Crystal structure of hetero hexameric 11S seed storage protein of hazelnut.

Edible plant seeds provide a relatively inexpensive source of protein and make up a large part of nutrients for humans. Plant seeds accumulate storage proteins during seed development. Seed storage proteins act as a reserve of nutrition for seed germination and seedling growth. However, seed storage proteins may be allergenic, and the prevalence of food allergy has increased rapidly in recent years. The 11S globulins account for a significant number of known major food allergens. They are of interest to the public and the agricultural industry because of food safety concerns and the need for crop enhancement. We sought to determine the crystal structure of Cor a 9, the 11 S storage protein of hazelnut and a food allergen. The structure was refined to 1.92 Å, and the R and Rfree for the refined structure are 17.6% and 22.5%, respectively. The structure of Cor a 9 showed a hetero hexamer of an 11S seed storage protein for the first time. The hexamer was two trimers associated back-to-back. Two long alpha helixes at the C-terminal end of the acidic domain of one of the Cor a 9 isoforms lay at the trimer-trimer interface's groove. These data provided much-needed information about the allergenicity of the 11S seed proteins. The information may also facilitate a better understanding of the folding and transportation of 11S seed storage proteins.

A roadmap of tissue culture and biotechnology in European hazelnut (Corylus avellana L.).

The increasing interest in European hazelnut (Corylus avellana L.) cultivation registered in the last years has led to a significant increase in worldwide hazelnut growing areas, also involving regions characterized by a marginal presence of hazelnut orchards. Despite this increasement, world production still relies on the cultivation of few varieties, most of which are particularly suitable to the environment where they have been selected. Therefore, it is necessary to develop new cultivars with high environmental plasticity capable of providing constant and high-quality productions in the new environments and under the climatic change conditions of traditional growing areas. Over the years, many molecular markers for genetic breeding programs have been developed and omics sciences also provided further information about the genetics of this species. These data could be of support to the application of new plant breeding techniques (NPBTs), which would allow the development of cultivars with the desired characteristics in a shorter time than traditional techniques. However, the application of these methodologies is subordinated to the development of effective regeneration protocols which, to date, have been set up exclusively for seed-derived explants. A further aspect to be exploited is represented by the possibility of cultivating hazelnut cells and tissues in vitro to produce secondary metabolites of therapeutic interest. This review aims to consolidate the state of the art on biotechnologies and in vitro culture techniques applied on this species, also describing the various studies that over time allowed the identification of genomic regions that control traits of interest.

Green synthesized Ag nanoparticles stimulate gene expression and paclitaxel production in Corylus avellana cells.

Synthesis of nanoparticles (NPs) through plant extracts has been suggested as an effective and nature-friendly method. Paclitaxel is one of the most valuable secondary metabolites with therapeutic uses, and hazelnut has been suggested as one of the sustainable resources for producing this metabolite. In the present study, we synthesized Ag NPs using the ethanolic extract of C. avellana leaves and were characterized using UV-visible, FTIR, XRD, EDX, DLS, SEM, and TEM analyses. In addition, we investigated the effect of green synthesized Ag (GS Ag) NPs (5 and 10 mg/L), para-aminobenzoic acid (PABA) (20 mg/L), and AgNO3 (10 mg/L) on cell viability, physiological characteristics, gene expression, and biosynthesis of secondary metabolites in hazelnut cell cultures. The results showed that 10 mg/L Ag NPs and AgNO3 significantly affected the cell viability, the content of ROS, peroxidation of lipids, antioxidant capacity, secondary metabolite production, and expression pattern of the genes involved in the taxanes biosynthesis pathway in the hazelnut cells. The cytotoxicity increased by increasing the GS Ag NPs concentration from 5 to 10 mg/L, which was associated with reduced membrane integrity and cell viability. Elicitation of the cells with 10 mg/L Ag NPs combined with 20 mg/L PABA (as a precursor) remarkably excited the expression of TAT and GGPPS genes and the production of secondary metabolites as well as paclitaxel. So that the highest expression of TAT and GGPPS genes (3.71 and 3.69) and the highest amount of taxol (230.21 µg g-1 FW) and baccatin (1025.8 µg g-1 FW) were observed in this treatment. KEY POINTS: • For the first time, we assessed and reported the molecular and physiological responses of C. avellana cells to GS Ag NPs, AgNO3, and PABA. • In hazel cells, GS Ag NPs stimulate several physiological and molecular responses. • In addition to increasing antioxidant activity, GS Ag NPs significantly increased the expression of genes involved in the paclitaxel biosynthesis pathway and the production of secondary metabolites.

Chemical Composition of Hazelnut Skin Food Waste and Protective Role against Advanced Glycation End-Products (AGEs) Damage in THP-1-Derived Macrophages.

Glycation and the accumulation of advanced glycation end-products (AGEs) are known to occur during aging, diabetes and neurodegenerative diseases. Increased glucose or methylglyoxal (MGO) levels in the blood of diabetic patients result in increased AGEs. A diet rich in bioactive food compounds, like polyphenols, has a protective effect. The aim of this work is to evaluate the capacity of hazelnut skin polyphenolic extract to protect THP-1-macrophages from damage induced by AGEs. The main polyphenolic subclass was identified and quantified by means of HPLC/MS and the Folin-Ciocalteu method. AGEs derived from incubation of bovine serum albumin (BSA) and MGO were characterized by fluorescence. Cell viability measurement was performed to evaluate the cytotoxic effect of the polyphenolic extract in macrophages. Reactive oxygen species' (ROS) production was assessed by the H2-DCF-DA assay, the inflammatory response by real-time PCR for gene expression, and the ELISA assay for protein quantification. We have shown that the polyphenolic extract protected cell viability from damage induced by AGEs. After treatment with AGEs, macrophages expressed high levels of pro-inflammatory cytokines and ROS, whereas in co-treatment with polyphenol extract there was a reduction in either case. Our study suggests that hazelnut skin polyphenol-rich extracts have positive effects and could be further investigated for nutraceutical applications.

Improving ACE inhibitory activity of hazelnut peptide modified by plastein: Physicochemical properties and action mechanism.

The plastein reaction can increase the activity of angiotensin-converting enzyme (ACE) inhibitory peptides, but the underlying mechanism is unknown. Hence, hazelnut protein hydrolysate and hazelnut peptide YLVR were used as substrate to explore the effect of plastein on physicochemical properties and the mechanism of structural change. The increase in turbidity and particle size and the decrease in free amino groups indicated that the reaction occurred via condensation. The modified products of YLVR were identified by NANO-HPLC-MS/MS, indicating that the N-terminal homologous amino acid aggregates in the plastein. Novel ACE inhibitory peptide YYLVR, YLLVR, and YYLLVR were synthesized and their inhibition rates were 66.35, 72.61, and 89.10 %, respectively, which were higher than that of YLVR (52.58 %). MD simulation showed that YYLLVR exhibited the lowest binding energies of -35.98 ± 2.30 kcal/mol to ACE. Taken together, plastein reaction is a promising strategy for inducing structural modifications to improve the activity of peptide.

De novo Transcriptome Analysis and Gene Expression Profiling of Corylus Species.

Hazelnut (Corylus), which has high commercial and nutritional benefits, is an important tree for producing nuts and nut oil consumed as ingredient especially in chocolate. While Corylus avellana L. (Euro-pean hazelnut, Betulaceae) and Corylus colurna L. (Turkish hazelnut, Betulaceae) are the two common hazelnut species in Europe, C. avellana L. (Tombul hazelnut) is grown as the most widespread hazelnut species in Turkey, and C. colurna L., which is the most important genetic resource for hazelnut breeding, exists naturally in Anatolia. We generated the transcriptome data of these two Corylus species and used these data for gene discovery and gene expression profiling. Total RNA from young leaves, flowers (male and female), buds, and husk shoots of C. avellana and C. colurna were used for two different libraries and were sequenced using Illumina HiSeq4000 with 100 bp paired-end reads. The transcriptome data 10.48 and 10.30 Gb of C. avellana and C. colurna, respectively, were assembled into 70,265 and 88,343 unigenes, respectively. These unigenes were functionally annotated using the TRAPID platform. We identified 25,312 and 27,051 simple sequen-ce repeats (SSRs) for C. avellana and C. colurna, respectively. TL1, GMPM1, N, 2MMP, At1g29670, CHIB1 unigenes were selected for validation with qPCR. The first de novo transcriptome data of C. co-lurna were used to compare data of C. avellana of commercial importance. These data constitute a valuable extension of the publicly available transcriptomic resource aimed at breeding, medicinal, and industrial research studies.

Biologically synthesized CuO nanoparticles induce physiological, metabolic, and molecular changes in the hazel cell cultures.

The utilization of plant extracts in nanoparticle (NP) synthesis has been suggested as a nature-friendly method and an efficient alternative to the conventional approaches such as physical and chemical methods. Taxol is a valuable medicinal compound, and hazelnut has been suggested as one of the sustainable resources for producing this metabolite. In the present research, copper oxide (CuO) nanoparticles (NPs) were biologically synthesized by utilizing hazelnut leaf extracts. FTIR, XRD, EDAX, DLS, and SEM analyses were used for characterizing and confirming the synthesized NPs. The effect of biosynthesized CuO NPs (10 and 90 ppm), para-aminobenzoic acid (PABA) (20 ppm), and CuSO4 (10 ppm) on the cell viability, biochemical properties, expression of TAT and GGPPS genes, and accumulation of taxol and baccatin III in hazelnut cell cultures was investigated. The results indicated that biosynthesized CuO NPs significantly influenced the cell viability, amount of ROS, antioxidant capacity, lipid peroxidation, secondary metabolite production, and expression pattern of the genes engaged in the biosynthesis pathway of taxanes in the C. avellana L. cells. The cytotoxicity of CuO NPs to cells was dose dependent and increased with increasing its concentration, as evidenced by a decline in the survival rate and cell membrane integrity. Furthermore, the utilization of 10 ppm CuSO4 caused more toxicity in the cells than the same concentration of CuO NPs. This result could be attributed to the fact that plant extracts components act as a coating for the NPs and reduce their toxicity. Treatment of the cell cultures with CuO (10 ppm) + PABA (20 ppm) and CuO (10 ppm) induced the highest radical scavenging activity. The activity of antioxidant enzymes was increased with increasing the copper oxide NPs level from 10 to 90 ppm. Contrariwise, the cell's survival rate, radical scavenging activity, and amount of secondary metabolites were significantly reduced in the higher levels of copper oxide NPs (90 ppm) compared to the 10 ppm. The combined utilization of 10 ppm copper oxide NPs and 20 ppm PABA considerably stimulated the TAT and GGPPS genes expression and produced the highest amount of taxol and baccatin III. KEY POINTS: • CuO NPs were biologically synthesized using the hazel leaf extracts and confirmed by FTIR, XRD, EDAX, DLS, and SEM analyses. • CuO NPs significantly affected the amount of ROS, antioxidant capacity, and lipid peroxidation in C. avellana L. cells. • Treatment of the hazel cells with CuO NPs increased the production of secondary metabolites including taxol and baccatin III and expression of the genes involved in taxol and baccatin III biosynthesis (TAT and GGPPS).

In silico study to predict potential precursors of human dipeptidyl peptidase-IV inhibitors from hazelnut.

Chinese hazelnut was chosen to become a probable precursor of biological active peptides via computer simulations in this article. There were a large number of bioactive peptides in Chinese hazelnut sequences according to analytical results from the BIOPEP database. The most prominent of these was the inhibitory peptide for dipeptidyl peptidase-IV (DPP-IV; EC 3.4.14.5), which can be used to treat type 2 diabetes, so the theoretical method to obtain DPP-IV inhibitory peptides by hydrolysis with a single or combination of enzymes was studied. Cytotoxicity analysis performed by ToxinPred showed that all of the DPP-IV inhibitory peptides generated from protein hydrolysis were not cytotoxic. Structural interaction fingerprint analysis revealed that Asp663 and Phe357 may be important residues for ligand binding. In order to further understand the inhibitory mechanism of peptide, VR with lowest half maximum inhibitory concentration (IC50) and IPI (inhibitors have been reported) were selected as ligand of DPP-IV to perform steered molecular dynamics simulations and PMF calculations. The results showed that P1 is the preferred (un)binding tunnel for the inhibitors obtained. Our findings help in the development of new DPP-IV inhibitors which were derived from common food.Communicated by Ramaswamy H. Sarma.

Choline Chloride-Lactic Acid-Based NADES As an Extraction Medium in a Response Surface Methodology-Optimized Method for the Extraction of Phenolic Compounds from Hazelnut Skin.

Deep eutectic solvents (DESs) are promising green solvents for the extraction of compounds from food byproducts. Hazelnut (Corylus avellana L.) is one of the most commonly cultivated tree nuts worldwide. The skin represents one of the major byproducts of the hazelnut industry and accounts for 2.5% of the total hazelnut kernel weight. It is a rich source of phenolic compounds like flavan-3-ols, flavonols, dihydrochalcones, and phenolic acids. In this work, fifteen DESs based on choline chloride and betaine, with different compositions, were studied in order to test their phenolic compounds extraction efficiency through the determination of their total concentration via Folin-Ciocalteu assay. A qualitative analysis of extracted phenolic compounds was assessed by HPLC with UV and MS detection. Using the DES with the best extraction efficiency, a new ultrasound-assisted solid liquid extraction (UA-SLE) method was optimized though the response surface methodology (RSM), taking into account some extraction parameters. Efficient recovery of extracted phenolic compounds was achieved using a 35% water solution of choline chloride and lactic acid (molar ratio 1:2) as an extraction solvent, working at 80 °C and with a solid-to-solvent ratio of 1:25 gmL-1. The optimized conditions made it possible to recover 39% more phenolic compounds compared to a classic organic solvent.

Effect of hot air and infrared roasting on hazelnut allergenicity.

Roasting is known to affect the protein profile and allergenicity of hazelnuts (Corylus avellana cv TGL). The aim of the study was to investigate whether roasting techniques based on different heat transfer methods (hot air and infrared), differently affect the protein solubility and the IgE-binding capacities of both the soluble and insoluble hazelnut protein fractions. The immune-reactivity of the Cor a 9, Cor a 11 and Cor a 14 allergens resulted to be stable after roasting at 140 °C, for both types of treatment, while roasting at 170 °C caused a reduction in IgE-binding, which was particularly noticeable after infrared processing, that led to an almost complete disappearance of allergenicity. Microscopical analyses showed that roasting caused cytoplasmic network disruption, with a loss of lipid compartmentalization, as well as an alteration of the structure of the protein bodies and of the cell wall organization.

An effective chromatographic fingerprinting workflow based on comprehensive two-dimensional gas chromatography - Mass spectrometry to establish volatiles patterns discriminative of spoiled hazelnuts (Corylus avellana L.).

The volatile fraction of hazelnuts encrypts information about: cultivar/geographical origin, post-harvest treatments, oxidative stability and sensory quality. However, sensory features could be buried under other dominant chemical signatures posing challenges to an effective classification based on pleasant/unpleasant notes. Here a novel workflow that combines Untargeted and Targeted (UT) fingerprinting on comprehensive two-dimensional gas-chromatographic patterns is developed to discriminate spoiled hazelnuts from those of acceptable quality. By flash-profiling, six hazelnut classes are defined: Mould, Mould-rancid-solvent, Rancid, Rancid-stale, Rancid-solvent, and Uncoded KO. Chromatographic fingerprinting on composite 2D chromatograms from samples belonging to the same class (i.e., composite class-images) enabled effective selection of chemical markers: (a) octanoic acid that guides the sensory classification being positively correlated to mould; (b) Æ´-nonalactone, Æ´-hexalactone, acetone, and 1-nonanol that are decisive to classify OK and rancid samples; (c) heptanoic and hexanoic acids and Æ´-octalactone present in high relative abundance in rancid-solvent and rancid-stale samples.

A chromosome-scale genome assembly of European hazel (Corylus avellana L.) reveals targets for crop improvement.

The European hazelnut (Corylus avellana L.) is a tree crop of economic importance worldwide, but especially for northern Turkey, where the majority of production takes place. Hazelnut production is currently challenged by environmental stresses, such as a recent outbreak of severe powdery mildew disease; furthermore, allergy to hazelnuts is an increasing health concern in some regions. In order to provide a foundation for using the available hazelnut genetic resources for crop improvement, we produced a fully assembled genome sequence and annotation for a hazelnut species, from C. avellana cv. 'Tombul', one of the most important Turkish varieties. A hybrid sequencing strategy, combining short reads, long reads and proximity ligation methods, enabled us to resolve heterozygous regions and produce a high-quality 370-Mb assembly that agrees closely with cytogenetic studies and genetic maps of the 11 C. avellana chromosomes, and covers 97.8% of the estimated genome size. The genome includes 27 270 high-confidence protein-coding genes, over 20 000 of which were functionally annotated based on homology with known plant proteins. We focused particularly on gene families encoding hazelnut allergens, and the Mildew resistance Locus O (MLO) proteins that are an important susceptibility factor for powdery mildew. The complete assembly enabled us to differentiate between members of these families and to identify homologues that may be important in mildew disease and hazelnut allergy. These findings provide examples of how the genome can be used to guide research and to develop effective strategies for crop improvement in C. avellana.

Hazelnut Modulates Neurobehaviour and Ameliorates Ageing-induced Oxidative Stress, and Caspase-3-Mediated Apoptosis in Mice.

BACKGROUND: Organismal aging has been associated with deleterious effects in different body tissues and organs, including the brain. There have been reports from ancient medicinal scripts of the beneficial effects of nuts like hazelnut in preventing aging induced-brain atrophy and memory loss. OBJECTIVES: This study examined the potential beneficial effects of a diet supplemented with two different (Italian and Turkish) cultivars of hazelnut on the brain of aged mice. METHODS: Aged (24 months old) mice were randomly assigned into 7 groups of ten mice each. Mice were grouped as standard diet (SD) control, three groups of Turkish and three groups of Italian hazelnut incorporated into SD at 2, 4 and 8% respectively. Animals were fed standard or hazelnut diet for 8 weeks. On day 56, behaviours in the elevated plus maze, radial-arm maze, open field, and Y-maze paradigms were monitored and scored, following which animals were euthanized. The brains were removed, weighed and homogenized for the assessment of specific biochemical tests. RESULT: Results showed that hazelnut-supplemented diet was associated with significantly increased weight gain, with the Italian hazelnut being associated with greater weight gain. The hazelnut- supplemented diet also increased behavioural parameters such as horizontal locomotion and grooming, while it decreased rearing activity. Working-memory also improved significantly with both cultivars of hazelnut, while anxiety indices were reduced at lower concentrations of Italian, and higher concentrations of Turkish hazelnut. Both hazelnut varieties were associated with a reduction in acetylcholinesterase activity, superoxide dismutase activity, nitric oxide levels, caspase- 3 level, but increased dopamine level. CONCLUSION: Overall, hazelnut cultivars have beneficial effects on the brain in aged mice; suggesting a possible role in the prevention or management of age-related neurodegenerative changes.

Modeling of paclitaxel biosynthesis elicitation in Corylus avellana cell culture using adaptive neuro-fuzzy inference system-genetic algorithm (ANFIS-GA) and multiple regression methods.

Paclitaxel as a microtubule-stabilizing agent is widely used for the treatment of a vast range of cancers. Corylus avellana cell suspension culture (CSC) is a promising strategy for paclitaxel production. Elicitation of paclitaxel biosynthesis pathway is a key approach for improving its production in cell culture. However, optimization of this process is time-consuming and costly. Modeling of paclitaxel elicitation process can be helpful to predict the optimal condition for its high production in cell culture. The objective of this study was modeling and forecasting paclitaxel biosynthesis in C. avellana cell culture responding cell extract (CE), culture filtrate (CF) and cell wall (CW) derived from endophytic fungus, either individually or combined treatment with methyl-ß-cyclodextrin (MBCD), based on four input variables including concentration levels of fungal elicitors and MBCD, elicitor adding day and CSC harvesting time, using adaptive neuro-fuzzy inference system (ANFIS) and multiple regression methods. The results displayed a higher accuracy of ANFIS models (0.94-0.97) as compared to regression models (0.16-0.54). The great accordance between the predicted and observed values of paclitaxel biosynthesis for both training and testing subsets support excellent performance of developed ANFIS models. Optimization process of developed ANFIS models with genetic algorithm (GA) showed that optimal MBCD (47.65 mM) and CW (2.77% (v/v)) concentration levels, elicitor adding day (16) and CSC harvesting time (139 h and 41 min after elicitation) can lead to highest paclitaxel biosynthesis (427.92 µg l-1). The validation experiment showed that ANFIS-GA method can be a promising tool for selecting the optimal conditions for maximum paclitaxel biosynthesis, as a case study.

Identification of vital candidate microRNA/mRNA pairs regulating ovule development using high-throughput sequencing in hazel.

BACKGROUND: Hazels (Corylus spp.) are economically important nut-producing species in which ovule development determines seed plumpness, one of the key parameters reflecting nut quality. microRNAs (miRNAs) play important roles in RNA silencing and the post-transcriptional regulation of gene expression. However, very little is currently known regarding the miRNAs involved in regulating ovule growth and development. RESULTS: In this study, we accordingly sought to determine the important miRNAs involved in ovule development and growth in hazel. We examined ovules at four developmental stages, namely, ovule formation (Ov1), early ovule growth (Ov2), rapid ovule growth (Ov3), and ovule maturity (Ov4). On the basis of small RNA and mRNA sequencing using the Illumina sequencing platform, we identified 970 miRNAs in hazel, of which 766 and 204 were known and novel miRNAs, respectively. In Ov1-vs-Ov2, Ov1-vs-Ov3, Ov1-vs-Ov4, Ov2-vs-Ov3, Ov2-vs-Ov4, and Ov3-vs-Ov4 paired comparisons, 471 differentially expressed microRNAs (DEmiRNAs) and their 3117 target differentially expressed messenger RNAs (DEmRNAs) formed 11,199 DEmiRNA/DEmRNA pairs, with each DEmiRNA changing the expression of an average of 6.62 target mRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of all DEmRNAs revealed 29 significantly enriched KEGG pathways in the six paired comparisons, including protein export (ko03060), fatty acid elongation (ko00062), starch and sucrose metabolism (ko00500), fatty acid biosynthesis (ko00061), and amino sugar and nucleotide sugar metabolism (ko00520). Our results indicate that DEmiRNA/DEmRNA pairs showing opposite change trends were related to stress tolerance, embryo and seed development, cell proliferation, auxin transduction, and the biosynthesis of proteins, starch, and fats may participate in ovule growth and development. CONCLUSIONS: These findings contribute to a better understanding of ovule development at the level of post-transcriptional regulation, and lay the foundation for further functional analyses of hazelnut ovule growth and development.

Whole fungal elicitors boost paclitaxel biosynthesis induction in Corylus avellana cell culture.

Paclitaxel is an effective natural-source chemotherapeutic agent commonly applied to treat a vast range of cancers. In vitro Corylus avellana culture has been reported as a promising and inexpensive system for paclitaxel production. Fungal elicitors have been made known as the most efficient strategy for the biosynthesis induction of secondary metabolites in plant in vitro culture. In this research, C. avellana cell suspension culture (CSC) was exposed to cell extract (CE) and culture filtrate (CF) derived from Camarosporomyces flavigenus, either individually or combined treatment, in mid and late log phase. There is no report on the use of whole fungal elicitors (the combined treatment of CE and CF) for the elicitation of secondary metabolite biosynthesis in plant in vitro culture. The combined treatment of CE and CF significantly led to more paclitaxel biosynthesis and secretion than the individual use of them. Also, multivariate statistical approaches including stepwise regression (SR), ordinary least squares regression (OLSR), principal component regression (PCR) and partial least squares regression (PLSR) were used to model and predict paclitaxel biosynthesis and secretion. Based on value account for (VAF), root mean square error (RMSE), coefficient of determination (R2), mean absolute percentage error (MAPE) and relative percent difference (RPD) can be concluded that mentioned regression models effectively worked only for modeling and predicting extracellular paclitaxel portion in C. avellana cell culture.

Comparative study of concatemer efficiency as an isotope-labelled internal standard for allergen quantification.

Mass spectrometry-based methods coupled with stable isotope dilution have become effective and widely used methods for the detection and quantification of food allergens. Current methods target signature peptides resulting from proteolytic digestion of proteins of the allergenic ingredient. The choice of appropriate stable isotope-labelled internal standard is crucial, given the diversity of encountered food matrices which can affect sample preparation and analysis. We propose the use of concatemer, an artificial and stable isotope-labelled protein composed of several concatenated signature peptides as internal standard. With a comparative analysis of three matrices contaminated with four allergens (egg, milk, peanut, and hazelnut), the concatemer approach was found to offer advantages associated with the use of labelled proteins, ideal but unaffordable, and circumvent certain limitations of traditionally used synthetic peptides as internal standards. Although used in the proteomic field for more than a decade, concatemer strategy has not yet been applied for food analysis.

Metabolomics and hormonomics to crack the code of filbert growth.

INTRODUCTION: Plants respond to changes in their environments through hormonal activation of a physiological cascade that redirects metabolic resources and growth. In filberts (Corylus sp.), chelated iron promotes the growth of new shoots but the mechanism(s) are not understood. OBJECTIVES: To use untargeted metabolomics and hormonomics approaches to generate novel hypotheses for the morphoregulatory role of ferric ethylenediamine-N,N'-di-(ortho-hydroxyphenyl) acetic acid (Fe-EDDHA) in filbert shoot organogenesis in vitro. METHODS: Data were generated using previously optimized standardized untargeted metabolomics protocols with time of flight mass spectrometry. Multivariate statistical tools (principal component and partial least squares discriminant analysis) did not detect significant differences. Discovery tools Significance Analysis of Microarrays (SAM), multiple linear regression analysis, Bayesian analysis, logical algorithms, machine learning, synthetic biotransformations, targeted hormonomics, and online resources including MetaboAnalyst were used. RESULTS: Starch/sucrose metabolism and shikimate pathway metabolites were increased. Dose dependent decreases were found in polyphenol metabolism, specifically ellagic acid and its methylated derivative 3,4,3'-tri-O-methylellagic acid. Hormonomics analysis revealed significant differences in phytohormones and their conjugates. FeEDDHA treatment reduced indole-3-acetic acid, abscisic acid, salicylic acid, jasmonic acid conjugates (JA-Trp, JA-Ile, OH-JA) and dihydrozeatinglucoside in regenerating explants. Serotonin (5HT) was decreased in FeEDDHA-treated regenerating tissues while the related metabolite melatonin was increased. Eight phenolic conjugates of 5HT and eight catabolites were affected by FeEDDHA indicating that metabolism to sequester, deactivate and metabolize 5HT was induced by Fe(III). Tryptophan was metabolized through kynurenine but not anthranilate. CONCLUSION: Seven novel hypotheses were generated to guide future studies to understand the regulatory control(s) of shoot organogenesis.

Fungal Cell Wall and Methyl-ß-Cyclodextrin Synergistically Enhance Paclitaxel Biosynthesis and Secretion in Corylus avellana Cell Suspension Culture.

Paclitaxel is the top-selling chemotherapeutic drug used for the treatment of lung, ovarian and breast cancer as well as Kaposi's sarcoma. Cell suspension culture (CSC) of Corylus avellana has been addressed as a promising alternative for producing paclitaxel. In this study, endophytic fungus strain YEF33 was isolated from Taxus baccata and identified as Coniothyrium palmarum. The effects of the elicitors derived from this fungus including cell extract, culture filtrate and cell wall (CW) and also chitin, alone or in combination with Methyl-ß-Cyclodextrin (MBCD), on paclitaxel biosynthesis in C. avellana CSC were assayed for the first time. CW of C. palmarum was the most efficient fungal elicitor for paclitaxel biosynthesis in C. avellana CSC. The results revealed that MBCD affected paclitaxel biosynthesis differently depending on fungal elicitor type and vice versa. MBCD, either alone or in combination with fungal elicitors, induced a high secretion of paclitaxel, suggesting the decrement of toxicity and retro-inhibition processes of paclitaxel for cells. The joint effects of C. palmarum CW (2.5% (v/v) on 17th day) and 50 mM MBCD synergistically enhanced paclitaxel biosynthesis (402.4 µg l-1; 5.8-fold), 78.6% of which (316.5 µg l-1) were secreted into culture medium, a level 146% higher than that in control.

NMR resonance assignments of the four isoforms of the hazelnut allergen Cor a 1.04.

In large parts of Europe, Northern America and China people are suffering from allergies after consuming certain kinds of fruits and vegetables. Typical allergic symptoms range from scratching and itching of the throat to severe symptoms like rhino conjunctivitis and anaphylaxis. For hazelnuts (Corylus avellana), these allergies result from initial sensitization to the birch (Betula verrucosa) pollen allergen Bet v 1 and subsequent development of allergic cross-reactions to proteins that are similar in their three-dimensional structure to the sensitizing protein Bet v 1. The cross-reactive proteins in hazelnut are the four isoforms Cor a 1.04 with a molecular weight of about 17.5 kDa. Significant differences regarding the immunologic behavior of these proteins have been reported. In this work we assigned backbone and side chain 1H, 13C, and 15N chemical shifts of these four isoforms, Cor a 1.0401, Cor a 1.0402, Cor a 1.0403, and Cor a 1.0404 by solution NMR spectroscopy. The chemical shift data confirm the characteristic Bet v onefold for all four isoforms, consisting of seven ß-strands that are separated by two short α-helices, along with a long C-terminal α-helix. These data provide the basis for a comparative structural and dynamic analysis of these proteins by NMR in order to characterize their different immunologic cross-reactivities on a molecular level.