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Powdery mildew (PM) is an economically important foliar disease of cultivated cereals worldwide. The cultivation of disease-resistant varieties is considered the most efficient, sustainable and economical strategy for disease management. The objectives of the current study were to fine map the chromosomal region harboring the wild emmer PM resistance locus Pm36 and to identify candidate genes by exploiting the improved tetraploid wheat genomic resources. A set of backcross inbred lines (BILs) of durum wheat were genotyped with the SNP 25K chip array and comparison of the PM-resistant and susceptible lines defined a 1.5 cM region (physical interval of 1.08 Mb) harboring Pm36. The genetic map constructed with F2:3 progenies derived by crossing the PM resistant line 5BIL-42 and the durum parent Latino, restricted to 0.3 cM the genetic distance between Pm36 and the SNP marker IWB22904 (physical distance 0.515 Mb). The distribution of the marker interval including Pm36 in a tetraploid wheat collection indicated that the positive allele was largely present in the domesticated and wild emmer Triticum turgidum spp. dicoccum and ssp. dicoccoides. Ten high-confidence protein coding genes were identified in the Pm36 region of the emmer, durum and bread wheat reference genomes, while three added genes showed no homologous in the emmer genome. The tightly linked markers can be used for marker-assisted selection in wheat breeding programs, and as starting point for the Pm36 map-based cloning.

Wheat is the most widely grown crop and represents the staple food for one third of the world&rsquo, s population. Wheat is attacked by a large variety of pathogens and the use of resistant cultivars is an effective and environmentally safe strategy for controlling diseases and eliminating the use of fungicides. In this study, a collection of wild and cultivated tetraploid wheats (Triticum turgidum) were evaluated for seedling resistance (SR) and adult plant resistance (APR) to powdery mildew (Blumeria graminis) and genotyped with a 90K single nucleotide polymorphism (SNP) array to identify new sources of resistance genes. The genome-wide association mapping detected 18 quantitative trait loci (QTL) for APR and 8 QTL for SR, four of which were identical or at least closely linked to four QTL for APR. Thirteen candidate genes, containing nucleotide binding sites and leucine-rich repeats, were localized in the confidence intervals of the QTL-tagging SNPs. The marker IWB6155, associated to QPm.mgb-1AS, was located within the gene TRITD1Av1G004560 coding for a disease resistance protein. While most of the identified QTL were described previously, five QTL for APR (QPm.mgb-1AS, QPm.mgb-2BS, QPm.mgb-3BL.1, QPm.mgb-4BL, QPm.mgb-7BS.1) and three QTL for SR (QPm.mgb-3BL.3, QPm.mgb-5AL.2, QPm.mgb-7BS.2) were mapped on chromosome regions where no resistance gene was reported before. The novel QTL/genes can contribute to enriching the resistance sources available to breeders.

The cyclic nucleotide cAMP (3′,5′-cyclic adenosine monophosphate) is nowadays recognised as an important signalling molecule in plants, involved in many molecular processes, including sensing and response to biotic and abiotic environmental stresses. The validation of a functional cAMP-dependent signalling system in higher plants has spurred a great scientific interest on the polyhedral role of cAMP, as it actively participates in plant adaptation to external stimuli, in addition to the regulation of physiological processes. The complex architecture of cAMP-dependent pathways is far from being fully understood, because the actors of these pathways and their downstream target proteins remain largely unidentified. Recently, a genetic strategy was effectively used to lower cAMP cytosolic levels and hence shed light on the consequences of cAMP deficiency in plant cells. This review aims to provide an integrated overview of the current state of knowledge on cAMP’s role in plant growth and response to environmental stress. Current knowledge of the molecular components and the mechanisms of cAMP signalling events is summarised.

Cyclic AMP plays important roles in different physiological processes, including plant defence responses. However, as little information is known on plant enzymes responsible for cAMP production/degradation, studies of cAMP functions have relied, to date, on non-specific pharmacological approaches. We therefore 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 wild-type (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 h 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 is discussed in this paper. Moreover, in silico analysis revealed that the promoter regions of the genes coding for proteins uniquely accumulating in WT plants shared the CGCG motif, a target of the calcium-calmodulin-binding transcription factor AtSR1 (Arabidopsis thaliana signal responsive1). Therefore, following pathogen perception, the low free cAMP content, altering timing and levels of defence signals, and likely acting in part through the mis-regulation of AtSR1 activity, affected the speed and strength of the immune response.

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.

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.

The presence of health promoting compounds in artichoke, such as polyphenols, is frequently a matter of study. Particularly mono- and di-caffeoylquinic acids (e.g., chlorogenic acid, cynarin), caffeic acid and flavonoids (e.g., luteolin-7-O-glucoside, naringerin) contribute to artichoke nutraceutical qualities. A combination of genetic and biochemical analyses was used to help identify genes involved in the accumulation of polyphenols and to characterize different artichoke varietal types diffused in the Mediterranean region by polyphenolic content. Six traditionally cultivated cultivars of artichoke (‘Mola’, ‘Tondo di Paestum’, ‘Sant’Erasmo’, ‘Bianco di Ostuni’, ‘Blanca de Tudela’ and ‘Violet de Provence’) were selected, and qualitative and quantitative evaluations of phenolic profile were carried out on different artichoke tissues (three bract orders and receptacle of flower heads and leaves) in several developmental stages, by HPLC and mass spectrometry analyses. To gain insight into the mechanisms which regulate polyphenol synthesis, in silico analysis of promoter regions of genes involved in the biosynthesis of chlorogenic acid were performed. These analyses led us to detected different binding sites recognized by MYB transcription factors (TF), suggesting that, as in other plant species, this class of TF might be involved in the regulation of these artichoke genes. Two putative artichoke MYB TF genes were isolated, starting from sequences of the ortholog MYB12 from other species (e.g., Arabidopsis, tomato) used to screen artichoke EST database.

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 improvement of wheat end-products.

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 anti-trypsin 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_BBI_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. Five BBI sequences, representing five different second reactive sites, were heterologously expressed in the yeast Pichia pastoris. The recombinant proteins demonstrated to be active against trypsin, while three of them were also active against chymotrypsin, and one against human leukocyte elastase. Comparative modeling and protein docking were used to further investigate interactions between two grass pea BBI isoforms and their target proteases. Thus two reliable 3D models have been proposed, representing two potential ternary complexes, each constituted of an inhibitor and its target enzymes.

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.

The Arabidopsis thaliana L. genome contains 58 membrane proteins belonging to the mitochondrial carrier family. Two mitochondrial carrier family members, here named AtNDT1 and AtNDT2, exhibit high structural similarities to the mitochondrial nicotinamide adenine dinucleotide (NAD(+)) carrier ScNDT1 from bakers' yeast. Expression of AtNDT1 or AtNDT2 restores mitochondrial NAD(+) transport activity in a yeast mutant lacking ScNDT. Localization studies with green fluorescent protein fusion proteins provided evidence that AtNDT1 resides in chloroplasts, whereas only AtNDT2 locates to mitochondria. Heterologous expression in Escherichia coli followed by purification, reconstitution in proteoliposomes, and uptake experiments revealed that both carriers exhibit a submillimolar affinity for NAD(+) and transport this compound in a counter-exchange mode. Among various substrates ADP and AMP are the most efficient counter-exchange substrates for NAD(+). Atndt1- and Atndt2-promoter-GUS plants demonstrate that both genes are strongly expressed in developing tissues and in particular in highly metabolically active cells. The presence of both carriers is discussed with respect to the subcellular localization of de novo NAD(+) biosynthesis in plants and with respect to both the NAD(+)-dependent metabolic pathways and the redox balance of chloroplasts and mitochondria.

Despite the fundamental importance and high level of compartmentation of mitochondrial nucleotide metabolism in plants, our knowledge concerning the transport of nucleotides across intracellular membranes remains far from complete. Study of a previously uncharacterized Arabidopsis (Arabidopsis thaliana) gene (At4g01100) revealed it to be a novel adenine nucleotide transporter, designated ADNT1, belonging to the mitochondrial carrier family. The ADNT1 gene shows broad expression at the organ level. Green fluorescent protein-based cell biological analysis demonstrated targeting of ADNT1 to mitochondria. While analysis of the expression of beta-glucuronidase fusion proteins suggested that it was expressed across a broad range of tissue types, it was most highly expressed in root tips. Direct transport assays with recombinant and reconstituted ADNT1 were utilized to demonstrate that this protein displays a relatively narrow substrate specificity largely confined to adenylates and their closest analogs. ATP uptake was markedly inhibited by the presence of other adenylates and general inhibitors of mitochondrial transport but not by bongkrekate or carboxyatractyloside, inhibitors of the previously characterized ADP/ATP carrier. Furthermore, the kinetics are substantially different from those of this carrier, with ADNT1 preferring AMP to ADP. Finally, isolation and characterization of a T-DNA insertional knockout mutant of ADNT1, alongside complementation and antisense approaches, demonstrated that although deficiency of this transporter did not seem to greatly alter photosynthetic metabolism, it did result in reduced root growth and respiration. These findings are discussed in the context of a potential function for ADNT1 in the provision of the energy required to support growth in heterotrophic plant tissues.

Cyclic adenosine 3',5'-monophosphate (cAMP) 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 not 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.

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.

Summary The malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspar- tate-glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identi- fied Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomy- ces cerevisiae . On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast.

La biodiversità rappresenta la variabilità di tutti gli esseri viventi e può essere riferita agli ecosistemi, alle specie, o a livelli di rango tassonomico infraspecifico, quali le varietà. In particolare, la biodiversità vegetale di interesse agrario è riferita alle specie 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 Puglia ha finanziato il progetto BiodiverSO - Biodiversità delle specie orticole della Puglia - con l"obiettivo primario di salvaguardare le risorse genetiche orticole della regione. Infatti, a tutt"oggi, sul territorio pugliese non è stata effettuata un"indagine capillare e sistematica delle risorse genetiche ortive presenti. Le azioni di conservazione del germoplasma sono spesso 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 ed Università. La rivalutazione di genotipi di varietà locali e/o il loro risanamento possono ampliare la base genetica del comparto orticolo consentendo migliore tolleranza agli stress biotici e abiotici, in un periodo in cui si assiste a notevoli cambiamenti climatici, per salvaguardare la salute del consumatore e l"ambiente, nonché per valorizzare alcune produzioni tipiche pugliesi. L"Italia meridionale rappresenta il centro di domesticazione e/o diversificazione di alcune colture, quali ad esempio alcune Brassicacee, il carciofo, il melone, e questo significa che le nostre regioni sono estremamente ricche di biodiversità di interesse agrario. La Puglia produce numerose varietà coltivate di ortaggi come la carota di Polignano e di Sant"Ippazio; la cipolla di Acquaviva e di Margherita; il cavolfiore, cavolo broccolo (cima nera, cavolo riccio, mugnoli) e cima di rapa; il melone immaturo (carosello e barattiere) e d"inverno; la cicoria (di Molfetta, di Galatina, di Otranto); il carciofo (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 da varietà moderne. La principale finalità del progetto integrato BiodiverSO è quella di contribuire a raggiungere una significativa riduzione del tasso attuale di erosione della biodiversità delle specie orticole pugliesi. Dopo aver reperito sul territorio pugliese le risorse genetiche orticole a rischio, esse saranno catalogate mediante strumenti informatizzati e caratterizzate sia dal punto di vista morfo-agronomico che nutrizionale, che genetico-molecolare. Il materiale genetico raccolto verrà conservato presso le banche del germoplasma del CNR-IBBR di Bari e dell"Università di Bari. La conservazione ex situ, oltre a garantire l"uso del germoplasma, avrà il ruolo di salvaguardare dall"estinzione le varietà minacciate di scomparsa per poterne tentare una successiva reintroduzione. Inoltre il progetto prevede la conservazione on farm delle risorse genetiche, affinché venga il più possibile assicurata la continuità della coltivazione e dell"utilizzo di tali varietà locali.

L"Italia possiede il più ampio pool genico di carciofo coltivato, includendo così una ricca biodiversità cinaricola. Le regioni centro-meridionali conservano una moltitudine di tipi varietali ed ecotipi che si differenziano sulla base di tratti morfologici e per composizione chimica, e di conseguenza presentano diverse proprietà nutrizionali e attività farmacologiche. Il carciofo è 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 di fondamentale importanza per la promozione e l"utilizzo di risorse genetiche autoctone di questa specie. A tale scopo sono state 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 Paesi del bacino del Mediterraneo. Analisi qualitative e quantitative (HPLC-DAD-ESI-MS/MS) del profilo polifenolico di ciascuna varietà hanno evidenziato che i polifenoli presenti in quantità maggiore sono acido clorogenico, cinarina, luteolin 7-O-rutinoside e luteolin 7-O-glucoside. Inoltre il contenuto varia a seconda del genotipo e del tessuto preso in considerazione (Negro et al. 2012). Uno studio approfondito è stato da tempo avviato su diversi geni coinvolti nella produzione di polifenoli (Sonnante et al. 2008, Sonnante et al. 2010) e, più di recente, sulla regolazione della biosintesi di tali composti nei diversi tessuti di carciofo. Da una libreria di cloni ricombinanti BAC sono stati isolati due geni codificanti per putativi fattori di trascrizione MYB coinvolti nella biosintesi di flavonoli e proantocianidine. Tali geni sono stati caratterizzati strutturalmente. La loro espressione è stata analizzata in diversi tessuti di carciofo tramite PCR quantitativa Real-Time. Le corrispondenti proteine ricombinanti sono state inoltre espresse in batteri, per stabilirne la funzione in vitro tramite 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) Plant Physiology 153: 1224-1238.

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.

Despite much study of the role of S-adenosylmethionine (SAM) in the methylation of DNA, RNA, and proteins, and as a cofactor for a wide range of biosynthetic processes, little is known concerning the intracellular transport of this essential metabolite. Screening of the Arabidopsis (Arabidopsis thaliana) genome yielded two potential homologs of yeast (Saccharomyces cerevisiae) and human SAM transporters, designated as SAMC1 and SAMC2, both of which belong to the mitochondrial carrier protein family. The SAMC1 gene is broadly expressed at the organ level, although only in specialized tissues of roots with high rates of cell division, and appears to be up-regulated in response to wounding stress, whereas the SAMC2 gene is very poorly expressed in all organs/tissues analyzed. Direct transport assays with the recombinant and reconstituted SAMC1 were utilized to demonstrate that this protein displays a very narrow substrate specificity confined to SAM and its closest analogs. Further experiments revealed that SAMC1 was able to function in uniport and exchange reactions and characterized the transporter as highly active, but sensitive to physiologically relevant concentrations of S-adenosylhomocysteine, S-adenosylcysteine, and adenosylornithine. Green fluorescent protein-based cell biological analysis demonstrated targeting of SAMC1 to mitochondria. Previous proteomic analyses identified this protein also in the chloroplast inner envelope. In keeping with these results, bioinformatics predicted dual localization for SAMC1. These findings suggest that the provision of cytosolically synthesized SAM to mitochondria and possibly also to plastids is mediated by SAMC1 according to the relative demands for this metabolite in the organelles.