The process of wine traceability/authentication is a key target for Italy, the world's first largest wine producer (around 4.6 million tons in 2010 according to FAOSTAT). Several European countries developed appellation systems, with their own unique labels and seals, trying to fight label fraud that misrepresented the true origins of the wine. Advancements on this topic were provided by Council Regulation (EC) No. 479/2008 on the common organisation of the market in wine. As reported by experts, it is suspected that as much as 5 % of the wine sold in secondary markets worldwide could be counterfeit. Despite the high number of traceability methods, commonly based on the use of time consuming and expensive techniques (SNIF-NMR and stable isotope-ratio mass spectrometry), few applications report the use of polymerase chain reaction directly in musts or in bottled wine. Information on genetic polymorphism given by simple sequence repeats (SSR; microsatellite markers) proved helpful when applied to grape and musts analysis. The limited quantity of amplifiable grape genomic DNA in wine represents the main issue for the application of such analytical approach. Musts are the first intermediate product to be checked, in order to exclude the unintentional or fraudulent contamination with foreign grape varieties. The aims of this work are (1) the selection of performing SSR markers able to discriminate 'Barbera' and 'Dolcetto' from 'Nebbiolo' and (2) the hyphenated use of capillary microelectrophoresis (lab-on-chip technology) for polymorphisms detection, to highlight the presence of foreign grape in 'Nebbiolo' musts produced in purity, as required by the designation disciplinary. Finally, we suggest using this approach by exploiting VVS2 marker in order to detect Barbera and Dolcetto grapes in Nebbiolo musts, waiting for more robust and powerful method to extract and amplify specific DNA from bottled wine.

In addition to the full-length transcript ARF8.1, a splice variant (ARF8.2) of the auxin response factor gene ARF8 has beenreported. Here, we identified an intron-retaining variant of ARF8.2, ARF8.4, whose translated product is imported into thenucleus and has tissue-specific localization in Arabidopsis thaliana. By inducibly expressing each variant in arf8-7 flowers, weshow that ARF8.4 fully complements the short-stamen phenotype of the mutant and restores the expression of AUX/IAA19,encoding a key regulator of stamen elongation. By contrast, the expression of ARF8.2 and ARF8.1 had minor or no effects onarf8-7 stamen elongation and AUX/IAA19 expression. Coexpression of ARF8.2 and ARF8.4 in both the wild type and arf8-7caused premature anther dehiscence: We show that ARF8.2 is responsible for increased expression of the jasmonic acidbiosynthetic gene DAD1 and that ARF8.4 is responsible for premature endothecium lignification due to precocious expressionof transcription factor gene MYB26. Finally, we show that ARF8.4 binds to specific auxin-related sequences in both the AUX/IAA19 and MYB26 promoters and activates their transcription more efficiently than ARF8.2. Our data suggest that ARF8.4 isa tissue-specific functional splice variant that controls filament elongation and endothecium lignification by directlyregulating key genes involved in these processes.

Plant cell cultures are valuable bioreactors for the production of bioactive compounds. Artemisia annua L. cell suspension cultures established for producing the antimalarial artemisinin (AN) were exploited to investigate the biosynthesis of other healthful phytochemicals. Culture supplementation with the putative elicitors ß-cyclodextrins (ß-CDs) was successful to increase intracellular and extracellular levels of AN. Other bioactive isoprenoid compounds, known for their antioxidant activity, were also investigated in the supplemented A. annua cell cultures by using metabolomic and transcriptomic analyses. In particular, the application of ß-CDs was useful to induce a considerable significant increase of lutein, quinones (Q9 and Q10) intracellular contents compared to untreated cell cultures. Moreover, the release of carotenoids and quinones into the culture medium of ß-CDs-treated cell cultures was significantly enhanced. Real Time PCR analysis revealed an up-regulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene, suggesting that the ß-CDs induced increase of isoprenoid compounds could be due to the enhancement of the plastidial isoprenoid biosynthetic route. In addition, protoplasts derived from Artemisia suspension cultures were explored to engineer subcellular compartmentalization using a modified SNARE protein as a molecular tool for biotechnological metabolite accumulation.

Plant cell cultures are valuable tools for the production of bioactive compounds. Recently, many efforts have been made in order to develop and optimize strategies for increasing the yields of the desired plant metabolites by eliciting their biosynthesis or improving the efficiency of product recovery. We established Artemisia annua L. (Asteraceae) cell suspension cultures to investigate the biosynthesis of the antimalarial artemisinin (AN) [1] and other healthful phytochemicals such as carotenoids and quinones. The use of culture supplementation with cyclic oligomers ?-cyclodextrins was successful to significantly increase intracellular and extracellular levels of AN, lutein, Q9 and Q10 [2, 3]. In order to investigate the mechanism underlying these effects, other oligosaccharides having a linear structure were explored as putative elicitors in A. annua cell suspension cultures. For this purpose, oligogalacturonides (OGA) were obtained by hydrolysis of polygalacturonic acid with pectolyase from Aspergillus japonicus. Various OGA fractions were used for culture medium supplementation at different concentrations. The results showed that the 4-5 OGA fraction induced significant increases of AN and the intermediate dihydroartemisinic acid. These results suggest that the ability of ?-CDs to elicit the production of bioactive compounds in A. annua cell cultures could be due to their chemical similarity to pectic oligosaccharides often occurring in plants after fungal infection.

Food authentication and traceability is one of the major concern to the food industry, strictly correlatedto food fraud and food adulteration. Rice (Oryza sativa L.) is one of the most important crops, supplyingfood for over half of the world's population. Authenticity of rice products has become a key issue in thefood industry addressed to protect the interests of quality conscious consumers, stakeholders, andimporting countries. DNA markers offer a powerful tool to address the validation of food authenticityand traceability of primary products. Progress in NGS technology has provided opportunities to detectlarge number of DNA polymorphisms, even in the closely related cultivars. In this study, a whole-genomesequencing of Italian rice cultivar Carnaroli has been carried out from genetically pure certified seed.The sequencing yielded about 22.5 million reads. After quality trimming 21.5 million reads were mappedonto the reference sequence of Oryza sativa ssp. japonica cv. Nipponbare (IRGSP-1.0), providing about90% coverage of the rice genome and an average coverage of 15.12x. Preliminary results, found 450,414candidate DNA polymophisms between cultivar Nipponbare and Carnaroli. These were classified into383,080 SNPs (85%) and 67,334 InDels (15%) by polymorphism types, 150,688 homozygous (85%) and299,726 (15%) heterozygous by zygosity type, 371,801 intergenic (82.5%) and 78613 (17.6%) by genomiclocation. The distribution of DNA polymorphisms was found to be uneven across and within the ricechromosomes. In particular, chromosome 8 and 10 showed the highest density of DNA polymorphisms(14.7% and 14.5%, respectively). This study represents the first report of whole genome sequencing ofItalian rice cultivar Carnaroli and will contribute to develop targeted and un-targeted method for riceauthentication and traceabilityThis work has been supported by the European project FOODINTEGRITY (FP7-KBBE-2013-single-stage, No 613688).

Artemisinin is a sesquiterpene antimalarialcompound produced, though at low levels (0.1-1% dryweight), in Artemisia annua in which it accumulates in theglandular trichomes of the plant. Due to its antimalarialproperties and short supply, efforts are being made toimprove our understanding of artemisinin biosynthesis andits production. Native ?-cyclodextrins, as well as thechemically modified heptakis(2,6-di-O-methyl)-?-cyclodextrin(DIMEB) and 2-hydroxypropyl-?-cyclodextrins,were added to the culture medium of A. annua suspensioncultures, and their effects on artemisinin production wereanalysed. The effects of a joint cyclodextrin and methyljasmonate treatment were also investigated. Fifty millimolarDIMEB, as well as a combination of 50 mMDIMEB and 100 ?M methyl jasmonate, was highlyeffective in increasing the artemisinin levels in the culturemedium. The observed artemisinin level (27 ?mol g-1 dryweight) was about 300-fold higher than that observed inuntreated suspensions. The influence of ?-cyclodextrinsand methyl jasmonate on the expression

The heavy metal cadmium (Cd) is a widespread environmental contaminant with harmful effects on living cells. In plants, phytochelatin (PC)-dependent Cd detoxification requires that PC-Cd complexes are transported into vacuoles. Here, it is shown that Arabidopsis thaliana seedlings defective in the ABCC transporter AtABCC3 (abcc3) have an increased sensitivity to different Cd concentrations, and that seedlings overexpressing AtABCC3 (AtABCC3ox) have an increased Cd tolerance. The cellular distribution of Cd was analysed in protoplasts from abcc3 mutants and AtABCC3 overexpressors grown in the presence of Cd, by means of the Cd-specific fluorochromes 5-nitrobenzothiazole coumarin (BTC-5N) and Leadmium (TM) Green AM dye. This analysis revealed that Cd is mostly localized in the cytosol of abcc3 mutant protoplasts whereas there is an increase in vacuolar Cd in protoplasts from AtABCC3ox plants. Overexpression of AtABCC3 in cad1-3 mutant seedlings defective in PC production and in plants treated with l-buthionine sulphoximine (BSO), an inhibitor of PC biosynthesis, had no effect on Cd tolerance, suggesting that AtABCC3 acts via PCs. In addition, overexpression of AtABCC3 in atabcc1 atabcc2 mutant seedlings defective in the Cd transporters AtABCC1 and AtABCC2 complements the Cd sensitivity of double mutants, but not in the presence of BSO. Accordingly, the level of AtABCC3 transcript in wild type seedlings was lower than that of AtABCC1 and AtABCC2 in the absence of Cd but higher after Cd exposure, and even higher in atabcc1 atabcc2 mutants. The results point to AtABCC3 as a transporter of PC-Cd complexes, and suggest that its activity is regulated by Cd and is co-ordinated with the activity of AtABCC1/AtABCC2.

Previous studies demonstrated that expression of the Arabidopsis phytochelatin (PC) biosynthetic gene AtPCS1 in Nicotiana tabacum plants increases the Cd tolerance in the presence of exogenous glutathione (GSH). In this paper, the Cd tolerance of Arabidopsis plants over-expressing AtPCS1 (AtPCSox lines) has been analysed and the differences between Arabidopsis and tobacco are shown. Based on the analysis of seedling fresh weight, primary root length, and alterations in root anatomy, evidence is provided that, at relatively low Cd concentrations, the Cd tolerance of AtPCSox lines is lower than the wild type, while AtPCS1 over-expressing tobacco is more tolerant to Cd than the wild type. At higher Cd concentrations, Arabidopsis AtPCSox seedlings are more tolerant to Cd than the wild type, while tobacco AtPCS1 seedlings are as sensitive as the wild type. Exogenous GSH, in contrast to what was observed in tobacco, did not increase the Cd tolerance of AtPCSox lines. The PC content in wild-type Arabidopsis at low Cd concentrations is more than three times higher than in tobacco and substantial differences were also found in the PC chain lengths. These data indicate that the differences in Cd tolerance and in its dependence on exogenous GSH between Arabidopsis and tobacco are due to species-specific differences in the endogenous content of PCs and GSH and may be in the relative abundance of PCs of different length.

The olive oil is an unfavorable substrate for microbial survival and growth. Only few microorganisms use olive oil fattyacids as carbon and energy sources, and survive in the presence of olive oil anti-microbial components. In this study,we have evaluated the occurrence of microorganisms in 1-year-stored extra-virgin olive oil samples. We detected thepresence of bacterial and yeast species with a recurrence of the bacterium Stenotrophomonas rhizophila and yeastSporobolomyces roseus. We then assayed the ability of all isolates to grow in a mineral medium supplemented with acommercial extra-virgin olive oil as a sole carbon and energy source, and analyzed the utilization of olive oil fatty acidsduring their growth. We finally focused on two bacterial isolates belonging to the species Pantoea septica. Both theseisolates produce carotenoids, and one of them synthesizes bioemulsifiers enabling the bacteria to better survive/growth in this unfavorable substrate. Analyses point to a mixture of glycolipids with glucose, galactose and xylose ascarbohydrate moieties whereas the lipid domain was constituted by C6-C10 ?-hydroxy carboxylic acids.

Food authentication and traceability is a complex problem, strictly correlated to fraud andadulteration detections that dramatically affect the consumer protection. Analysis of protein,metabolite and DNA represents robust tools for food authentication. In particular, DNA-basedmethods are more reliable, thanks to the stability of DNA under production and processing techniquesapplied along the food-chain. Therefore, DNA markers offer a powerful tool to address the validationof food authenticity and traceability of primary products. Single nucleotide polymorphism (SNP)markers have become the most used markers in genetic characterization studies as well as intranslational genomic even in plants. SNP are, in fact, the most abundant forms of genetic variationamong individuals of a species. In particular, SNP analysis by next generation sequencing (NGS)(e.g.genotyping by sequencing (GBS) and double-digest restriction site-associated DNA sequencing(ddRAD-Seq) or by high resolution melting analysis (HRM), e.g. single-base variants and smallinsertions or deletions, have rapidly become popular due to their flexibility and relatively low cost.The ddRAD-Seq technology has the advantage over GBS of high accuracy read mapping by paired-endsequencing of identical loci. Progress in NGS technology has led to the availability of several plantgenomes. This situation makes it possible to simulate ddRAD-Seqin silico, allowing prediction of thenumbers, sizes, and genome positions of digested fragments. However, few reports have evaluatedthe in silico predictions by comparative experiments using several combinations of restriction enzymesand multiple samples with different SNP density. HRM analysis has several advantages over traditionalmethods for gene scanning and genotyping, making it faster, less laborious and more suitable for highsample throughput. In this study, two approaches are proposed for the authentication of the Italianrice cultivars Carnaroli and Roma: in silico and empirical ddRAD-Seq analysis and HRM analysistargeting an A/C SNP in exon 6, responsible for the Wxin allele. The ddRAD-Seq approach consisted ofa workflow, as follows:(i) in silico prediction of optimum restriction enzymes from the reference ricegenome,(ii) verification of the prediction by ddRAD-Seq data of Carnaroli and Roma genomes (iii)establishment of a computational data processing pipeline for high confidence SNP calling, and (iv)validation of SNP accuracy. In silico prediction prior to sequencing analysis will contribute tooptimization of the experimental conditions for ddRAD-Seq and could help to accelerate the detectionof DNA markers useful for the authentication of rice cultivars Carnaroli and Roma. Preliminary resultsof HRM analysis show potential for rice cultivar differentiation since Carnaroli was distinguished fromRoma, among others (Carnise/Karnak, Gladio, Sant'Andrea and others) with high level of confidence(>98%). Acknowledgments: This work has

Artemisinin is an effective antimalarial compound obtained by thè aerial parts of thè herbalplant Artemisia annua L. Unfortunately, thè production of artemisinin in plants is very low (0.1-1% on a dry weight basis) and its chemical synthesis is very difficult. In recent yearsbiotechnological attempts to improve artemisinin production either in plant or geneticallyengineered microrganisms were made. A. annua celi and tissue cultures were also exploredfor thè production of artemisinin, although thè yieids obtained have been so far quite low. Wehave recently established A. annua celi cultures able to biosynthesize artemisinin and torespond to thè elicitation by methyl jasmonate (MeJA). Interestingly, amounts of artemisininproduced by these cultures were also found in thè spent culture medium. Cyclodextrins (CDs)are known to increase thè water solubility of various compounds, by forming host-guestinclusion complexes. Chemically modified CDs, such as 2,6 dimethyl-p-cyclodextrins (DIMEB)have been successfully used to improve thè production of health-promoting plant metabolitessuch as resveratrol in grape celi cultures. The aim of this work was to evaluate thè ability ofDIMEB to enhance artemisinin production in A. annua suspension celi cultures. HPLCanalysis of artemisinin and Real Time PCR gene expression analysis were carried out inDIMEB treated and untreated cultures. DIMEB induced a 300-fold increase of artemisininlevels in thè spent culture medium after a three-day-treatment. The addition of MeJA induceda further increase of thè artemisinin amounts. The expression levels of artemisininbiosynthetic genes (cytochrome P450 monoxygenase, cytochrome P450 reductase andartemisinic aldehyde A11 reductase) were not altered by thè DIMEB treatment. These resultssuggest that DIMEB improved artemisinin yieids of A. annua celi cultures, probably notaffecting thè biosynthetic pathway but enhancing thè solubility and stability of artemisininmolecules.

Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs) for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-?-cyclodextrins (DIMEB) on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold), Q9 (3-fold) and Q10 (2.5-fold). Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules.

Genetic distances evaluated via SSR-based profiling can be usefully assessed by using capillary electrophoresis. In order to set up a method to distinguish pure Italian rice varieties from imported Asian blends, seven Italian rice genotypes and seven uncharacterized rice samples coming from outside Italy were studied using a classical SSR polymorphism analysis coupled with Lab-on-a-chip microcapillary electrophoresis. A special algorithm for the elaboration of the raw outputs provided by the software was generated, thus overcoming the problems connected to the instrument intrinsic limits of resolution. The results showed that even considering just the smallest verifiable genetic distance between the employed samples, locally cultivated Italian rice varieties clustered separately from other foreign cultivars. Moreover, it was possible to clearly identify an artificial blend formed by Venere rice mixed with a black variety from Thailand, thus confirming the uSefulness of this new postanalysis approach. (C) 2014 Published by Elsevier Ltd.

The polyphenol oxidase (PPO) enzyme, which can catalyze the oxidation of phenolics to quinones, has been reported to be involved in undesirable browning in many plant foods. This phenomenon is particularly severe in artichoke headswounded during the manufacturing process. A full-length cDNA encoding for a putative polyphenol oxidase (designated as CsPPO) along with a 1432 bp sequence upstream of the starting ATG codon was characterized for the first time from [Cynara cardunculus var. scolymus (L.) Fiori]. The 1764 bp CsPPO sequence encodes a putative protein of 587 amino acids with a calculated molecular mass of 65,327 Da and an isoelectric point of 5.50. Analysis of the promoter region revealed the presence of cis-acting elements, some of which are putatively involved in the response to light and wounds. Expression analysis of the gene in wounded capitula indicated that CsPPO was significantly induced after 48 h, even though the browning process had started earlier. This suggests that the early browning event observed in artichoke heads was not directly related to de novo mRNA synthesis. Finally, we provide the complete gene sequence encoding for polyphenol oxidase and the upstream regulative region in artichoke

Artemisia annua L., is a herbal plant traditionally used for the treatment of fever. The glandular trichomes of this plant accumulate, although at low levels, artemisinin which is highly effective against malaria. Due to the great importance of this compound many efforts have been made to improve the knowledge on artemisinin production both in plants and in cell cultures. In this study A. annua suspension cultures were established in order to investigate the effects of methyl jasmonate (MeJA) and miconazole treatments on artemisinin biosynthesis. Twenty-two mM MeJA induced a three-fold increase of artemisinin production soon after 30 min. Two hundred mM miconazole induced a 2.5 fold increase of artemisinin production after 24 h treatment but had severe effects on cell viability. The influence of these treatments on the expression of biosynthetic genes was also investigated. MeJA induced an up-regulation of CYP71AV1 while miconazole induced an up-regulation of CPR and DBR2.

A short partial sequence of 28 amino acids is all the information we have so far about the putative allergen 2S albumin from almond. The aim of this work was to analyze this information using mainly bioinformatics tools, in order to verify its rightness. Based on the results reported in the paper describing this allergen from almond, we analyzed the original data of amino acids sequencing through available software. The degree of homology of the almond 12 kDa protein with any other known 2S albumin appears to be much lower than the one reported in the paper that firstly described it. In a publicly available cDNA library we discovered an expressed sequence tag which translation generates a protein that perfectly matches both of the sequencing outputs described in the same paper. A further analysis indicated that the latter protein seems to belong to the vicilin superfamily rather than to the prolamin one. The fact that also vicilins are seed storage proteins known to be highly allergenic would explain the IgE reactivity originally observed. Based on our observations we suggest that the IgE reactive 12 kDa protein from almond currently known as Pru du 25 albumin is in reality the cleaved N-terminal region of a 7S vicilin like protein. (C) 2015 Elsevier Ltd. All rights reserved.

Dittrichia viscosa (L.) è una pianta sempreverde, cespugliosa, della famiglia delle Asteraceaemolto comune nelle regioni mediterranee. E' in grado di tollerare la presenza di diversimetalli tossici nel suolo e accumulare nei propri tessuti fogliari concentrazioni di zinco (Zn) epiombo (Pb) al di sopra dei livelli di tossicità critica per le piante, rispettivamente 300 e 28?g g-1, senza mostrare sintomi o alterazione dello stato nutrizionale (Lopez-Orenes et al.,2018).I metalli pesanti sono noti inquinanti ambientali che hanno un effetto negativo sulla vita ditutti gli organismi quando presenti in concentrazioni tossiche (Nriagu, 1996). Essi si dividonoin essenziali, quali ferro (Fe), rame (Cu) o zinco (Zn) in quanto svolgono un ruolo all'internodella cellula, e non essenziali, quali cadmio (Cd), piombo (Pb) o mercurio (Hg) che nonsvolgono alcuna funzione fisiologica e risultano quindi altamente tossici anche a basseconcentrazioni.L' assorbimento nelle piante è la principale causa di ingresso dei metalli pesanti nella catenaalimentare e questa una delle fonti di esposizione per l'uomo alla loro tossicità. Conoscere imeccanismi molecolari alla base dei processi di assorbimento, ritenzione e traslocazione deidiversi metalli nelle piante permetterebbe di ottenere: (i) cultivar con un ridotto contenuto dimetalli tossici da utilizzare per l'alimentazione; (ii) cultivar in grado di tollerarne elevatilivelli da utilizzare nei processi di bonifica.

Pine nuts are a known source of food allergens and several cases of adverse immunological reaction after ingestion have been reported. To protect allergic consumers, methods to unequivocally detect the presence of pine nuts in complex matrices must be developed. A Taqman-based real time PCR method for the detection of Pinus spp. was set up. A homemade pesto spiked at known concentration of pine nut powder was used as model food. Moreover, DNA was purified from commercial foods declaring or not the presence of pine nuts. The method displayed a very high efficiency and specificity for the genus Pinus. The intrinsic LOD was 1 pg of DNA, while the practical LOD evaluated on model foods was 0.1 ppm of pine nuts powder, the lowest ever registered for the detection of food allergens via real-time PCR. Finally, the declared presence/absence of pine nut in commercial foods was confirmed. (C) 2015 Elsevier Ltd. All rights reserved.

Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging.Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from Endoplasmic Reticulum to Vacuole trafficking.The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.