paesidel bacino del Mediterraneo. Analisi qualitative e quantitative (HPLC-DAD-ESI-MS/MS) del profilo polifenolico diciascuna varietà hanno evidenziato che i polifenoli presenti in quantità maggiore sono acido clorogenico, cinarina, luteolin7-O-rutinoside e luteolin 7-O-glucoside. Inoltre il contenuto varia a seconda del genotipo e del tessuto preso inconsiderazione (Negro et al. 2012). Uno studio approfondito è stato da tempo avviato su diversi geni coinvolti nellaproduzione di polifenoli (Sonnante et al. 2008, Sonnante et al. 2010) e, più di recente, sulla regolazione della biosintesi ditali composti nei diversi tessuti di carciofo. Da una libreria di cloni ricombinanti BAC sono stati isolati due geni codificantiper putativi fattori di trascrizione MYB coinvolti nella biosintesi di flavonoli e proantocianidine. Tali geni sono staticaratterizzati strutturalmente. La loro espressione è stata analizzata in diversi tessuti di carciofo tramite PCR quantitativaReal-Time. Le corrispondenti proteine ricombinanti sono state inoltre espresse in batteri, per stabilirne la funzione in vitrotramite lo studio delle interazioni proteine/DNA mediante saggi di mobilità elettroforetica. Bibliografia: Negro et al. (2012)Journal of Food Science 77: 244-252. Sonnante et al. (2008) Physiologia Plantarum 132: 33-43. Sonnante et al. (2010) PlantPhysiology 153: 1224-1238.