Distinct structural variants and repeat landscape shape the genomes of the ancient grapes Aglianico and Falanghina.

Mounting evidence recognizes structural variations (SVs) and repetitive DNA sequences as crucial players in shaping the existing grape phenotypic diversity at intra- and inter-species levels. To deepen our understanding on the abundance, diversity, and distribution of SVs and repetitive DNAs, including transposable elements (TEs) and tandemly repeated satellite DNA (satDNAs), we re-sequenced the genomes of the ancient grapes Aglianico and Falanghina. The analysis of large copy number variants (CNVs) detected candidate polymorphic genes that are involved in the enological features of these varieties. In a comparative analysis of Aglianico and Falanghina sequences with 21 publicly available genomes of cultivated grapes, we provided a genome-wide annotation of grape TEs at the lineage level. We disclosed that at least two main clusters of grape cultivars could be identified based on the TEs content. Multiple TEs families appeared either significantly enriched or depleted. In addition, in silico and cytological analyses provided evidence for a diverse chromosomal distribution of several satellite repeats between Aglianico, Falanghina, and other grapes. Overall, our data further improved our understanding of the intricate grape diversity held by two Italian traditional varieties, unveiling a pool of unique candidate genes never so far exploited in breeding for improved fruit quality.

Functional characterization of DcMYB11, an R2R3 MYB associated with the purple pigmentation of carrot petiole.

MAIN CONCLUSION: DcMYB11, an R2R3 MYB gene associated with petiole anthocyanin pigmentation in carrot, was functionally characterized. A putative enhancer sequence is able to increase DcMYB11 activity. The accumulation of anthocyanin pigments can exhibit different patterns across plant tissues and crop varieties. This variability allowed the investigation of the molecular mechanisms behind the biosynthesis of these pigments in several plant species. Among crops, carrots have a well-defined anthocyanin pigmentation pattern depending on the genic background. In this work, we report on the discovery of DNA structural differences affecting the activity of an R2R3 MYB (encoded by DcMYB11) involved in anthocyanin regulation in carrot petiole. To this end, we first verified the function of DcMYB11 using heterologous systems and identified three different alleles which may explain differences in petiole pigmentation. Characterization of the DcMYB11 alleles at the 5' upstream sequence unveiled a sequence that functions as a putative enhancer. In conclusion, this study provides novel insight into the molecular mechanisms controlling anthocyanin accumulation in carrot. By these outcomes, we expanded our knowledge on the cis-regulatory sequences in plants.

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.

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.

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.

Cistus incanus: a natural source of antimicrobial metabolites.

The discovery of natural molecules with antimicrobial properties has become an urgent need for the global treatment of bacterium and virus infections. Cistus incanus, a Mediterranean shrub species, represents a valuable source of phytochemicals with an interesting wide-spectrum antimicrobial potential. In this study, we analysed the spectrum of molecules composing a commercial hydroalcoholic extract of C. incanus finding ellagitannins as the most abundant. The effect of the extract and its main constituents (gallic acid, ellagic acid and punicalin) was assessed as co-treatment during viral (HSV-1, HCoV-229E, SARS-CoV-2) and bacterial infection (Staphylococcus aureus and Escherichia coli) of cells and as pre-treatment before virus infections. The results indicated a remarkable antiviral activity of punicalin against SARS-CoV-2 by pre-treating both the viral and the host cells, and a major sensitivity of S. aureus to the C. incanus extract compared to E. coli. The present study highlights broad antimicrobial potential of C. incanus extract.

Targeted mutagenesis of StISAC stabilizes the production of anthocyanins in potato cell culture.

To increase the production of decorated anthocyanins in potato cell cultures, we knocked out a novel potato gene, named Inducer Silencing of Anthocyanins in Cell culture (StISAC), using CRISPR-Cas9 editing. Our results provided evidence that mutant cell lines doubled the accumulation level of anthocyanins biosynthesized. Moreover, the production of these important pigments was stabilized over time. Our study overcame important challenges in the efficient biotechnological production of these valuable pigments and reported the function of a novel anthocyanin biosynthesis repressor gene.

Biochemical Characterization and Effects of Cooking Methods on Main Phytochemicals of Red and Purple Potato Tubers, a Natural Functional Food.

Potato is a staple food crop and an important source of dietary energy. Its tubers contain several essential amino acids, vitamins, minerals and phytochemicals that contribute to the nutritional value of this important product. Recently, scientific interest has focused on purple and red potatoes that, due to the presence of anthocyanins, may be considered as natural powerful functional food. The aim of this study was to evaluate the characteristics of pigmented varieties, the types of anthocyanins accumulated and the level of both beneficial phytochemicals (vitamin C and chlorogenic acids, CGAs) and anti-nutritional compounds (glycoalkaloids) following various cooking methods. The analyses described the presence of a mix of several acylated anthocyanins in pigmented tubers along with high level of CGA. The amount of antioxidants was differently affected by heat treatments according to the type of molecule and the cooking methods used. In some cases, the beneficial compounds were made more available by heat treatments for the analytical detection as compared to raw materials. Data reported here describe both the agronomic properties of these pigmented varieties and the effects of food processing methods on bioactive molecules contained in this natural functional food. They may provide useful information for breeders aiming to develop new varieties that could include desirable agronomical and industrial processing traits.

Abiotic Stresses Elicitation Potentiates the Productiveness of Cardoon Calli as Bio-Factories for Specialized Metabolites Production.

Cultivated cardoon (Cynara cardunculus L. var altilis) is a Mediterranean traditional food crop. It is adapted to xerothermic conditions and also grows in marginal lands, producing a large biomass rich in phenolic bioactive metabolites and has therefore received attention for pharmaceutical, cosmetic and innovative materials applications. Cardoon cell cultures can be used for the biotechnological production of valuable molecules in accordance with the principles of cellular agriculture. In the current study, we developed an elicitation strategy on leaf-derived cardoon calli for boosting the production of bioactive extracts for cosmetics. We tested elicitation conditions that trigger hyper-accumulation of bioactive phenolic metabolites without compromising calli growth through the application of chilling and salt stresses. We monitored changes in growth, polyphenol accumulation, and antioxidant capability, along with transcriptional variations of key chlorogenic acid and flavonoids biosynthetic genes. At moderate stress intensity and duration (14 days at 50-100 mM NaCl) salt exerted the best eliciting effect by stimulating total phenols and antioxidant power without impairing growth. Hydroalcoholic extracts from elicited cardoon calli with optimal growth and bioactive metabolite accumulation were demonstrated to lack cytotoxicity by MTT assay and were able to stimulate pro-collagen and aquaporin production in dermal cells. In conclusion, we propose a "natural" elicitation system that can be easily and safely employed to boost bioactive metabolite accumulation in cardoon cell cultures and also in pilot-scale cell culture production.

Specialized Metabolites and Valuable Molecules in Crop and Medicinal Plants: The Evolution of Their Use and Strategies for Their Production.

Plants naturally produce a terrific diversity of molecules, which we exploit for promoting our overall well-being. Plants are also green factories. Indeed, they may be exploited to biosynthesize bioactive molecules, proteins, carbohydrates and biopolymers for sustainable and large-scale production. These molecules are easily converted into commodities such as pharmaceuticals, antioxidants, food, feed and biofuels for multiple industrial processes. Novel plant biotechnological, genetics and metabolic insights ensure and increase the applicability of plant-derived compounds in several industrial sectors. In particular, synergy between disciplines, including apparently distant ones such as plant physiology, pharmacology, 'omics sciences, bioinformatics and nanotechnology paves the path to novel applications of the so-called molecular farming. We present an overview of the novel studies recently published regarding these issues in the hope to have brought out all the interesting aspects of these published studies.

Anthocyanins are Key Regulators of Drought Stress Tolerance in Tobacco.

Abiotic stresses will be one of the major challenges for worldwide food supply in the near future. Therefore, it is important to understand the physiological mechanisms that mediate plant responses to abiotic stresses. When subjected to UV, salinity or drought stress, plants accumulate specialized metabolites that are often correlated with their ability to cope with the stress. Among them, anthocyanins are the most studied intermediates of the phenylpropanoid pathway. However, their role in plant response to abiotic stresses is still under discussion. To better understand the effects of anthocyanins on plant physiology and morphogenesis, and their implications on drought stress tolerance, we used transgenic tobacco plants (AN1), which over-accumulated anthocyanins in all tissues. AN1 plants showed an altered phenotype in terms of leaf gas exchanges, leaf morphology, anatomy and metabolic profile, which conferred them with a higher drought tolerance compared to the wild-type plants. These results provide important insights for understanding the functional reason for anthocyanin accumulation in plants under stress.

Genome-Wide HMG Family Investigation and Its Role in Glycoalkaloid Accumulation in Wild Tuber-Bearing Solanum commersonii.

Steroidal glycoalkaloids (SGAs) are a class of nitrogen-containing glycosides occurring in several plant families and biosynthesized through a specific pathway. HMG-CoA reductase is the first enzyme of this pathway, and its transcription can be regulated by biotic and abiotic stressors and even in a tissue-specific manner. This study aimed to characterize the HMG genes family in a tuber-bearing potato species, Solanum commersonii, using transcriptional and functional approaches. Our results provided evidence that four ScHMGs with different tissue-specificities represent the HMG gene family in S. commersonii and that they originated from ScHMG1 through segmental duplications. Phylogenetic analysis suggests that ScHMG1 is the direct ortholog of AtHMG1, which is associated with SGAs accumulation in plants. Its overexpression in S. commersonii revealed that this gene plays a key role in the accumulation of glycoalkaloids regulating the production of dehydrocommersonine.

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.

A basic Helix-Loop-Helix (SlARANCIO), identified from a Solanum pennellii introgression line, affects carotenoid accumulation in tomato fruits.

Carotenoid accumulation in tomato (Solanum lycopersicum) fruits is influenced by environmental stimuli and hormonal signals. However, information on the relative regulatory mechanisms are scanty since many molecular players of the carotenoid biosynthetic pathway are still unknown. Here, we reported a basic Helix-Loop-Helix transcription factor, named SlARANCIO (SlAR), whose silencing influences carotenoid accumulation in tomato fruits. The SlAR gene was found in the S. pennellii introgression line (IL) 12-4SL that holds the carotenoid QTL lyc12.1. We observed that the presence of the wild region in a cultivated genetic background led to a decrease in total carotenoid content of IL12-4SL fruits. To get insights into the function of SlAR, a quick reverse genetic approach was carried out. Virus-induced gene silencing of SlAR in S. lycopersicum M82 and MicroTom fruits reproduced the same phenotype observed in IL12-4SL, i.e. decreased content of lycopene and total carotenoids. Vice versa, the overexpression of SlAR in Nicotiana benthamiana leaves increased the content of total carotenoids and chlorophylls. Our results, combined with public transcriptomic data, highly suggest that SlAR acts indirectly on the carotenoid pathway and advances current knowledge on the molecular regulators controlling lyc12.1 and, potentially, precursors of carotenoid biosynthesis.

Relationships between composition, microstructure and cooking performances of six potato varieties.

Potatoes tubers are the raw materials of many processed food, such as cooked potatoes in hot water, baked potatoes and the most popular fried potatoes. The objective of this work was to study the impact of boiling, baking and frying on microstructure and properties of six potato varieties (Agata, Agria, Innovator, Lady Rosetta, Musica and Spunta) with different origin. Scanning Electron Microscopy revealed significant differences between varieties and tuber microstructure changes following all cooking processes. Differential Scanning Calorimeter analysis showed that the transition temperatures (ranging between 60 °C and 85 °C) and enthalpies of gelatinization (2.1 J/g-3.9 J/g) of tubers were also variety dependent. In addition, the elasticity modulus of cooked samples depended on process type and followed the order: baked potatoes > boiled > fried potatoes. In particular, baked Lady Rosetta (224.3 kPa) showed the least decrease in rigidity between thermal processes. Fried Agria and Spunta, (56.3 and 61 kPa, respectively) had the smallest value of Young's modulus. Molecular marker analyses provided a genetic fingerprinting of our varieties, allowing the identification of diagnostic markers. Innovator revealed an important genetic distance from the other varieties. Such distance corresponded to its exclusive phenotypic traits, that are known to affect thermochemical properties. The information obtained in this work may be useful to further study and associate genetic sequences with appreciable food technological traits.

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.