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The cultivated peanut ( Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR-Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR-Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2 , and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

Background: Specific IgE against a peanut 2S albumin (Ara h 2 or 6) is the best predictor of clinically relevant peanut sensitization. However, sIgE levels of peanut allergic and those of peanut sensitized but tolerant patients partly overlap, highlighting the need for improved diagnostics to prevent incorrect diagnosis and consequently unnecessary food restrictions. Thus, we sought to explore differences in V(D)J gene transcripts coding for peanut 2S albumin-specific monoclonal antibodies (mAbs) from allergic and sensitized but tolerant donors., Methods: 2S albumin-binding B-cells were single-cell sorted from peripheral blood of peanut allergic (n=6) and tolerant (n=6) donors sensitized to Ara h2 and/or 6 (≥ 0.1 kU/l) and non-atopic controls (n=5). h 2 and/or 6 (≥ 0.1 kU/l). Corresponding h heavy and light chain gene transcripts were heterologously expressed as mAbs and tested for specificity to native Ara h2 and 6. HCDR3 sequence motifs were identified by Levenshtein distances and hierarchically clustering., Results: The frequency of 2S albumin-binding B cells was increased in allergic (median: 0.01%) compared to tolerant (median: 0.006%) and non-atopic donors (median: 0.0015%, p = 0.008). The majority of mAbs (74%, 29/39) bound specifically to Ara h 2 and/or 6. Non-specific mAbs (9/10) were mainly derived from non-atopic controls. In allergic donors, 89% of heavy chain gene transcripts consisted of VH3 family genes, compared with only 54% in sensitized but tolerant and 63% of non-atopic donors. Additionally, certain HCDR3 sequence motifs were associated with allergy (n = 4) or tolerance (n = 3) upon hierarchical clustering of their Levenshtein distances., Conclusions: Peanut allergy is associated with dominant VH3 family gene usage and certain public antibody sequences (HCDR3 motifs)., (© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

2S albumins are important peanut allergens. Within this protein family, Ara h 2 and Ara h 6 have been described in detail, but Ara h 7 has received little attention. We now describe the first purification of Ara h 7 and its characterization. Two Ara h 7 isoforms were purified from peanuts. Mass spectrometry revealed that both the isoforms have a post-translation cleavage, a hydroxyproline modification near the N -terminus, and four disulfide bonds. The secondary structure of both Ara h 7 isoforms is highly comparable to those of Ara h 2 and Ara h 6. Both Ara h 7 isoforms bind IgE, and Ara h 7 is capable of inhibiting the binding between Ara h 2 and IgE, suggesting at least partially cross-reactive IgE epitopes. Ara h 7 was found in all main market types of peanut, at comparable levels. This suggests that Ara h 7 is a relevant allergen from the peanut 2S albumin protein family.

Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy., Competing Interests: The authors have declared that no competing interests exist.

Transcription infidelity (TI) is a mechanism that increases RNA and protein diversity. We found that single-base omissions (i.e., gaps) occurred at significantly higher rates in the RNA of highly allergenic legumes. Transcripts from peanut, soybean, sesame, and mite allergens contained a higher density of gaps than those of nonallergens. Allergen transcripts translate into proteins with a cationic carboxy terminus depleted in hydrophobic residues. In mice, recombinant TI variants of the peanut allergen Ara h 2, but not the canonical allergen itself, induced, without adjuvant, the production of anaphylactogenic specific IgE (sIgE), binding to linear epitopes on both canonical and TI segments of the TI variants. The removal of cationic proteins from bovine lactoserum markedly reduced its capacity to induce sIgE. In peanut-allergic children, the sIgE reactivity was directed toward both canonical and TI segments of Ara h 2 variants. We discovered 2 peanut allergens, which we believe to be previously unreported, because of their RNA-DNA divergence gap patterns and TI peptide amino acid composition. Finally, we showed that the sIgE of children with IgE-negative milk allergy targeted cationic proteins in lactoserum. We propose that it is not the canonical allergens, but their TI variants, that initiate sIgE isotype switching, while both canonical and TI variants elicit clinical allergic reactions.

Immunoglobulin E (IgE) is pivotal for manifestation and persistence of most immediate-type allergies and some asthma phenotypes. Consequently, IgE represents a crucial target for both, diagnostic purposes as well as therapeutic approaches. In fact, allergen-specific immunotherapy - aiming to re-route an IgE-based inflammatory response into an innocuous immune reaction against the allergen - is the only curative approach for IgE-mediated allergic diseases known so far. However, this requires the cognate allergen to be known. Unfortunately, even in well-characterized allergics or asthmatics, often just a small fraction of total IgE can be assigned to specific target allergens. To overcome this knowledge gap, we have devised an analytical platform for unbiased IgE target epitope detection. The system relies on chemically produced random peptide libraries immobilized on polystyrene beads ("one-bead-one-compound (OBOC) libraries") capable to present millions of different peptide motifs simultaneously to immunoglobulins from biological samples. Beads binding IgE are highlighted with a fluorophore-labeled anti-IgE antibody allowing fluorescence-based detection and isolation of positives, which then can be characterized by peptide sequencing. Setting-up this platform required an elaborate optimization process including proper choice of background suppressants, secondary antibody and fluorophore label as well as incubation conditions. For optimal performance our procedure involves a sophisticated pre-adsorption step to eliminate beads that react nonspecifically with anti-IgE secondary antibodies. This step turned out to be important for minimizing detection of "false positive" motifs that otherwise would erroneously be classified as IgE epitopes. In validation studies we were able to retrieve artificial test-peptide beads spiked into our library by using IgE directed against those test-peptides at physiological concentrations (≤20 IU/ml of specific IgE), and disease-relevant bead-bound epitopes of the major peanut allergen Ara h 2 by screening with sera from peanut allergics. Thus, we established a platform with which one can find and validate new immunoglobulin targets using patient material which displays a largely unknown immunoglobulin repertoire., (Copyright © 2020 Krause, Röckendorf, Meckelein, Sinnecker, Schwager, Möckel, Jappe and Frey.)

The tradeoff between protein and oil storage in oilseed crops has been tested here in oilseed rape ( Brassica napus ) by analyzing the effect of suppressing key genes encoding protein storage products (napin and cruciferin). The phenotypic outcomes were assessed using NMR and mass spectrometry imaging, microscopy, transcriptomics, proteomics, metabolomics, lipidomics, immunological assays, and flux balance analysis. Surprisingly, the profile of storage products was only moderately changed in RNA interference transgenics. However, embryonic cells had undergone remarkable architectural rearrangements. The suppression of storage proteins led to the elaboration of membrane stacks enriched with oleosin (sixfold higher protein abundance) and novel endoplasmic reticulum morphology. Protein rebalancing and amino acid metabolism were focal points of the metabolic adjustments to maintain embryonic carbon/nitrogen homeostasis. Flux balance analysis indicated a rather minor additional demand for cofactors (ATP and NADPH). Thus, cellular plasticity in seeds protects against perturbations to its storage capabilities and, hence, contributes materially to homeostasis. This study provides mechanistic insights into the intriguing link between lipid and protein storage, which have implications for biotechnological strategies directed at improving oilseed crops., (© 2020 American Society of Plant Biologists. All rights reserved.)

Aim: To study the prophylactic effect of recombinant Lactococcus lactis (rLl) harbouring Ara h 2.02 peanut allergen, in sensitized and challenged mice., Methods and Results: Ara h 2.02 cDNA was cloned into pNZ8048 for heterologous expression in L. lactis. The purified recombinant allergen showed IgE binding comparable with native Ara h 2. Balb/c mice were fed with either recombinant (rLl), nonrecombinant L. lactis (Ll) or NaHCO 3 (Sham) prior to sensitization and challenged with rAra h 2.02, whereas the baseline group was only fed with Ll. Allergen-specific immunoglobulin and splenocyte cytokines responses were determined for each mouse. Mice fed with either Ll or rLl showed significant alleviation of IgE and IgG 1 compared to the Sham group. Despite no significant decrease in Th2 (IL-4, IL-13, IL-6) or increase in Th1 (IFN-γ) cytokines, both groups showed lower IL-10 level, while the IL-4 : IFN-γ ratio was significantly lower for rLl compared to Ll group., Conclusions: Oral administration of rLl harbouring Ara h 2.02 demonstrated alleviation of Th2-associated responses in allergen-challenged mice and a possible added allergen-specific prophylactic effect., Significance and Impact of the Study: Ara h 2.02 coupled with the intrinsic properties of probiotic L. lactis as a delivery vehicle can be explored for the development of a commercially scalable vaccine., (© 2019 The Society for Applied Microbiology.)

Background: To date, no safe allergen-specific immunotherapy for patients with peanut allergy is available. Previous trials were associated with severe side effects., Objective: We sought to determine the relative importance of conformational and linear IgE-binding epitopes of the major peanut allergen Ara h 2 and to produce a hypoallergenic variant with abolished anaphylactogenic activity., Methods: Wild-type Ara h 2 and a mutant lacking the loops containing linear IgE epitopes were produced in insect cells. Conformational IgE epitopes were removed by unfolding these proteins through reduction and alkylation. IgE binding was tested by means of ELISA with sera from 48 Ara h 2-sensitized patients with peanut allergy. Basophil activation and T-cell proliferation were tested with blood samples from selected patients. Anaphylactogenic potency was tested by using intraperitoneal challenge of mice sensitized intragastrically to peanut extract., Results: Patients' IgE recognized conformational and linear epitopes in a patient-specific manner. The unfolded mutant lacking both types of epitopes displayed significantly lower IgE binding (median ELISA OD, 0.03; interquartile range, 0.01-0.06) than natural Ara h 2 (median ELISA OD, 0.99; interquartile range, 0.90-1.03; P < .01). Basophil activation by unfolded mutant Ara h 2 was low (median area under the curve, 72 vs 138 for native wild-type Ara h 2; P < .05), but its ability to induce T-cell proliferation was retained. Unfolded mutants without conformational epitopes did not induce anaphylaxis in peanut-sensitized mice., Conclusions: By removing conformational and linear IgE epitopes, a hypoallergenic Ara h 2 mutant with abolished IgE binding and anaphylactogenic potency but retained T-cell activation was generated., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Biosynthesis of plant seed oil is accomplished through the coordinate action of multiple enzymes in multiple subcellular compartments. Fatty acid (FA) has to be transported from plastid to endoplasmic reticulum (ER) for TAG synthesis. However, the role of plastid FA transportation during seed oil accumulation has not been evaluated. AtFAX1 (Arabidopsis fatty acid export1) mediated the FA export from plastid. In this study, we overexpressed AtFAX1 under the control of a seed specific promoter in Arabidopsis. The resultant overexpression lines (OEs) produced seeds which contained 21-33% more oil and 24-30% more protein per seed than those of the wild type (WT). The increased oil content was probably because of the enhanced FA and TAG synthetic activity. The seed size and weight were both increased accordingly. In addition, the seed number per silique and silique number per plant had no changes in transgenic plants. Taken together, our results demonstrated that seed specific overexpression of AtFAX1 could promote oil accumulation in Arabidopsis seeds and manipulating FA transportation is a feasible strategy for increasing the seed oil content., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Peanut ( Arachis hypogaea ) is one of the most common food allergens that can induce fatal anaphylaxis, and Ara h 2 is one of the major allergen components involved in peanut allergy. The aim of this study was to develop a quantitative method for detecting peanut allergen using monoclonal antibodies against Ara h 2. The splenocytes of immunized mice were fused with myeloma cells (SP2/0), and stable mAb-producing clones were obtained by limiting dilution. mAbs against Ara h 2 were isolated from mouse ascites, and specificity was confirmed by immunoblotting. Five mAbs with high purity and specific reactivity were obtained, which were referred to as 1-2E10, 2-1D5, 3-1C5, 4-1C2, and 5-1G4, respectively. After screening different mAb combinations for development of a sandwich ELISA, we selected 5-1G4 as the capture antibody and 1-2E10 as the detection antibody for the measurement of Ara h 2 from which an optimal correlation between the Ara h 2 concentration and the OD value was obtained. This sandwich ELISA could specifically detect Ara h 2 in peanut extract at concentrations as low as 5 ng/mL and up to 10  μ g/mL. These mAbs can, therefore, serve as quantitative diagnostic reagents for peanut and peanut product risk assessment.

Cytosolic phospholipase A2α (cPLA2α) is implicated in the progression of excitotoxic neuronal injury and cerebral ischemia. Previous work suggests that cPLA2α increases aberrant electrophysiologic events through attenuating K channel functions. Nevertheless, which K channels are affected by cPLA2α needs to be determined. Here we examined K channels-mediated electrophysiologic responses in hippocampal CA1 pyramidal neurons from wild-type and cPLA2α mice using simultaneous patch-clamp recording and confocal Ca imaging. After the exposure to the blockers of Ca-sensitive and A-type K channels, all CA1 neurons developed spike broadening and increased dendritic Ca transients. These effects were occluded in CA1 neurons from cPLA2α mice. Therefore, cPLA2α modulates the functions of Ca-sensitive and A-type K channels in neurotoxicity.

This study aimed to report the complete sequence of a 2S albumin purified from pine nut and to analyze its allergenic properties. Individual recognition of this protein by serum IgE from pine nut-allergic patients was assessed. IgE cross-linking capacity was analyzed in a basophil activation test. Inhibition of IgE-binding and stability to heating was also assessed. The complete nucleotide sequence was obtained and a phylogenetic study was carried out. 2S albumin from pine nut (registered as Pin p 1.0101) was recognized by IgE of 75% of sera. The allergen was heat-stable and had a robust capacity to inhibit IgE-binding to whole pine nut extract. The IgE cross-linking capacity of Pin p 1 on basophils was also demonstrated. Despite the low homology of Pin p 1 sequence with other allergenic 2S albumins from angiosperms, Pin p 1 contains the typical skeleton of 8 cysteine residues, important for its α-helixes enriched structure., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Peanut allergy is an IgE-mediated adverse reaction to a subset of proteins found in peanuts. Immunotherapy aims to desensitize allergic patients through repeated and escalating exposures for several months to years using extracts or flours. The complex mix of proteins and variability between preparations complicates immunotherapy studies. Moreover, peanut immunotherapy is associated with frequent negative side effects and patients are often at risk of allergic reactions once immunotherapy is discontinued. Allergen-specific approaches using recombinant proteins are an attractive alternative because they allow more precise dosing and the opportunity to engineer proteins with improved safety profiles. We tested whether Ara h 1 and Ara h 2, two major peanut allergens, could be produced using chloroplast of the unicellular eukaryotic alga, Chlamydomonas reinhardtii. C. reinhardtii is novel host for producing allergens that is genetically tractable, inexpensive and easy to grow, and is able to produce more complex proteins than bacterial hosts. Compared to the native proteins, algal-produced Ara h 1 core domain and Ara h 2 have a reduced affinity for IgE from peanut-allergic patients. We further found that immunotherapy using algal-produced Ara h 1 core domain confers protection from peanut-induced anaphylaxis in a murine model of peanut allergy., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Key Message: We have used a combination of genomic, transcriptomic, and proteomic approaches to identify the napin-type albumin genes in sunflower and define their contributions to the seed albumin pool. Seed protein content is determined by the expression of what are typically large gene families. A major class of seed storage proteins is the napin-type, water soluble albumins. In this work we provide a comprehensive analysis of the napin-type albumin content of the common sunflower (Helianthus annuus) by analyzing a draft genome, a transcriptome and performing a proteomic analysis of the seed albumin fraction. We show that although sunflower contains at least 26 genes for napin-type albumins, only 15 of these are present at the mRNA level. We found protein evidence for 11 of these but the albumin content of mature seeds is dominated by the encoded products of just three genes. So despite high genetic redundancy for albumins, only a small sub-set of this gene family contributes to total seed albumin content. The three genes identified as producing the majority of sunflower seed albumin are potential future candidates for manipulation through genetics and breeding.

Background: The prevalence of food allergy has been increasing, but treatment is very limited. DNA vaccination has been recognized as a promising method for the treatment of allergic diseases; however, poor immunogenicity has hindered its application., Methods: BALB/c mice were intradermally injected with plasmid DNA encoding the peanut protein Ara h 2 (pAra h 2) or pAra h 2 pretreated with poly-L- lysine (PLL) before or after sensitization with Ara h 2 protein. Ara h 2-specific antibodies were measured by ELISA. CD207+ dendritic cells (DCs) and Treg cells in draining lymph nodes were analyzed by flow cytometry after DNA immunization, and cytokine production in splenocytes was also analyzed., Results: In the prophylactic study, pretreatment with pAra h 2 or PLL-pAra h 2 resulted in lower levels of Ara h 2-specific IgG1, IgG2a, and IgE after sensitization with Ara h 2 protein, and mice in the PLL-pAra h 2 group had a significantly lower level of antibodies than those in the pAra h 2 group. In the treatment study, intradermal injection with pAra h 2 or PLL-pAra h 2 after Ara h 2 protein sensitization significantly decreased the level of Ara h 2-specific antibodies, and PLL- pAra h 2 had stronger effects than pAra h 2. There were increased numbers of CD207+ DCs and Treg cells in the mice receiving intradermal injection with PLL-pAra h 2, and splenocytes from PLL-pAra h 2-treated mice secreted increased levels of IFN-γ and IL-10., Conclusions: Modification of pAra h 2 with PLL improved its prophylactic and therapeutic effects in peanut-allergic mice., (© 2017 S. Karger AG, Basel.)

Current diagnostic tools for peanut allergy using crude peanut extract showed low predictive value and reduced specificity for detection of peanut allergen-specific immunoglobulin E (IgE). The Ara h 2.02, an isoform of the major peanut allergen Ara h 2, contains three IgE epitope recognition sequence of 'DPYSPS' and may be a better reagent for component resolve diagnosis. This research aimed to generate a codon-optimised Ara h 2.02 gene for heterologous expression in Escherichia coli and allergenicity study of this recombinant protein. The codon-optimised gene was generated by PCR using overlapping primers and cloned into the pET-28a (+) expression vector. Moderate expression of a 22.5 kDa 6xhistidine-tagged recombinant Ara h 2.02 protein (6xHis-rAra h 2.02) in BL21 (DE3) host cells was observed upon induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The insoluble recombinant protein was purified under denaturing condition using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and refolded by dialysis in decreasing urea concentration, amounting to a yield of 74 mg/l of expression culture. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and immunoblot analysis confirmed the production of the recombinant 6xHis-rAra h 2.02. The refolded recombinant 6xHis-rAra h 2.02, with or without adjuvant, was able to elicit comparable level of allergen-specific IgE and IgG1 in sensitised Balb/c mice. In addition, the specific IgE antibodies raised against the recombinant protein were able to recognise the native Ara h 2 protein, demonstrating its allergenicity and potential as a reagent for diagnosis and therapeutic study.

This study investigated the structural stability of yellow mustard (YM, Sinapis alba L.) napin and the changes of its Sin a 1 anti-epitope antibody-binding ability during myrosinase enzyme inactivation process. The food industry uses myrosinase-inactive non-pungent YM for uses beyond spice applications. Napin was isolated from seeds received from an industrial processor before (YM + M) and after (YM - M) myrosinase inactivation. Secondary and tertiary structural features and surface hydrophobicity parameters of napin were analyzed. The Sin a 1 content in YM seeds and the stability of Sin a 1-containing napin during simulated in vitro gastrointestinal (GI) digestion were determined by a non-competitive indirect enzyme-linked immunosorbent assay using the Sin a 1 anti-epitope antibody (AE-Ab) as the primary Ab. YM napin retained the dominant alpha-helical components of secondary and tertiary structure folds during this process. YM - M napin showed changes in hydrophobicity parameters of the molecules and binding ability of AE-Ab: 2.19 ± 0.48 g per 100 g of YM - M seeds vs. 1.49 ± 0.16 g per 100 g YM + M seeds. YM - M proteins were more susceptible for in vitro GI digestion and also showed a 30% reduction in AE-Ab binding ability upon digestion of napins. This suggests that the myrosinase inactivation process has induced the surface modification of napin, exposing Sin a 1 epitope, leading to an increase in AE-Ab binding. However, the epitope region of YM - M napin showed improved susceptibility for hydrolysis during GI digestion resulting in fewer available epitope regions, suggesting a possible reduction in napin immune reactivity.

Mo-CBP3 is a chitin-binding protein from M. oleifera seeds that inhibits the germination and mycelial growth of phytopathogenic fungi. This protein is highly thermostable and resistant to pH changes, and therefore may be useful in the development of new antifungal drugs. However, the relationship of MoCBP3 with the known families of carbohydrate-binding domains has not been established. In the present study, full-length cDNAs encoding 4 isoforms of Mo-CBP3 (Mo-CBP3-1, Mo-CBP3-2, Mo-CBP3-3 and Mo-CBP3-4) were cloned from developing seeds. The polypeptides encoded by the Mo-CBP3 cDNAs were predicted to contain 160 (Mo-CBP3-3) and 163 amino acid residues (Mo-CBP3-1, Mo-CBP3-2 and Mo-CBP3-4) with a signal peptide of 20-residues at the N-terminal region. A comparative analysis of the deduced amino acid sequences revealed that Mo-CBP3 is a typical member of the 2S albumin family, as shown by the presence of an eight-cysteine motif, which is a characteristic feature of the prolamin superfamily. Furthermore, mass spectrometry analysis demonstrated that Mo-CBP3 is a mixture of isoforms that correspond to different mRNA products. The identification of Mo-CBP3 as a genuine member of the 2S albumin family reinforces the hypothesis that these seed storage proteins are involved in plant defense. Moreover, the chitin-binding ability of Mo-CBP3 reveals a novel functionality for a typical 2S albumin.

Genes encoding seed storage proteins (SSPs) are specifically and highly expressed during seed maturation. In Arabidopsis, chromatin-based mechanisms involved in the repression of SSPs during germination have been proposed. However, epigenetic regulation involved in repressing SSPs in vegetative tissues of peanut is not well understood. Histone deacetylase (HDAC) is a chromatin-remodelling factor that contributes to transcriptional repression in eukaryotes. To address whether histone deacetylation modification is involved in the repression of SSP genes during germination in peanut, we generated an Ara h 2.02pro : β-glucuronidase (GUS) construct by fusing the 1972 bp Ara h 2.02 promoter of peanut (from -1972 to -1) to the GUS reporter gene and transformed it into wild-type Arabidopsis plants and HDAC mutants. GUS staining revealed that the mutation in HISTONE DEACETYLASE19 (HDA19) resulted in the ectopic expression of peanut SSP gene Ara h 2.02 in seedlings. In addition, Chromatin immunoprecipitation (ChIP) assays showed that the ectopic expression of Ara h 2.02 was accompanied by histone hyperacetylation during germination. These results suggest that histone deacetylation modification may play a vital role in repressing embryonic properties during the peanut vegetative growth., (© 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Transgenic peanut (Arachis hypogaea L.) containing a gene designed for RNA interference (RNAi) showed stable complete silencing of Ara h 2 and partial silencing of Ara h 6, two potent peanut allergens/proteins, along with minimal collateral changes to other allergens, Ara h 1 and Ara h 3, across three generations (T3, T4, and T5) under field conditions. Different soil sulfur levels (0.012, 0.3, and 3.0 mM) differentially impacted sulfur-rich (Ara h 2, Ara h 3, and Ara h 6) versus sulfur-poor (Ara h 1) proteins in non-transgenic versus transgenic peanut. The sulfur level had no effect on Ara h 1, whereas low sulfur led to a significant reduction of Ara h 3 in transgenic and non-transgenic seeds and Ara h 2 and Ara h 6 in non-transgenic but not in transgenic peanuts because these proteins already were reduced by gene silencing. These results demonstrate stability of transgene expression and the potential utility of RNAi in allergen manipulation.

A new selective electrochemical genosensor has been developed for the detection of an 86-mer DNA peanut sequence encoding part of the allergen Ara h 2 (conglutin-homolog protein). The method is based on a sandwich format, which presents two advantages: it permits shortening the capture probe and avoids labeling of the target. Screen-printed gold electrodes have been used as platform for the immobilization of oligonucleotides by the well-known S-Au bond. Mixed self-assembled monolayers (SAM), including thiol-modified capture probe and mercaptohexanol, were prepared to achieve an organized, homogeneous and not too compact SAM in which unspecific adsorption of the capture probe would be prevented. The optimization of the sensing phase was carried out using the Design of Experiments (DoE) approach. Traditionally, response optimization is achieved by changing the value of one factor at a time until there is no further improvement. However, DoE involves regulating the important factors so that the result becomes optimal. Optimized conditions were found to be 1.34 µM for capture probe concentration and 3.15 mM for mercaptohexanol (spacer) concentration. When the optimal conditions were employed the analytical performance of the proposed genosensor improved significantly, showing a sensitivity as high as 3 µA/nM, with a linear range from 5×10(-11) to 5×10(-8) M and a detection limit of 10 pM., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The sesame 2S albumin (2Salb) promoter was evaluated for its capacity to express the reporter gusA gene encoding β-glucuronidase in transgenic tobacco seeds relative to the soybean fad3C gene promoter element. Results revealed increased expression of gusA gene in tobacco seed tissue when driven by sesame 2S albumin promoter. Prediction based deletion analysis of both the promoter elements confirmed the necessary cis-acting regulatory elements as well as the minimal promoter element for optimal expression in each case. The results also revealed that cis-regulatory elements might have been responsible for high level expression as well as spatio-temporal regulation of the sesame 2S albumin promoter. Transgenic over-expression of a fatty acid desaturase (fad3C) gene of soybean driven by 2S albumin promoter resulted in seed-specific enhanced level of α-linolenic acid in sesame. The present study, for the first time helped to identify that the sesame 2S albumin promoter is a promising endogenous genetic element in genetic engineering approaches requiring spatio-temporal regulation of gene(s) of interest in sesame and can also be useful as a heterologous genetic element in other important oil seed crop plants in general for which seed oil is the harvested product. The study also established the feasibility of fatty acid metabolic engineering strategy undertaken to improve quality of edible seed oil in sesame using the 2S albumin promoter as regulatory element.

Key Message: Cruciferin (cru) and napin (nap) were negatively correlated and the cru/nap ratio was closely negative correlated with glucosinolate content indicating a link between the two biosynthetic pathways. Canola-type oilseed rape (Brassica napus L.) is an economically important oilseed crop in temperate zones. Apart from the oil, the canola protein shows potential as a value-added food and nutraceutical ingredient. The two major storage protein groups occurring in oilseed rape are the 2 S napins and 12 S cruciferins. The aim of the present study was to analyse the genetic variation and the inheritance of napin and cruciferin content of the seed protein in the winter oilseed rape doubled haploid population Express 617 × R53 and to determine correlations to other seed traits. Seed samples were obtained from field experiments performed in 2 years at two locations with two replicates in Germany. A previously developed molecular marker map of the DH population was used to map quantitative trait loci (QTL) of the relevant traits. The results indicated highly significant effects of the year and the genotype on napin and cruciferin content as well as on the ratio of cruciferin to napin. Heritabilities were comparatively high with 0.79 for napin and 0.77 for cruciferin. Napin and cruciferin showed a significant negative correlation (-0.36**) and a close negative correlation of the cru/nap ratio to glucosinolate content was observed (-0.81**). Three QTL for napin and two QTL for cruciferin were detected, together explaining 47 and 35 % of the phenotypic variance. A major QTL for glucosinolate content was detected on linkage group N19 whose confidence interval overlapped with QTL for napin and cruciferin content. Results indicate a relationship between seed protein composition and glucosinolate content.

Seeds are capable of accumulating high levels of seed storage proteins (SSP), as well as heterologous proteins under certain conditions. Arabidopsis thaliana was used to develop a strategy to deplete seeds of an endogenous SSP and then replenish them with the same protein fused to a heterologous protein. In several other studies, competition with endogenous SSP for space and metabolic resources was shown to affect the accumulation of recombinant proteins in seeds. We used RNAi to reduce the expression of the five napin genes and deplete the seeds of this SSP. Targeting a recombinant protein to a vacuole or structure within the seed where it can be protected from cytosolic proteases can also promote its accumulation. To achieve this, a synthetic Brassica napus napin gene (Bn napin) was designed that was both impervious to the A. thaliana napin (At napin) RNAi construct and permitted fusion to a heterologous protein, in this case green fluorescent protein (GFP). GFP was placed in several strategic locations within Bn napin with consideration to maintaining structure, processing sites and possible vacuolar targeting signals. In transgenic A. thaliana plants, GFP was strongly localized to the seed protein storage vacuole in all Bn napin fusion configurations tested, but not when expressed alone. This SSP depletion-replenishment strategy outlined here would be applicable to expression of recombinant proteins in industrial crops that generally have large repertoires of endogenous SSP genes., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

The similarities of two major peanut allergens, Ara h 2 and Ara h 6, in molecular size, amino acid sequence, and structure have made it difficult to obtain natural Ara h 6 free of Ara h 2. The objectives of this study were to purify natural Ara h 6 that is essentially free of Ara h 2 and to compare its IgE reactivity and potency in histamine release assays to Ara h 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the highly purified allergen (<0.01% Ara h 2) revealed a single 14.5 kD band, and the identity of Ara h 6 was confirmed by liquid chromatography-tandem mass spectrometry. Ara h 6 showed a higher seroprevalence in chimeric IgE enzyme-linked immunosorbent assay (n = 54) but a weaker biological activity in basophil histamine release assays than Ara h 2. Purified Ara h 6 will be useful for diagnostic IgE antibody assays as well as molecular and cellular studies to investigate the immunological mechanisms of peanut allergy.

Conglutins, the major peanut allergens, Ara h 2 and Ara h 6, are highly structured proteins stabilized by multiple disulfide bridges and are stable towards heat-denaturation and digestion. We sought a way to reduce their potent allergenicity in view of the development of immunotherapy for peanut allergy. Isoforms of conglutin were purified, reduced with dithiothreitol and subsequently alkylated with iodoacetamide. The effect of this modification was assessed on protein folding and IgE-binding. We found that all disulfide bridges were reduced and alkylated. As a result, the secondary structure lost α-helix and gained some β-structure content, and the tertiary structure stability was reduced. On a functional level, the modification led to a strongly decreased IgE-binding. Using conditions for limited reduction and alkylation, partially reduced and alkylated proteins were found with rearranged disulfide bridges and, in some cases, intermolecular cross-links were found. Peptide mass finger printing was applied to control progress of the modification reaction and to map novel disulfide bonds. There was no preference for the order in which disulfides were reduced, and disulfide rearrangement occurred in a non-specific way. Only minor differences in kinetics of reduction and alkylation were found between the different conglutin isoforms. We conclude that the peanut conglutins Ara h 2 and Ara h 6 can be chemically modified by reduction and alkylation, such that they substantially unfold and that their allergenic potency decreases., (© 2013.)

This study investigated the subcellular location of mung bean (Vigna radiata) 8S globulin in transient expression systems as well as in tobacco (Nicotiana tabacum) BY-2 cells and different tissues from a transgenic Arabidopsis (Arabidopsis thaliana) line stably expressing this storage globulin. When transiently expressed in protoplasts from both BY-2 cells and Arabidopsis suspension cultured cells, the 8S globulin located to structures that were neither Golgi nor pre-vacuolar compartments (PVCs). Immunogold electron microscopy of the transgenics reveals the 8S globulin-positive structures to be small, spherical, ribosome-covered endoplasmic reticulum (ER)-derived bodies. In BY-2 cells and all vegetative cells, the 8S globulin was present as a pro-form. However, in Arabidopsis embryos, with the onset of endogenous storage protein synthesis, the 8S globulin exited the ER and passed through the PVC to the protein storage vacuole where it was processed to its smaller mature form. These results clearly demonstrated that, when taken out of context and expressed in vegetative cells, the mung bean 8S storage globulin cannot exit the ER, and indicate that natural targeting of storage proteins to the vacuole should be better studied in the maturing seed.

Background: Conventional immunotherapy for peanut allergy using crude peanut extracts is not recommended because of the unacceptably high risk of anaphylaxis. Allergen-specific immunotherapy is not currently undertaken for peanut allergy., Objectives: The objective of this study was to develop a novel peanut-human fusion protein to block peanut-induced anaphylaxis., Methods: We genetically designed and expressed a novel plant-human fusion protein composed of the major peanut allergen Ara h 2 and human IgG Fcγ1. We tested the Ara h 2-Fcγ fusion protein (AHG2)'s function in purified human basophils. Transgenic mice expressing human FcεRIα and a murine peanut allergy model were used., Results: AHG2 inhibited histamine release induced by whole peanut extract (WPE) from basophils of patients with peanut allergy, whereas the fusion protein itself did not induce mediator release. AHG2 inhibited the WPE-induced, peanut-specific, IgE-mediated passive cutaneous anaphylaxis in hFcεRIα transgenic mice. AHG2 also significantly inhibited acute anaphylactic reactivity, including the prototypical decrease in body temperature in WPE-sensitized mice challenged with crude peanut extract. Histologic evaluation of the airways showed that AHG2 decreased peanut-induced inflammation, whereas the fusion protein itself did not induce airway inflammation in peanut-sensitized mice. AHG2 did not exert an inhibitory effect in mice lacking FcγRII., Conclusion: AHG2 inhibited peanut-specific IgE-mediated allergic reactions in vitro and in vivo. Linking specific peanut allergen to Fcγ can provide a new approach for the allergen immunotherapy of peanut allergy., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Scope: Ara h 6 has recently been recognized as an important peanut allergen. Recombinant allergens have been used for analysis of IgE binding, but have not been used to analyze the allergic effector activity that is more relevant to allergic reactions., Methods and Results: Ara h 6 was expressed as a recombinant protein in both Escherichia coli and Pichia pastoris (rAra h 6-E. coli and rAra h 6-Pichia, respectively). Effector activity was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from patients with severe peanut allergy. Compared to native Ara h 6 (nAra h 6), rAra h 6-Pichia had intact effector function whereas rAra h 6-E. coli had significantly reduced function. The lower effector activity in rAra h 6-E. coli compared to nAra h 6 and rAra h 6-Pichia did not appear to be due to differences in posttranslational modifications (analyzed by mass spectrometry and staining for carbohydrates) and may be due to subtle alteration(s) of folding seen on CD analysis and on nonreduced gels. Finally, we introduced point mutations in four important IgE-binding linear epitopes of Ara h 6 and found dramatically reduced allergic effector activity., Conclusion: Our studies demonstrate the utility of fully functional rAra h 6-Pichia as a starting point for analysis of specific mutations that adversely affect allergic effector function., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: A one-step polymerase chain reaction (PCR) method for the simultaneous detection of the major allergens of pecan and Brazil nuts was developed. Primer pairs for the amplification of partial sequences of genes encoding the allergens were designed and tested for their specificity on a range of food components., Results: The targeted amplicon size was 173 bp of Ber e 1 gene of Brazil nuts and 72 bp of vicilin-like seed storage protein gene in pecan nuts. The primer pair detecting the noncoding region of the chloroplast DNA was used as the internal control of amplification. The intrinsic detection limit of the PCR method was 100 pg mL(-1) pecan or Brazil nuts DNA. The practical detection limit was 0.1% w/w (1 g kg(-1)). The method was applied for the investigation of 63 samples with the declaration of pecans, Brazil nuts, other different nut species or nuts generally. In 15 food samples pecans and Brazil nuts allergens were identified in the conformity with the food declaration., Conclusion: The presented multiplex PCR method is specific enough and can be used as a fast approach for the detection of major allergens of pecan or Brazil nuts in food., (Copyright © 2011 Society of Chemical Industry.)

Background: Peanut allergy affects 1% of the population and causes the most fatal food-related anaphylactic reactions. The protein Ara h 2 is the most potent peanut allergen recognized by 80-90% of peanut allergic patients., Methods: The crystal structure of the major peanut allergen Ara h 2 was determined for the first time at 2.7 Å resolution using a customized maltose-binding protein (MBP)-fusion system. IgE antibody binding to the MBP fusion construct vs the natural allergen was compared by ELISA using sera from peanut allergic patients., Results: The structure of Ara h 2 is a five-helix bundle held together by four disulfide bonds and related to the prolamin protein superfamily. The fold is most similar to other amylase and trypsin inhibitors. The MBP--Ara h 2 fusion construct was positively recognized by IgE from 76% of allergic patients (25/33). Two populations of patients could be identified. Subpopulation 1 (n = 14) showed an excellent correlation of IgE antibody binding to natural vs recombinant Ara h 2. Subpopulation 2 (n = 15) showed significantly reduced IgE binding to the MBP fusion protein. Interestingly, about 20% of the IgE binding in subpopulation 2 could be recovered by increasing the distance between MBP and Ara h 2 in a second construct., Discussion: The reduced IgE binding to the MBP--Ara h 2 of subpopulation 2 indicates that the MBP molecule protects an immunodominant epitope region near the first helix of Ara h 2. Residues involved in the epitope(s) are suggested by the crystal structure. The MBP--Ara h 2 fusion constructs will be useful to further elucidate the relevance of certain epitopes to peanut allergy., (© Published 2011. This article is a US Government work and is in the public domain in the USA.)

Background: Allergic reactions to peanuts (Arachis hypogaea L.) can cause severe symptoms and in some cases can be fatal, but avoidance is difficult due to the prevalence of peanut-derived products in processed foods. One strategy of reducing the allergenicity of peanuts is to alter or eliminate the allergenic proteins through mutagenesis. Other seed quality traits could be improved by altering biosynthetic enzyme activities. Targeting Induced Local Lesions in Genomes (TILLING), a reverse-genetics approach, was used to identify mutations affecting seed traits in peanut., Results: Two similar copies of a major allergen gene, Ara h 1, have been identified in tetraploid peanut, one in each subgenome. The same situation has been shown for major allergen Ara h 2. Due to the challenge of discriminating between homeologous genes in allotetraploid peanut, nested PCR was employed, in which both gene copies were amplified using unlabeled primers. This was followed by a second PCR using gene-specific labeled primers, heteroduplex formation, CEL1 nuclease digestion, and electrophoretic detection of labeled fragments. Using ethyl methanesulfonate (EMS) as a mutagen, a mutation frequency of 1 SNP/967 kb (3,420 M2 individuals screened) was observed. The most significant mutations identified were a disrupted start codon in Ara h 2.02 and a premature stop codon in Ara h 1.02. Homozygous individuals were recovered in succeeding generations for each of these mutations, and elimination of Ara h 2.02 protein was confirmed. Several Ara h 1 protein isoforms were eliminated or reduced according to 2D gel analyses. TILLING also was used to identify mutations in fatty acid desaturase AhFAD2 (also present in two copies), a gene which controls the ratio of oleic to linoleic acid in the seed. A frameshift mutation was identified, resulting in truncation and inactivation of AhFAD2B protein. A mutation in AhFAD2A was predicted to restore function to the normally inactive enzyme., Conclusions: This work represents the first steps toward the goal of creating a peanut cultivar with reduced allergenicity. TILLING in peanut can be extended to virtually any gene, and could be used to modify other traits such as nutritional properties of the seed, as shown in this study.

Background: Peanut allergy is a life-threatening condition; there is currently no cure. Although whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions, and even fatalities, in peanut allergy., Objective: This study aimed to identify short, T-cell epitope-based peptides that target allergen-specific CD4(+) T cells but do not bind IgE as candidates for safe peanut-specific immunotherapy., Methods: Multiple CD4(+) T-cell lines specific for the major peanut allergen Ara h 2 were generated from PBMCs of 16 HLA-diverse subjects with peanut allergy by using 5,6-carboxyfluorescein diacetate succinimidylester-based methodology. Proliferation and ELISPOT assays were used to identify dominant epitopes recognized by T-cell lines and to confirm recognition by peripheral blood T cells of epitope-based peptides modified for therapeutic production. HLA restriction of core epitope recognition was investigated by using anti-HLA blocking antibodies and HLA genotyping. Serum-IgE peptide-binding was assessed by dot-blot., Results: Five dominant CD4(+) T-cell epitopes were identified in Ara h 2. In combination, these were presented by HLA-DR, HLA-DP, and HLA-DQ molecules and recognized by T cells from all 16 subjects. Three short peptide variants containing these T-cell epitopes were designed with cysteine-to-serine substitutions to facilitate stability and therapeutic production. Variant peptides showed HLA-binding degeneracy, did not bind peanut-specific serum IgE, and could directly target T(H)2-type T cells in peripheral blood of subjects with allergy., Conclusion: Short CD4(+) T-cell epitope-based Ara h 2 peptides were identified as novel candidates for a T-cell-targeted peanut-specific immunotherapy for an HLA-diverse population., (Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Double-blind placebo-controlled food challenge (DBPCFC) is currently considered the gold standard for peanut allergy diagnosis. However, this procedure that requires the hospitalization of patients, mostly children, in specialized centers for oral exposure to allergens may cause severe reactions requiring emergency measures. Thus, a simpler and safer diagnosis procedure is needed. The aim of this study was to evaluate the diagnostic performance of a new set of in vitro blood tests for peanut allergy., Methods: The levels of IgE directed towards peanut extract and recombinant peanut allergens Ara h 1, Ara h 2, Ara h 3, Ara h 6, Ara h 7, and Ara h 8 were measured in 3 groups of patients enrolled at 2 independent centers: patients with proven peanut allergy (n=166); pollen-sensitized subjects without peanut allergy (n=61), and control subjects without allergic disease (n=10)., Results: Seventy-nine percent of the pollen-sensitized patients showed IgE binding to peanut, despite their tolerance to peanut. In contrast, combining the results of specific IgE to peanut extract and to recombinant Ara h 2 and Ara h 6 yielded a peanut allergy diagnosis with a 98% sensitivity and an 85% specificity at a positivity threshold of 0.10 kU/l. Use of a threshold of 0.23 kU/l for recombinant Ara h 2 increased specificity (96%) at the cost of sensitivity (93%)., Conclusion: A simple blood test can be used to diagnose peanut allergy with a high level of precision. However, DBPCFC will remain useful for the few cases where immunological and clinical observations yield conflicting results., (Copyright © 2010 S. Karger AG, Basel.)

Peanut is one of the most popular foods in the world due to its high nutrition; however, it contains multiple seed storage proteins which are identified as allergens and hence are the most common cause of life-threatening, IgE-mediated anaphylaxis among the hypersensitive individuals. Three peanut proteins, Arachis hypogaea allergy 1, 2, 3 (Ara h 1, Ara h 2 and Ara h 3), which have the common biochemical characteristics like resistance to proteases and heat, are considered as the major allergens because they are recognized by serum IgE from a peanut-allergic patient population. The linear IgE-binding epitopes in the allergens lay the foundation of the anaphylaxis in the peanut-allergic individuals. Peanut allergy is often a life-long problem, so many investigators are focusing on decreasing clinical reactivity. In this review, the latest advances in the researches on biochemical characteristics, structure and function of the three major allergens were described and particular attention was given to the immunity properties of the three allergens. The future research directions were also discussed.

Scope: 2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity., Methods: 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP-HPLC. After N-terminal sequencing, degenerated and poly-d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut-allergic patients., Results: N-terminal sequencing of a approximately 10 kDa peak from size exclusion chromatography/RP-HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N-terminal and a 10 aa internal peptide. The obtained clone (441 bp) encoded a 147 aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses., Conclusion: We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.

In recent years, several novel relevant peanut allergens have been identified. Among those, a new member of the conglutin family was cloned by a phage display approach and initially annotated as Ara h 7.0101. Later, however, recloning of Ara h 7 revealed an alternate isoform, termed Ara h 7.0201. Because the natural Ara h 7 counterpart had not been found at the protein level in peanut extracts, the aim of the present study was to search for authentic natural Ara h 7 protein(s). To this end, enriched low molecular mass proteins (<20 kDa) from peanut extracts were separated by 2D electrophoresis and subjected to mass spectrometric analyses. Fifty of 65 analyzed spots were identified. Interestingly, Ara h 7.0101 was not identified, but Ara h 7.0201 and Ara h 7.0202, a different Ara h 7 isoallergen containing an additional pro-peptide cleavage site, were. In accordance with the conserved cysteine pattern of conglutins, Ara h 7.0201 possesses eight cysteine residues, in contrast to the six cysteines present in the previously cloned Ara h 7.0101. Moreover, a putative cleavage site in the Ara h 7.0202 isoform points to the characteristic biological function of conglutins as amylase/trypsin inhibitors.

Here we report the production of marker-free transgenic plants expressing phenolic compounds with high pharmacological value. Our strategy consisted in simultaneous delivery of lox-target and cre-containing constructs into the plant genome by cotransformation. In the Cre-vector, the cre recombinase gene was controlled by a seed-specific napin promoter. In the lox-target construct the selectable bar gene was placed between two lox sites in direct orientation, while a napin promoter driven vstI gene was inserted outside of the lox sites. Upon seed-specific cre induction the bar expression cassette was excised from the tobacco genome. Genetic and molecular analysis of T1 progeny plants indicated DNA excision in all 10 transgenic lines tested. RP-HPLC analysis demonstrated that the expression of the vstI gene resulted in accumulation of trans-resveratrol and its glycosylated derivative piceid in seeds of all marker free lines. These findings indicate that the seed-specific marker gene excision did not interfere with the expression of the gene of interest. Our data demonstrated the feasi of a developmentally controlled cre gene to mediate site-specific excision in tobacco very efficiently.

Many commodity crops are grown for human consumption, and the resulting food products usually contain proteins, some of which may be allergenic. The legumes, peanut and soybean, as well as tree nuts and some cereal grains are well recognized sources of food allergens. In peanut, there are 11 documented allergenic proteins, although the major allergens are considered to be Ara h 1 and Ara h 2, both of which are seed storage proteins. Methods to reduce or eliminate these proteins from seeds are available and allow the feasibility of this approach to be tested. Greatly reduced amounts of Ara h 2 can be achieved by RNA silencing in transgenic peanut; however, mutagenesis is a more viable and socially acceptable approach to allergen elimination. Although the techniques for mutagenesis are not new, methods for mutant detection at the molecular level have recently been developed. However, these methods are dependent on genome sequence. These methods will facilitate discovery of spontaneous and induced mutations that may be useful over the long term to eliminate certain allergens from peanut.

Jug r 1, the 2S albumin allergen from walnut, was isolated from ripe nuts as a native allergen and expressed in Escherichia coli using the Gateway technology as a recombinant allergen. The recombinant Jug r 1 (15 kDa) differs from the native allergen by the absence of cleavage of the polypeptide chain in two covalently associated light (3.5 kDa) and heavy (8 kDa) chains. Recombinant rJug r 1 adopts the canonical alpha-helical fold of plant 2S albumins as checked on CD spectra. Four IgE-binding epitopic stretches were identified along the amino acid sequence of Jug r 1 and localized on the molecular surface of the modeled allergen. Both native and recombinant allergens exhibit similar IgE-binding activity and similarly trigger the degranulation of a FcepsilonRI-expressing rat basophilic leukaemia cell line previously treated by IgE-containing sera. Native Jug r 1 resists to heat denaturation and to the proteolytic attack of trypsin and chymotrypsin but is readily hydrolyzed in the presence of pepsin at acidic pH after 1 h of incubation at 37 degrees C in vitro. Recombinant Jug r 1 could be used for a component-resolved diagnosis of food-allergy.

An amaranth (Amaranthus hypochondriacus) albumin gene, encoding the 35-kDa AmA1 protein of the seed, with a high content of essential amino acids, was used in the biolistic transformation of bread wheat (Triticum aestivum L.) variety Cadenza. The transformation cassette carried the ama1 gene under the control of a powerful wheat endosperm-specific promoter (1Bx17 HMW-GS). Southern-blot analysis of T(1) lines confirmed the integration of the foreign gene, while RT-PCR and Western-blot analyses of the samples confirmed the transcription and translation of the transgene. The effects of the extra albumin protein on the properties of flour, produced from bulked T(2) seeds, were calculated using total protein and essential amino acid content analysis, polymeric/monomeric protein and HMW/LMW glutenin subunit ratio measurements. The results indicated that not only can essential amino acid content be increased, but some parameters associated with functional quality may also be improved because of the expression of the AmA1 protein.

Transcription of Arabidopsis thaliana seed maturation (MAT) genes is controlled by members of several transcription factor families, such as basic leucine zippers (bZIPs), B3s, MYBs, and DOFs. In this work, we identify Arabidopsis bZIP53 as a novel transcriptional regulator of MAT genes. bZIP53 expression in developing seeds precedes and overlaps that of its target genes. Gain- and loss-of-function approaches indicate a correlation between the amount of bZIP53 protein and MAT gene expression. Specific in vivo and in vitro binding of bZIP53 protein to a G-box element in the albumin 2S2 promoter is demonstrated. Importantly, heterodimerization with bZIP10 or bZIP25, previously described bZIP regulators of MAT gene expression, significantly enhances DNA binding activity and produces a synergistic increase in target gene activation. Full-level target gene activation is strongly correlated with the ratio of the correspondent bZIP heterodimerization partners. Whereas bZIP53 does not interact with ABI3, a crucial transcriptional regulator in Arabidopsis seeds, ternary complex formation between the bZIP heterodimers and ABI3 increases the expression of MAT genes in planta. We therefore propose that heterodimers containing bZIP53 participate in enhanceosome formation to produce a dramatic increase in MAT gene transcription.

Peanut (Arachis hypogaea L.), can elicit type I allergy becoming the most common cause of fatal food-induced anaphylactic reactions. Strict avoidance is the only effective means of dealing with this allergy. Ara h 2, a peanut seed storage protein, has been identified as the most potent peanut allergen and is recognized by approximately 90% of peanut hypersensitive individuals in the US. Because peanut has limited genetic variation, wild relatives are a good source of genetic diversity. After screening 30 Arachis duranensis accessions by EcoTILLing, we characterized five different missense mutations in ara d 2.01. None of these polymorphisms induced major conformational modifications. Nevertheless, a polymorphism in the immunodominant epitope #7 (S73T) showed a 56-99% reduction in IgE-binding activity and did not affect T cell epitopes, which must be retained for effective immunotherapy. The identification of natural hypoallergenic isoforms positively contributes to future immunological and therapeutic studies and peanut cultivar development.

The most potent peanut allergens, Ara h 2 and Ara h 6, were silenced in transgenic plants by RNA interference. Three independent transgenic lines were recovered after microprojectile bombardment, of which two contained single, integrated copies of the transgene. The third line contained multiple copies of the transgene. Ara h 2 expression was significantly suppressed in all three lines, whereas Ara h 6 was reduced in two lines. Expression of peanut allergens Ara h 1 and Ara h 3 was not noticeably affected. Significant reduction of human IgE binding to Ara h 2 and Ara h 6 also was observed. Seed weight and germination data from transgenic and nontransgenic segregants showed no significant differences. Data collected from in vitro Aspergillus flavus infection indicate no significant difference in fungal growth between the transgenic lines and the nontransgenic controls. These data suggest that silencing Ara h 2 and Ara h 6 is a feasible approach to produce hypoallergenic peanut.

Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.