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.

CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars.

Phytophthora infestans, the causal agent of late blight (LB) in tomato (Solanum lycopersicum L.), is a devastating disease and a serious concern for plant productivity. The presence of susceptibility (S) genes in plants facilitates pathogen proliferation; thus, disabling these genes may help provide a broad-spectrum and durable type of tolerance/resistance. Previous studies on Arabidopsis and tomato have highlighted that knock-out mutants of the PMR4 susceptibility gene are tolerant to powdery mildew. Moreover, PMR4 knock-down in potato has been shown to confer tolerance to LB. To verify the same effect in tomato in the present study, a CRISPR-Cas9 vector containing four single guide RNAs (sgRNAs: sgRNA1, sgRNA6, sgRNA7, and sgRNA8), targeting as many SlPMR4 regions, was introduced via Agrobacterium-tumefaciens-mediated transformation into two widely grown Italian tomato cultivars: 'San Marzano' (SM) and 'Oxheart' (OX). Thirty-five plants (twenty-six SM and nine OX) were selected and screened to identify the CRISPR/Cas9-induced mutations. The different sgRNAs caused mutation frequencies ranging from 22.1 to 100% and alternatively precise insertions (sgRNA6) or deletions (sgRNA7, sgRNA1, and sgRNA8). Notably, sgRNA7 induced in seven SM genotypes a -7 bp deletion in the homozygous status, whereas sgRNA8 led to the production of fifteen SM genotypes with a biallelic mutation (-7 bp and -2 bp). Selected edited lines were inoculated with P. infestans, and four of them, fully knocked out at the PMR4 locus, showed reduced disease symptoms (reduction in susceptibility from 55 to 80%) compared to control plants. The four SM lines were sequenced using Illumina whole-genome sequencing for deeper characterization without exhibiting any evidence of mutations in the candidate off-target regions. Our results showed, for the first time, a reduced susceptibility to Phytophtora infestans in pmr4 tomato mutants confirming the role of KO PMR4 in providing broad-spectrum protection against pathogens.

Dual catalytic activity of hydroxycinnamoyl-coenzyme A quinate transferase from tomato allows it to moonlight in the synthesis of both mono- and dicaffeoylquinic acids.

Tomato (Solanum lycopersicum), like other Solanaceous species, accumulates high levels of antioxidant caffeoylquinic acids, which are strong bioactive molecules and protect plants against biotic and abiotic stresses. Among these compounds, the monocaffeoylquinic acids (e.g. chlorogenic acid [CGA]) and the dicaffeoylquinic acids (diCQAs) have been found to possess marked antioxidative properties. Thus, they are of therapeutic interest both as phytonutrients in foods and as pharmaceuticals. Strategies to increase diCQA content in plants have been hampered by the modest understanding of their biosynthesis and whether the same pathway exists in different plant species. Incubation of CGA with crude extracts of tomato fruits led to the formation of two new products, which were identified by liquid chromatography-mass spectrometry as diCQAs. This chlorogenate:chlorogenate transferase activity was partially purified from ripe fruit. The final protein fraction resulted in 388-fold enrichment of activity and was subjected to trypsin digestion and mass spectrometric sequencing: a hydroxycinnamoyl-Coenzyme A:quinate hydroxycinnamoyl transferase (HQT) was selected as a candidate protein. Assay of recombinant HQT protein expressed in Escherichia coli confirmed its ability to synthesize diCQAs in vitro. This second activity (chlorogenate:chlorogenate transferase) of HQT had a low pH optimum and a high Km for its substrate, CGA. High concentrations of CGA and relatively low pH occur in the vacuoles of plant cells. Transient assays demonstrated that tomato HQT localizes to the vacuole as well as to the cytoplasm of plant cells, supporting the idea that in this species, the enzyme catalyzes different reactions in two subcellular compartments.

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.

The Population Structure of a Globe Artichoke Worldwide Collection, as Revealed by Molecular and Phenotypic Analyzes.

The knowledge of the organization of the domesticated gene pool of crop species is an essential requirement to understand crop evolution, to rationalize conservation programs, and to support practical decisions in plant breeding. Here, we integrate simple sequence repeat (SSR) analysis and phenotypic characterization to investigate a globe artichoke collection that comprises most of the varieties cultivated worldwide. We show that the cultivated gene pool of globe artichoke includes five distinct genetic groups associated with the major phenotypic typologies: Catanesi (which based on our analysis corresponds to Violetti di Provenza), Spinosi, Violetti di Toscana, Romaneschi, and Macau. We observed that 17 and 11% of the molecular and phenotypic variance, respectively, is between these groups, while within groups, strong linkage disequilibrium and heterozygote excess are evident. The divergence between groups for quantitative traits correlates with the average broad-sense heritability within the groups. The phenotypic divergence between groups for both qualitative and quantitative traits is strongly and positively correlated with SSR divergence (FST) between groups. All this implies a low population size and strong bottleneck effects, and indicates a long history of clonal propagation and selection during the evolution of the domesticated gene pool of globe artichoke. Moreover, the comparison between molecular and phenotypic population structures suggests that harvest time, plant architecture (i.e., plant height, stem length), leaf spininess, head morphology (i.e., head shape, bract shape, spininess) together with the number of heads per plant were the main targets of selection during the evolution of the cultivated germplasm. We emphasize our findings in light of the potential exploitation of this collection for association mapping studies.

In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation.

Gene editing has already proved itself as an invaluable tool for the generation of mutants for crop breeding, yet its ultimate impact on agriculture will depend on how crops generated by gene editing technologies are regulated, and on our ability to characterize the impact of mutations on plant phenotype. A starting operational strategy for evaluating gene editing-based approaches to plant breeding might consist of assessing the effect of the induced mutations in a crop- and locus-specific manner: this involves the analysis of editing efficiency in different cultivars of a crop, the assessment of potential off-target mutations, and a phenotypic evaluation of edited lines carrying different mutated alleles. Here, we targeted the GREENFLESH (GF) locus in two tomato cultivars ('MoneyMaker' and 'San Marzano') and evaluated the efficiency, specificity and mutation patterns associated with CRISPR/Cas9 activity for this gene. The GF locus encodes a Mg-dechelatase responsible for initiating chlorophyll degradation; in gf mutants, ripe fruits accumulate both carotenoids and chlorophylls. Phenotypic evaluations were conducted on two transgene-free T2 'MoneyMaker' gf lines with different mutant alleles (a small insertion of 1 nucleotide and a larger deletion of 123 bp). Both lines, in addition to reduced chlorophyll degradation, showed a notable increase in carotenoid and tocopherol levels during fruit ripening. Infection of gf leaves and fruits with Botrytis cinerea resulted in a significant reduction of infected area and pathogen proliferation compared to the wild type (WT). Our data indicates that the CRISPR/Cas9-mediated mutation of the GF locus in tomato is efficient, specific and reproducible and that the resulting phenotype is robust and consistent with previously characterized greenflesh mutants obtained with different breeding techniques, while also shedding light on novel traits such as vitamin E overaccumulation and pathogen resistance. This makes GF an appealing target for breeding tomato cultivars with improved features for cultivation, as well as consumer appreciation and health.

Grafting vigour is associated with DNA de-methylation in eggplant.

In horticulture, grafting is a popular technique used to combine positive traits from two different plants. This is achieved by joining the plant top part (scion) onto a rootstock which contains the stem and roots. Rootstocks can provide resistance to stress and increase plant production, but despite their wide use, the biological mechanisms driving rootstock-induced alterations of the scion phenotype remain largely unknown. Given that epigenetics plays a relevant role during distance signalling in plants, we studied the genome-wide DNA methylation changes induced in eggplant (Solanum melongena) scion using two interspecific rootstocks to increase vigour. We found that vigour was associated with a change in scion gene expression and a genome-wide hypomethylation in the CHH context. Interestingly, this hypomethylation correlated with the downregulation of younger and potentially more active long terminal repeat retrotransposable elements (LTR-TEs), suggesting that graft-induced epigenetic modifications are associated with both physiological and molecular phenotypes in grafted plants. Our results indicate that the enhanced vigour induced by heterografting in eggplant is associated with epigenetic modifications, as also observed in some heterotic hybrids.

Production of novel antioxidative phenolic amides through heterologous expression of the plant's chlorogenic acid biosynthesis genes in yeast.

Phenolic esters like chlorogenic acid play an important role in therapeutic properties of many plant extracts. We aimed to produce phenolic esters in baker's yeast, by expressing tobacco 4CL and globe artichoke HCT. Indeed yeast produced phenolic esters. However, the primary product was identified as N-(E)-p-coumaroyl-3-hydroxyanthranilic acid by NMR. This compound is an amide condensation product of p-coumaric acid, which was supplied to the yeast, with 3-hydroxyanthranilic acid, which was unexpectedly recruited from the yeast metabolism by the HCT enzyme. N-(E)-p-coumaroyl-3-hydroxyanthranilic acid has not been described before, and it shows structural similarity to avenanthramides, a group of inflammation-inhibiting compounds present in oat. When applied to mouse fibroblasts, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid induced a reduction of intracellular reactive oxygen species, indicating a potential therapeutic value for this novel compound.

MCSeEd (Methylation Context Sensitive Enzyme ddRAD): A New Method to Analyze DNA Methylation.

Methylation context sensitive enzyme ddRAD (MCSeEd) is a NGS-based method for genome-wide investigations of DNA methylation at different contexts requiring only low to moderate sequencing depth. It is particularly useful for identifying methylation changes in experimental systems challenged by biotic or abiotic stresses or at different developmental stages.

Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.

Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T1 and T2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.

Identification of a new R3 MYB type repressor and functional characterization of the members of the MBW transcriptional complex involved in anthocyanin biosynthesis in eggplant (S. melongena L.).

Here we focus on the highly conserved MYB-bHLH-WD repeat (MBW) transcriptional complex model in eggplant, which is pivotal in the transcriptional regulation of the anthocyanin biosynthetic pathway. Through a genome-wide approach performed on the recently released Eggplant Genome (cv. 67/3) previously identified, and reconfirmed by us, members belonging to the MBW complex (SmelANT1, SmelAN2, SmelJAF13, SmelAN1) were functionally characterized. Furthermore, a regulatory R3 MYB type repressor (SmelMYBL1), never reported before, was identified and characterized as well. Through a qPCR approach, we revealed specific transcriptional patterns of candidate genes in different plant tissue/organs at two stages of fruit development. Two strategies were adopted for investigating the interactions of bHLH partners (SmelAN1, SmelJAF13) with MYB counterparts (SmelANT1, SmelAN2 and SmelMYBL1): Yeast Two Hybrid (Y2H) and Bimolecular Fluorescent Complementation (BiFC) in A. thaliana mesophylls protoplast. Agro-infiltration experiments highlighted that N. benthamiana leaves transiently expressing SmelANT1 and SmelAN2 showed an anthocyanin-pigmented phenotype, while their co-expression with SmelMYBL1 prevented anthocyanin accumulation. Our results suggest that SmelMYBL1 may inhibits the MBW complex via the competition with MYB activators for bHLH binding site, although this hypothesis requires further elucidation.

The isolation and mapping of a novel hydroxycinnamoyltransferase in the globe artichoke chlorogenic acid pathway.

BACKGROUND: The leaves of globe artichoke and cultivated cardoon (Cynara cardunculus L.) have significant pharmaceutical properties, which mainly result from their high content of polyphenolic compounds such as monocaffeoylquinic and dicaffeoylquinic acid (DCQ), and a range of flavonoid compounds. RESULTS: Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase (HQT) encoding genes have been isolated from both globe artichoke and cultivated cardoon (GenBank accessions DQ915589 and DQ915590, respectively) using CODEHOP and PCR-RACE. A phylogenetic analysis revealed that their sequences belong to one of the major acyltransferase groups (anthranilate N-hydroxycinnamoyl/benzoyltransferase). The heterologous expression of globe artichoke HQT in E. coli showed that this enzyme can catalyze the esterification of quinic acid with caffeoyl-CoA or p-coumaroyl-CoA to generate, respectively, chlorogenic acid (CGA) and p-coumaroyl quinate. Real time PCR experiments demonstrated an increase in the expression level of HQT in UV-C treated leaves, and established a correlation between the synthesis of phenolic acids and protection against damage due to abiotic stress. The HQT gene, together with a gene encoding hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT) previously isolated from globe artichoke, have been incorporated within the developing globe artichoke linkage maps. CONCLUSION: A novel acyltransferase involved in the biosynthesis of CGA in globe artichoke has been isolated, characterized and mapped. This is a good basis for our effort to understand the genetic basis of phenylpropanoid (PP) biosynthesis in C. cardunculus.

Proteomics in globe artichoke: protein extraction and sample complexity reduction by PEG fractionation.

Here, we report the first leaf proteome analysis for globe artichoke. Three protein extraction protocols were tested and a reproducible Mg/NP-40-based method was established. Ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO) is a highly abundant leaf protein, and its presence masks co-localizing, less abundant proteins. To remove RuBisCO from the sample, and thereby improve spot resolution, a PEG fractionation approach was elaborated. 2-DE profiles of various PEG fractions showed that the fractionation procedure was successful in excluding most of the RuBisCO, allowing for the detection of many low-abundance proteins. Western blot analysis was able to confirm the reduction in RuBisCO content achieved by PEG fractionation. In all, 841 distinct protein spots were detected, and 40 of these, selected from the RuBisCO region of the 2-DE profile, were successfully identified by MS. A number of homologues of these proteins also co-localize with RuBisCO in Arabidopsis thaliana.

Isolation and mapping of a C3'H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress.

Globe artichoke represents a natural source of phenolic compounds with dicaffeoylquinic acids along with their biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the predominant molecules. We report the isolation and characterization of a full-length cDNA and promoter of a globe artichoke p-coumaroyl ester 3'-hydroxylase (CYP98A49), which is involved in both chlorogenic acid and lignin biosynthesis. Phylogenetic analyses demonstrated that this gene belongs to the CYP98 family. CYP98A49 was also heterologously expressed in yeast, in order to perform an enzymatic assay with p-coumaroylshikimate and p-coumaroylquinate as substrates. Real Time quantitative PCR analysis revealed that CYP98A49 expression is induced upon exposure to UV-C radiation. A single nucleotide polymorphism in the CYP98A49 gene sequence of two globe artichoke varieties used for genetic mapping allowed the localization of this gene to linkage group 10 within the previously developed maps.

Methylation content sensitive enzyme ddRAD (MCSeEd): a reference-free, whole genome profiling system to address cytosine/adenine methylation changes.

Methods for investigating DNA methylation nowadays either require a reference genome and high coverage, or investigate only CG methylation. Moreover, no large-scale analysis can be performed for N6-methyladenosine (6 mA) at an affordable price. Here we describe the methylation content sensitive enzyme double-digest restriction-site-associated DNA (ddRAD) technique (MCSeEd), a reduced-representation, reference-free, cost-effective approach for characterizing whole genome methylation patterns across different methylation contexts (e.g., CG, CHG, CHH, 6 mA). MCSeEd can also detect genetic variations among hundreds of samples. MCSeEd is based on parallel restrictions carried out by combinations of methylation insensitive and sensitive endonucleases, followed by next-generation sequencing. Moreover, we present a robust bioinformatic pipeline (available at https://bitbucket.org/capemaster/mcseed/src/master/ ) for differential methylation analysis combined with single nucleotide polymorphism calling without or with a reference genome.

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.

Identification of DNA methyltransferases and demethylases in Solanum melongena L., and their transcription dynamics during fruit development and after salt and drought stresses.

DNA methylation through the activity of cytosine-5-methyltransferases (C5-MTases) and DNA demethylases plays important roles in genome protection as well as in regulating gene expression during plant development and plant response to environmental stresses. In this study, we report on a genome-wide identification of six C5-MTases (SmelMET1, SmelCMT2, SmelCMT3a, SmelCMT3b, SmelDRM2, SmelDRM3) and five demethylases (SmelDemethylase_1, SmelDemethylase_2, SmelDemethylase_3, SmelDemethylase_4, SmelDemethylase_5) in eggplant. Gene structural characteristics, chromosomal localization and phylogenetic analyses are also described. The transcript profiling of both C5-MTases and demethylases was assessed at three stages of fruit development in three eggplant commercial F1 hybrids: i.e. 'Clara', 'Nite Lady' and 'Bella Roma', representative of the eggplant berry phenotypic variation. The trend of activation of C5-MTases and demethylase genes varied in function of the stage of fruit development and was genotype dependent. The transcription pattern of C5MTAses and demethylases was also assessed in leaves of the F1 hybrid 'Nite Lady' subjected to salt and drought stresses. A marked up-regulation and down-regulation of some C5-MTases and demethylases was detected, while others did not vary in their expression profile. Our results suggest a role for both C5-MTases and demethylases during fruit development, as well as in response to abiotic stresses in eggplant, and provide a starting framework for supporting future epigenetic studies in the species.

A chromosome-anchored eggplant genome sequence reveals key events in Solanaceae evolution.

With approximately 450 species, spiny Solanum species constitute the largest monophyletic group in the Solanaceae family, but a high-quality genome assembly from this group is presently missing. We obtained a chromosome-anchored genome assembly of eggplant (Solanum melongena), containing 34,916 genes, confirming that the diploid gene number in the Solanaceae is around 35,000. Comparative genomic studies with tomato (S. lycopersicum), potato (S. tuberosum) and pepper (Capsicum annuum) highlighted the rapid evolution of miRNA:mRNA regulatory pairs and R-type defense genes in the Solanaceae, and provided a genomic basis for the lack of steroidal glycoalkaloid compounds in the Capsicum genus. Using parsimony methods, we reconstructed the putative chromosomal complements of the key founders of the main Solanaceae clades and the rearrangements that led to the karyotypes of extant species and their ancestors. From 10% to 15% of the genes present in the four genomes were syntenic paralogs (ohnologs) generated by the pre-γ, γ and T paleopolyploidy events, and were enriched in transcription factors. Our data suggest that the basic gene network controlling fruit ripening is conserved in different Solanaceae clades, and that climacteric fruit ripening involves a differential regulation of relatively few components of this network, including CNR and ethylene biosynthetic genes.

Isolation and functional characterization of a cDNA coding a hydroxycinnamoyltransferase involved in phenylpropanoid biosynthesis in Cynara cardunculus L.

BACKGROUND: Cynara cardunculus L. is an edible plant of pharmaceutical interest, in particular with respect to the polyphenolic content of its leaves. It includes three taxa: globe artichoke, cultivated cardoon, and wild cardoon. The dominating phenolics are the di-caffeoylquinic acids (such as cynarin), which are largely restricted to Cynara species, along with their precursor, chlorogenic acid (CGA). The scope of this study is to better understand CGA synthesis in this plant. RESULTS: A gene sequence encoding a hydroxycinnamoyltransferase (HCT) involved in the synthesis of CGA, was identified. Isolation of the gene sequence was achieved by using a PCR strategy with degenerated primers targeted to conserved regions of orthologous HCT sequences available. We have isolated a 717 bp cDNA which shares 84% aminoacid identity and 92% similarity with a tobacco gene responsible for the biosynthesis of CGA from p-coumaroyl-CoA and quinic acid. In silico studies revealed the globe artichoke HCT sequence clustering with one of the main acyltransferase groups (i.e. anthranilate N-hydroxycinnamoyl/benzoyltransferase). Heterologous expression of the full length HCT (GenBank accession DQ104740) cDNA in E. coli demonstrated that the recombinant enzyme efficiently synthesizes both chlorogenic acid and p-coumaroyl quinate from quinic acid and caffeoyl-CoA or p-coumaroyl-CoA, respectively, confirming its identity as a hydroxycinnamoyl-CoA: quinate HCT. Variable levels of HCT expression were shown among wild and cultivated forms of C. cardunculus subspecies. The level of expression was correlated with CGA content. CONCLUSION: The data support the predicted involvement of the Cynara cardunculus HCT in the biosynthesis of CGA before and/or after the hydroxylation step of hydroxycinnamoyl esters.

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.