Aglianico Grape Seed Semi-Polar Extract Exerts Anticancer Effects by Modulating MDM2 Expression and Metabolic Pathways.

Grapevine (Vitis vinifera L.) seeds are rich in polyphenols including proanthocyanidins, molecules with a variety of biological effects including anticancer action. We have previously reported that the grape seed semi-polar extract of Aglianico cultivar (AGS) was able to induce apoptosis and decrease cancer properties in different mesothelioma cell lines. Concomitantly, this extract resulted in enriched oligomeric proanthocyanidins which might be involved in determining the anticancer activity. Through transcriptomic and metabolomic analyses, we investigated in detail the anticancer pathway induced by AGS. Transcriptomics analysis and functional annotation allowed the identification of the relevant causative genes involved in the apoptotic induction following AGS treatment. Subsequent biological validation strengthened the hypothesis that MDM2 could be the molecular target of AGS and that it could act in both a p53-dependent and independent manner. Finally, AGS significantly inhibited tumor progression in a xenograft mouse model of mesothelioma, confirming also in vivo that MDM2 could act as molecular player responsible for the AGS antitumor effect. Our findings indicated that AGS, exerting a pro-apoptotic effect by hindering MDM2 pathway, could represent a novel source of anticancer molecules.

Genetic and epigenetic dynamics affecting anthocyanin biosynthesis in potato cell culture.

Anthocyanins are antioxidant pigments widely used in drugs and food preparations. Flesh-coloured tubers of the cultivated potato Solanum tuberosum are important sources of different anthocyanins. Due to the high degree of decoration achieved by acylation, anthocyanins from potato are very stable and suitable for the food processing industry. The use of cell culture allows to extract anthocyanins on-demand, avoiding seasonality and consequences associated with land-based-tuber production. However, a well-known limit of cell culture is the metabolic instability and loss of anthocyanin production during successive subcultures. To get a general picture of mechanisms responsible for this instability, we explored both genetic and epigenetic regulation that may affect anthocyanin production in cell culture. We selected two clonally related populations of anthocyanin-producing (purple) and non-producing (white) potato cells. Through targeted molecular investigations, we identified and functionally characterized an R3-MYB, here named StMYBATV. This transcription factor can interact with bHLHs belonging to the MBW (R2R3-MYB, bHLH and WD40) anthocyanin activator complex and, potentially, may interfere with its formation. Genome methylation analysis revealed that, for several genomic loci, anthocyanin-producing cells were more methylated than clonally related white cells. In particular, we localized some methylation events in ribosomal protein-coding genes. Overall, our study explores novel molecular aspects associated with loss of anthocyanins in cell culture systems.

WRKY genes family study reveals tissue-specific and stress-responsive TFs in wild potato species.

Wild potatoes, as dynamic resource adapted to various environmental conditions, represent a powerful and informative reservoir of genes useful for breeding efforts. WRKY transcription factors (TFs) are encoded by one of the largest families in plants and are involved in several biological processes such as growth and development, signal transduction, and plant defence against stress. In this study, 79 and 84 genes encoding putative WRKY TFs have been identified in two wild potato relatives, Solanum commersonii and S. chacoense. Phylogenetic analysis of WRKY proteins divided ScWRKYs and SchWRKYs into three Groups and seven subGroups. Structural and phylogenetic comparative analyses suggested an interspecific variability of WRKYs. Analysis of gene expression profiles in different tissues and under various stresses allowed to select ScWRKY045 as a good candidate in wounding-response, ScWRKY055 as a bacterial infection triggered WRKY and ScWRKY023 as a multiple stress-responsive WRKY gene. Those WRKYs were further studied through interactome analysis allowing the identification of potential co-expression relationships between ScWRKYs/SchWRKYs and genes of various pathways. Overall, this study enabled the discrimination of WRKY genes that could be considered as potential candidates in both breeding programs and functional studies.

Comparative Phytochemical Characterization, Genetic Profile, and Antiproliferative Activity of Polyphenol-Rich Extracts from Pigmented Tubers of Different Solanum tuberosum Varieties.

Plants produce a vast array of biomolecules with beneficial effects for human health. In this study, polyphenol and anthocyanin-rich extracts (PAE) from pigmented tubers of Solanum tuberosum L. varieties "Blue Star", "Magenta Love", and "Double Fun" in comparison with the more extensively studied "Vitelotte" were evaluated and compared for antiproliferative effects in human leukemia cells, and their phytochemical and genetic profiles were determined. In U937 cells, upon treatment with PAE, it was possible to reveal the expression of specific apoptotic players, such as caspase 8, 9, 3, and poly (ADP-ribose) polymerase (PARP), as well as the induction of monocyte and granulocyte differentiation. A liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) investigation revealed the presence of polyphenolic compounds in all the varieties of potatoes analyzed, among which caffeoyl and feruloyl quinic acid derivatives were the most abundant, as well as several acylated anthocyanins. Each pigmented variety was genotyped by DNA-based molecular markers, and flavonoid-related transcription factors were profiled in tubers in order to better characterize these outstanding resources and contribute to their exploitation in breeding. Interesting biological activities were observed for "Blue Star" and "Vitelotte" varieties with respect to the minor or no effect of the "Double Fun" variety.

LTR-TEs abundance, timing and mobility in Solanum commersonii and S. tuberosum genomes following cold-stress conditions.

MAIN CONCLUSION: Copia/Ale is the youngest lineage in both Solanum tuberosum and S. commersonii. Within it, we identified nightshade, a new LTR element active in the cultivated potato. From an evolutionary perspective, long-terminal repeat retrotransposons (LTR-RT) activity during stress may be viewed as a mean by which organisms can keep up rates of genetic adaptation to changing conditions. Potato is one of the most important crop consumed worldwide, but studies on LTR-RT characterization are still lacking. Here, we assessed the abundance, insertion time and activity of LTR-RTs in both cultivated Solanum tuberosum and its cold-tolerant wild relative S. commersonii genomes. Gypsy elements were more abundant than Copia ones, suggesting that the former was somehow more successful in colonizing potato genomes. However, Copia elements, and in particular, the Ale lineage, are younger than Gypsy ones, since their insertion time was in average ~ 2 Mya. Due to the ability of LTR-RTs to be circularized by the host DNA repair mechanisms, we identified via mobilome-seq a Copia/Ale element (called nightshade, informal name used for potato family) active in S. tuberosum genome. Our analyses represent a valuable resource for comparative genomics within the Solanaceae, transposon-tagging and for the design of cultivar-specific molecular markers in potato.

Dicer-like and RNA-dependent RNA polymerase gene family identification and annotation in the cultivated Solanum tuberosum and its wild relative S. commersonii.

MAIN CONCLUSION: We provide advances in DCL and RDR gene diversity in Solanaceae. We also shed light on DCL and RDR gene expression in response to cold stress. DICER-like (DCL) and RNA-dependent RNA polymerase (RDR) genes form the core components to trigger small non-coding RNA (ncRNA) production. In spite of this, little is known about the two gene families in non-model plant species. As their genome sequences are now available, the cultivated potato (Solanum tuberosum) and its cold-tolerant wild relative Solanum commersonii offer a valuable opportunity to advance our understanding of the above genes. To determine the extent of diversification and evolution of DCLs and RDRs in these species, we performed a comparative analysis. Seven DCLs were identified in the two species, whereas seven and six RDR genes were found in S. tuberosum and S. commersonii, respectively. Based on phylogenetic analysis with DCLs and RDRs from several species, we provide evidence for an increase in their number in both potato species. We also disclosed that tandem duplications played a major role in the evolution of these gene families in Solanaceae. DCL and RDR expression was investigated in different tissues and under cold and virus stresses, with divergent profiles of the tandem duplicated genes being found in different tissues. DCL paralogs showed a contrasting expression in S. tuberosum and S. commersonii following cold stress and virus infection. By contrast, no change in RDR transcript activity was detected following both stresses. Overall, this study provides the first comparative genomic analysis of the core components of the RNAi machinery in Solanaceae and offers a scaffold for future functional analysis of these gene families.

Subfunctionalization of duplicate MYB genes in Solanum commersonii generated the cold-induced ScAN2 and the anthocyanin regulator ScAN1.

Wild potato species are useful sources of allelic diversity and loci lacking in the cultivated potato. In these species, the presence of anthocyanins in leaves has been associated with a greater tolerance to cold stress. However, the molecular mechanisms that allow potatoes to withstand cold exposure remain unclear. Here, we show that the expression of AN2, a MYB transcription factor, is induced by low temperatures in wild, cold-tolerant Solanum commersonii, and not in susceptible Solanum tuberosum varieties. We found that AN2 is a paralog of the potato anthocyanin regulator AN1, showing similar interaction ability with basic helix-loop-helix (bHLH) co-partners. Their sequence diversity resulted in a different capacity to promote accumulation of phenolics when tested in tobacco. Indeed, functional studies demonstrated that AN2 is less able to induce anthocyanins than AN1, but nevertheless it has a strong ability to induce accumulation of hydroxycinnamic acid derivatives. We propose that the duplication of R2R3 MYB genes resulted in subsequent subfunctionalization, where AN1 specialized in anthocyanin production and AN2 conserved the ability to respond to cold stress, inducing mainly the synthesis of hydroxycinnamic acid derivatives. These results contribute to understanding the evolutionary significance of gene duplication on phenolic compound regulation.

Microstructure and tuber properties of potato varieties with different genetic profiles.

The objectives of this research were to study tuber starch characteristics and chemical - thermal properties of 21 potato varieties, and to determine their genetic diversity through SSR markers. Starch granular size varied among samples, with a wide diameter distribution (5-85µm), while granule shapes were similar. Differential Scanning Calorimeter analysis showed that the transition temperatures (69°C-74°C) and enthalpies of gelatinization (0.9J/g-3.8J/g) of tubers were also variety dependent. SSR analysis allowed the detection of 157 alleles across all varieties, with an average value of 6.8 alleles per locus. Variety-specific alleles were also identified. SSR-based cluster analysis revealed that varieties with interesting quality attributes were distributed among all clusters and sub-clusters, suggesting that the genetic basis of traits analyzed may differ among our varieties. The information obtained in this study may be useful to identify and develop varieties with slowly digestible starch.

Dating the beginning of the Roman viticultural model in the Western Mediterranean: The case study of Chianti (Central Italy).

Although domestication of the grapevine (Vitis vinifera L.) has been extensively documented, the history of genotype selection and evolution of vineyard management remain relatively neglected fields of study. The find of 454 waterlogged grapevine pips from a well-dated Etrusco-Roman site in the Chianti district (Tuscany, Central Italy) is an extraordinary chance to gain insights into the progress of viticulture occurring in a key historical period in one of the world's most famous wine regions. The molecular and geometrical analyses of grape seeds showed (a) the presence in the site of different grapevine individuals and (b) a sudden increase in pip size, occurring at around 200 BC, whic explainable by the selection and introduction of new varieties. In this period, the Etruscans settlers in Chianti were stimulated by northward-expanding Roman culture to use novel vineyard management practices. We hypothesize that one of the most important innovations may have been the introduction of pruning, inducing vine physiological conditions more favorable to pip growth. Such changes were the consequence of specific entrepreneurial choices made by the Romans in a period of economic investment in grape cultivation and wine making to satisfy the increased trade demand after the conquest of the Central-Western Mediterranean basin.

Comparative metabolite and genome analysis of tuber-bearing potato species.

The cultivated potato Solanum tuberosum is unrivalled among crop plants for its wild relatives, which potentially represent an important source of genetic diversity to improve the nutritional value of potato varieties and understand metabolism regulation. The main aim of this research was to profile human health-related metabolites in a number of clones from 13 Solanum species. Results from HPLC-DAD and LC-ESI-MS analyses highlighted a high interspecific variability in the level of metabolites analysed. Ascorbic acid was confirmed to be the most abundant antioxidant in potato and chlorogenic acid the primary polyphenol. Generally, metabolite-based hierarchical clustering (HCL) and correlation networks did not group clones of identical species in the same cluster. This might be due to various factors, including the outcrossing nature of potato species, gene expression level and metabolic profiling techniques. Access to the genome sequence of S. tuberosum and S. commersonii allowed comparison of the genes involved in ascorbic acid, aromatic amino acid, phenylpropanoid and glycoalkaloid biosynthesis and helped interpret their respective pathways.

Transcriptome and metabolome of synthetic Solanum autotetraploids reveal key genomic stress events following polyploidization.

Polyploids are generally classified as autopolyploids, derived from a single species, and allopolyploids, arising from interspecific hybridization. The former represent ideal materials with which to study the consequences of genome doubling and ascertain whether there are molecular and functional rules operating following polyploidization events. To investigate whether the effects of autopolyploidization are common to different species, or if species-specific or stochastic events are prevalent, we performed a comprehensive transcriptomic and metabolomic characterization of diploids and autotetraploids of Solanum commersonii and Solanum bulbocastanum. Autopolyploidization remodelled the transcriptome and the metabolome of both species. In S. commersonii, differentially expressed genes (DEGs) were highly enriched in pericentromeric regions. Most changes were stochastic, suggesting a strong genotypic response. However, a set of robustly regulated transcripts and metabolites was also detected, including purine bases and nucleosides, which are likely to underlie a common response to polyploidization. We hypothesize that autopolyploidization results in nucleotide pool imbalance, which in turn triggers a genomic shock responsible for the stochastic events observed. The more extensive genomic stress and the higher number of stochastic events observed in S. commersonii with respect to S. bulbocastanum could be the result of the higher nucleoside depletion observed in this species.

Combined Use of Molecular Markers and High-Resolution Melting (HRM) to Assess Chromosome Dosage in Potato Hybrids.

In plants, the most widely used cytological techniques to assess parental genome contributions are based on in situ hybridization (FISH and GISH), but they are time-consuming and need specific expertise and equipment. Recent advances in genomics and molecular biology have made PCR-based markers a straightforward, affordable technique for chromosome typing. Here, we describe the development of a molecular assay that uses single-copy conserved ortholog set II (COSII)-based single nucleotide polymorphisms (SNPs) and the high-resolution melting (HRM) technique to assess the chromosome dosage of interspecific hybrids between a Solanum phureja-S. tuberosum diploid (2n = 2x = 24) hybrid and its wild relative S. commersonii. Screening and analysis of 45 COSII marker sequences allowed S. commersonii-specific SNPs to be identified for all 12 chromosomes. Combining the HRM technique with the establishment of synthetic DNA hybrids, SNP markers were successfully used to predict the expected parental chromosome ratio of 5 interspecific triploid hybrids. These results demonstrate the ability of this strategy to distinguish diverged genomes from each other, and to estimate chromosome dosage. The method could potentially be applied to any species as a tool to assess paternal to maternal ratios in the framework of a breeding program or following transformation techniques.

The Solanum commersonii Genome Sequence Provides Insights into Adaptation to Stress Conditions and Genome Evolution of Wild Potato Relatives.

Here, we report the draft genome sequence of Solanum commersonii, which consists of ∼830 megabases with an N50 of 44,303 bp anchored to 12 chromosomes, using the potato (Solanum tuberosum) genome sequence as a reference. Compared with potato, S. commersonii shows a striking reduction in heterozygosity (1.5% versus 53 to 59%), and differences in genome sizes were mainly due to variations in intergenic sequence length. Gene annotation by ab initio prediction supported by RNA-seq data produced a catalog of 1703 predicted microRNAs, 18,882 long noncoding RNAs of which 20% are shown to target cold-responsive genes, and 39,290 protein-coding genes with a significant repertoire of nonredundant nucleotide binding site-encoding genes and 126 cold-related genes that are lacking in S. tuberosum. Phylogenetic analyses indicate that domesticated potato and S. commersonii lineages diverged ∼2.3 million years ago. Three duplication periods corresponding to genome enrichment for particular gene families related to response to salt stress, water transport, growth, and defense response were discovered. The draft genome sequence of S. commersonii substantially increases our understanding of the domesticated germplasm, facilitating translation of acquired knowledge into advances in crop stability in light of global climate and environmental changes.

A DArT marker-based linkage map for wild potato Solanum bulbocastanum facilitates structural comparisons between Solanum A and B genomes.

BACKGROUND: Wild potato Solanum bulbocastanum is a rich source of genetic resistance against a variety of pathogens. It belongs to a taxonomic group of wild potato species sexually isolated from cultivated potato. Consistent with genetic isolation, previous studies suggested that the genome of S. bulbocastanum (B genome) is structurally distinct from that of cultivated potato (A genome). However, the genome architecture of the species remains largely uncharacterized. The current study employed Diversity Arrays Technology (DArT) to generate a linkage map for S. bulbocastanum and compare its genome architecture with those of potato and tomato. RESULTS: Two S. bulbocastanum parental linkage maps comprising 458 and 138 DArT markers were constructed. The integrated map comprises 401 non-redundant markers distributed across 12 linkage groups for a total length of 645 cM. Sequencing and alignment of DArT clones to reference physical maps from tomato and cultivated potato allowed direct comparison of marker orders between species. A total of nine genomic segments informative in comparative genomic studies were identified. Seven genome rearrangements correspond to previously-reported structural changes that have occurred since the speciation of tomato and potato. We also identified two S. bulbocastanum genomic regions that differ from cultivated potato, suggesting possible chromosome divergence between Solanum A and B genomes. CONCLUSIONS: The linkage map developed here is the first medium density map of S. bulbocastanum and will assist mapping of agronomical genes and QTLs. The structural comparison with potato and tomato physical maps is the first genome wide comparison between Solanum A and B genomes and establishes a foundation for further investigation of B genome-specific structural chromosome rearrangements.

High AN1 variability and interaction with basic helix-loop-helix co-factors related to anthocyanin biosynthesis in potato leaves.

AN1 is a regulatory gene that promotes anthocyanin biosynthesis in potato tubers and encodes a R2R3 MYB transcription factor. However, no clear evidence implicates AN1 in anthocyanin production in leaves, where these pigments might enhance environmental stress tolerance. In our study we found that AN1 displays intraspecific sequence variability in both coding/non-coding regions and in the promoter, and that its expression is associated with high anthocyanin content in leaves of commercial potatoes. Expression analysis provided evidence that leaf pigmentation is associated to AN1 expression and that StJAF13 acts as putative AN1 co-regulator for anthocyanin gene expression in leaves of the red leaf variety 'Magenta Love,' while a concomitant expression of StbHLH1 may contribute to anthocyanin accumulation in leaves of 'Double Fun.' Yeast two-hybrid experiments confirmed that AN1 interacts with StbHLH1 and StJAF13 and the latter interaction was verified and localized in the cell nucleus by bimolecular fluorescence complementation assays. In addition, transgenic tobacco (Nicotiana tabacum) overexpressing a combination of either AN1 with StJAF13 or AN1 with StbHLH1 showed deeper purple pigmentation with respect to AN1 alone. This further confirmed AN1/StJAF13 and AN1/StbHLH1 interactions. Our findings demonstrate that the classical loci identified for potato leaf anthocyanin accumulation correspond to AN1 and may represent an important step to expand our knowledge on the molecular mechanisms underlying anthocyanin biosynthesis in different plant tissues.

Biochemical features of native red wines and genetic diversity of the corresponding grape varieties from Campania region.

Campania region has always been considered one of the most appreciated Italian districts for wine production. Wine distinctiveness arises from their native grapevines. To better define the chemical profile of Campania autochthonous red grape varieties, we analysed the phenolic composition of Aglianico di Taurasi, Aglianico del Vulture, Aglianico del Taburno, Piedirosso wines, and a minor native variety, Lingua di Femmina in comparison with Merlot and Cabernet Sauvignon, as reference cultivars. A genetic profiling was also carried out using microsatellite molecular markers with high polymorphic and unambiguous profiles. Principal component analysis applied to 72 wines based on the 18 biochemical parameters, explained 77.6% of the total variance and highlighted important biological entities providing insightful patterns. Moreover, comparison of SSR-based data with phenylpropanoid molecules exhibited a statistically significant correlation. Our approach might be reasonably adopted for future characterisations and traceability of grapevines and corresponding wines.

Impact of ploidy change on secondary metabolites and photochemical efficiency in Solanum bulbocastanum.

Plants are well known for producing a wide diversity of natural compounds and several strategies have been proposed to enhance their production. Among them, somatic chromosome doubling may represent an effective and inexpensive method. The objective of the current study was to investigate the effect of polyploidization on the leaf metabolic profile and content of tetraploids produced from a wild diploid (2n=2x=24) potato species, Solanum bulbocastanum Dun. Photochemical efficiency of tetraploids was also analyzed. Results from HPLC-DAD and LC/MS analyses provided evidence that tetraploid genotypes displayed either a similar or a lower phenylpropanoids, tryptophan, tyrosine and a-chaconine content compared with the diploid parent. Similarly, no significant differences were found among genotypes both for measures of gas and for chlorophyll fluorescence, except for non-photochemical quenching (NPQ). Steroidal saponins content revealed superiority of some tetraploids with respect to the diploid parent, suggesting perturbations in the mechanism regulating the biosynthesis of such compounds following polyploidization. Lack of superiority may be attributed to the time required for adjustment, adaptation and evolution after the genomic shock induced by polyploidization, as well as the fact that an optimum ploidy level for each species may be crucial. Our results suggest that polyploidization as a strategy to enhance metabolite production cannot be generalized.

Stochastic changes affect Solanum wild species following autopolyploidization.

Polyploidy is very common within angiosperms, and several studies are in progress to ascertain the effects of early polyploidization at the molecular, physiological, and phenotypic level. Extensive studies are available only in synthetic allopolyploids. By contrast, less is known about the consequences of autopolyploidization. The current study aimed to assess the occurrence and extent of genetic, epigenetic, and anatomical changes occurring after oryzaline-induced polyploidization of Solanum commersonii Dunal and Solanum bulbocastanum Dunal, two diploid (2n=2×=24) potato species widely used in breeding programmes. Microsatellite analysis showed no polymorphisms between synthetic tetraploids and diploid progenitors. By contrast, analysis of DNA methylation levels indicated that subtle alterations at CG and CHG sites were present in tetraploids of both species. However, no change occurred concurrently in all tetraploids analysed with respect to their diploid parent, revealing a stochastic trend in the changes observed. The morpho-anatomical consequences of polyploidization were studied in leaf main veins and stomata. With only a few exceptions, analyses showed no clear superiority of tetraploids in terms of leaf thickness and area, vessel number, lumen size and vessel wall thickness, stomata pore length and width, guard cell width, and stomatal density compared with their diploid progenitors. These results are consistent with the hypothesis that there are no traits systematically associated with autopolyploidy.

Secondary metabolite profile in induced tetraploids of wild Solanum commersonii Dun.

The main aim of this work was to study the leaf secondary metabolite profiles of artificially induced tetraploids (2n=4x=48) of Solanum commersonii, a diploid (2n=2x=24) wild potato species. The tetraploid genotypes of S. commersonii were produced by oryzalin treatment. Both HPLC-UV and LC/MS analyses revealed that there were no qualitative differences in the metabolite profiles between the diploid S. commersonii and its tetraploids. By contrast, the results showed that the phenylpropanoid content was generally significantly higher in the tetraploids than in the diploid S. commersonii. Concerning the glycoalkaloids (GAs), the results provided evidence that the content of minor GAs (solanidenediol triose, solanidadienol lycotetraose, and solanidenol lycotetraose) was higher in tetraploids than in the diploid progenitor, while the content of major GAs (dehydrodemissine and dehydrocommersonine) was significantly higher in diploid S. commersonii than in its tetraploid genotypes. The results are discussed from the practical perspective of potato biodiversity enhancement.

Glycoalkaloid profile in potato haploids derived from solanum tuberosum-S. bulbocastanum somatic hybrids.

Cultivated and wild potato species synthesize a wide variety of steroidal glycoalkaloids (GA) that may affect either human health or biotic stress resistance. Therefore, GA composition must be a major criterion in the evaluation of breeding products when species genomes are merged and/or manipulated. This work reports the results of GA analysis performed on unique haploid (2n=2x=24) plants obtained from tetraploid (2n=4x=48) Solanum bulbocastanum-S. tuberosum hybrids through in vitro anther culture. Glycoalkaloids were extracted from tubers and analyzed by HPLC. Haploids generally showed the occurrence of parental GA. However, in several cases loss of parental GA and gain of new GA lacking in the parents was observed. It may be hypothesized that new GA profiles of our haploids is the result of either genetic recombination or combinatorial biochemistry events. To highlight differences between haploids and parents, soluble proteins and antioxidant activities were also determined. Both were always higher in haploids compared to their parents. The nature of the newly formed GAs will be further investigated, because they may represent new metabolites that can be used against pest and diseases, or are useful for human health.