Extracellular DNA as an elicitor of broad-spectrum resistance in Arabidopsis thaliana.

Like in mammals, the plant immune system has evolved to perceive damage. Damaged-associated molecular patterns (DAMPs) are endogenous signals generated in wounded or infected tissue after pathogen or insect attack. Although extracellular DNA (eDNA) is a DAMP signal that induces immune responses, plant responses after eDNA perception remain largely unknown. Here, we report that signaling defenses but not direct defense responses are induced after eDNA applications enhancing broad-range plant protection. A screening of defense signaling and hormone biosynthesis marker genes revealed that OXI1, CML37 and MPK3 are relevant eDNA-Induced Resistance markers (eDNA-IR). Additionally, we observed that eDNA from several Arabidopsis ecotypes and other phylogenetically distant plants such as citrus, bean and, more surprisingly, a monocotyledonous plant such as maize upregulates eDNA-IR marker genes. Using 3,3'-Diaminobenzidine (DAB) and aniline blue staining methods, we observed that H2O2 but not callose was strongly accumulated following self-eDNA treatments. Finally, eDNA resulted in effective induced resistance in Arabidopsis against the pathogens Hyaloperonospora arabidopsidis, Pseudomonas syringae, and Botrytis cinerea and against aphid infestation, reducing the number of nymphs and moving forms. Hence, the unspecificity of DNA origin and the wide range of insects to which eDNA can protect opens many questions about the mechanisms behind eDNA-IR.

[A CASE OF A 7-YEAR-OLD GIRL WITH ANAPHYLAXIS CAUSED BY PECTIN IN ALBEDO OF CITRUS UNSHIU BUT NOT BY THE FRUIT ITSELF].

Pectin is used in several foods as an additive and a thickner. But some cases of anaphylaxis have been reported. Most of these are induced by occasional exposures; however, no cases of anaphylaxis after eating a Citrus unshiu, the albedo of which is rich in pectin, have been reported.A 7-year-old girl developed barking cough and pruritus approximately two hours after eating a frozen Citrus unshiu. She had a history of anaphylaxis induced by consuming cashew nuts. Skin testing and basophil activation tests were performed using a commercially available pectin product. Both tests were positive. In an oral food challenge test, she felt abdominal pain and nausea only after eating fruit, along with the albedo, of Citrus unshiu. We concluded that this case was induced by pectin present in the albedo of Citrus unshiu, but not by the fruit itself. We should consider that patients with cashew nut allergies have a possibility of pectin allergies as well, and that pectin in the albedo of Citrus unshiu may induce anaphylaxis.

Tyramine Pathways in Citrus Plant Defense: Glycoconjugates of Tyramine and Its N-Methylated Derivatives.

Glucosylated forms of tyramine and some of its N-methylated derivatives are here reported for the first time to occur in Citrus genus plants. The compounds tyramine-O-ß-d-glucoside, N-methyltyramine-O-ß-d-glucoside, and N,N-dimethyltyramine-O-ß-d-glucoside were detected in juice and leaves of sweet orange, bitter orange, bergamot, citron, lemon, mandarin, and pomelo. The compounds were identified by mass spectrometric analysis, enzymatic synthesis, and comparison with extracts of Stapelia hirsuta L., a plant belonging to the Apocynaceae family in which N,N-dimethyltyramine-O-ß-d-glucoside was identified by others. Interestingly, in Stapelia hirsuta we discovered also tyramine-O-ß-d-glucoside, N-methyltyramine-O-ß-d-glucoside, and the tyramine metabolite, N,N,N-trimethyltyramine-O-ß-glucoside. However, the latter tyramine metabolite, never described before, was not detected in any of the Citrus plants included in this study. The presence of N-methylated tyramine derivatives and their glucosylated forms in Citrus plants, together with octopamine and synephrine, also deriving from tyramine, supports the hypothesis of specific biosynthetic pathways of adrenergic compounds aimed to defend against biotic stress.

Ponciretin attenuates ethanol-induced gastric damage in mice by inhibiting inflammatory responses.

BACKGROUND: Poncirin (PO) and isosakuranetin (or ponciretin [PT]) are compounds found in fruits of the genus Citrus. They are frequently used in traditional Chinese medicine for the treatment of inflammation and asthma. Therefore, we examined their anti-gastritis effects in vitro and in vivo. METHODS: The anti-inflammatory effects of PO and PT were examined using ethanol- or LPS-stimulated KATO III cells. Gastritis was induced in ICR mice via intragastric injection of absolute ethanol. Levels of inflammatory markers were measured by enzyme-linked immunosorbent assay, immunoblotting, and quantitative polymerase chain reaction. RESULTS: Treatment with PT or PO inhibited the secretion of interleukin (IL)-8 and tumor necrosis factor (TNF) in ethanol- or LPS-stimulated KATO III cells. They also reduced the activation of nuclear factor kappa B (NF-κB). Pre-treatment with PT or PO significantly protected against ethanol-induced hemorrhagic gastritis, characterized by edema, tissue erosions, and mucosal friability in mice. Treatment with PT or PO suppressed ethanol-induced NF-κB activation and the release of TNF, IL-8, and IFN-γ. The protective effect of PT was greater than that of PO and comparable to ranitidine, a positive control. CONCLUSION: PT may attenuate ethanol-induced gastritis by inhibiting the infiltration of immune cells, including neutrophils, via the regulation of CXCL4 (or IL-8) secretion and the activation NF-κB.

Comparative proteomic and metabolomic profiling of citrus fruit with enhancement of disease resistance by postharvest heat treatment.

BACKGROUND: From field harvest to the consumer's table, fresh citrus fruit spends a considerable amount of time in shipment and storage. During these processes, physiological disorders and pathological diseases are the main causes of fruit loss. Heat treatment (HT) has been widely used to maintain fruit quality during postharvest storage; however, limited molecular information related to this treatment is currently available at a systemic biological level. RESULTS: Mature 'Kamei' Satsuma mandarin (Citrus unshiu Marc.) fruits were selected for exploring the disease resistance mechanisms induced by HT during postharvest storage. Proteomic analyses based on two-dimensional gel electrophoresis (2-DE), and metabolomic research based on gas chromatography coupled to mass spectrometry (GC-MS), and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) were conducted. The results show resistance associated proteins were up-regulated in heat treated pericarp, such as beta-1, 3-glucanase, Class III chitinase, 17.7 kDa heat shock protein and low molecular weight heat-shock protein. Also, redox metabolism enzymes were down-regulated in heat treated pericarp, including isoflavone reductase, oxidoreductase and superoxide dismutase. Primary metabolic profiling revealed organic acids and amino acids were down-regulated in heat treated pericarp; but significant accumulation of metabolites, including tetradecanoic acid, oleic acid, ornithine, 2-keto-d-gluconic acid, succinic acid, turanose, sucrose, galactose, myo-inositol, glucose and fructose were detected. Noticeably, H2O2 content decreased, while, lignin content increased in heat treated pericarp compared to the control, which might increase fruit resistibility in response to external stress. Also, flavonoids, substances which are well-known to be effective in reducing external stress, were up-regulated in heat treated pericarp. CONCLUSIONS: This study provides a broad picture of differential accumulation of proteins and metabolites in postharvest citrus fruit, and gives new insights into HT improved fruit disease resistance during subsequent storage of 'Kamei' Satsuma mandarin. Interpretation of the data for the proteins and metabolites revealed reactive oxygen species (ROS) and lignin play important roles in heat treatment induced fruit resistance to pathogens and physiological disorders.

Citrus allergy from pollen to clinical symptoms.

Allergy to citrus fruits is often associated with pollinosis and sensitization to other plants due to a phenomenon of cross-reactivity. The aims of the present study were to highlight the cross-reactivity among citrus and the major allergenic pollens/fruits, throughout clinical and molecular investigations, and to evaluate the sensitization frequency to citrus fruits in a population of children and adults with pollinosis. We found a relevant percentage of sensitisation (39%) to citrus fruits in the patients recruited and in all of them the IgE-mediated mechanism has been confirmed by the positive response to the prick-to-prick test. RT-PCR experiments showed the expression of Cit s 1, Cit s 3 and a profilin isoform, already described in apple, also in Citrus clementine pollen. Data of multiple sequence alignments demonstrated that Citrus allergens shared high percentage identity values with other clinically relevant species (i.e. Triticum aestivum, Malus domestica), confirming the possible cross-allergenicity citrus/grasses and citrus/apple. Finally, a novelty of the present work has been the expression of two phospholipaseA2 isoforms (PLA2 α and ß) in Citrus as well as in Triticum pollens; being PLA2 able to generate pro-inflammatory factors, this enzyme could participate in the activation of the allergenic inflammatory cascade.

Bergamot (Citrus bergamia Risso) fruit extracts and identified components alter expression of interleukin 8 gene in cystic fibrosis bronchial epithelial cell lines.

BACKGROUND: Cystic fibrosis (CF) airway pathology is a fatal, autosomal, recessive genetic disease characterized by extensive lung inflammation. After induction by TNF-α, elevated concentrations of several pro-inflammatory cytokines (i.e. IL-6, IL-1ß) and chemokines (i.e. IL-8) are released from airway epithelial cells. In order to reduce the excessive inflammatory response in the airways of CF patients, new therapies have been developed and in this respect, medicinal plant extracts have been studied. In this article we have investigated the possible use of bergamot extracts (Citrus bergamia Risso) and their identified components to alter the expression of IL-8 associated with the cystic fibrosis airway pathology. METHODS: The extracts were chemically characterized by 1H-NMR (nuclear magnetic resonance), GC-FID (gas chromatography-flame ionization detector), GC-MS (gas chromatography-mass spectrometry) and HPLC (high pressure liquid chromatography). Both bergamot extracts and main detected chemical constituents were assayed for their biological activity measuring (a) cytokines and chemokines in culture supernatants released from cystic fibrosis IB3-1 cells treated with TNF-α by Bio-Plex cytokine assay; (b) accumulation of IL-8 mRNA by real-time PCR. RESULTS: The extracts obtained from bergamot (Citrus bergamia Risso) epicarps contain components displaying an inhibitory activity on IL-8. Particularly, the most active molecules were bergapten and citropten. These effects have been confirmed by analyzing mRNA levels and protein release in the CF cellular models IB3-1 and CuFi-1 induced with TNF-α or exposed to heat-inactivated Pseudomonas aeruginosa. CONCLUSIONS: These obtained results clearly indicate that bergapten and citropten are strong inhibitors of IL-8 expression and could be proposed for further studies to verify possible anti-inflammatory properties to reduce lung inflammation in CF patients.

Anaphylaxis to lemon soap: citrus seed and peanut allergen cross-reactivity.

BACKGROUND: Many individuals allergic to peanuts have multiple allergen sensitivity. OBJECTIVE: To report the first case, to our knowledge, of a peanut allergic patient who exhibited cosensitivity to citrus seeds and who had experienced anaphylaxis to lemon soap. METHODS: Extracts of peanut and seeds from different varieties of citrus fruit (orange, lemon, and mandarin) were prepared and resolved with 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Direct and inhibition immunoblotting of the patient's serum on the extracts was used to examine the pattern of IgE reactivity and the presence of cross-reactive allergens. RESULTS: Numerous IgE reactive proteins were demonstrated in each citrus seed extract and the peanut extract. Complete IgE cross-reactivity was demonstrated among the different citrus seed extracts. Partial cross-reactivity was demonstrated between the peanut and orange seed extracts. CONCLUSIONS: Citrus seeds contain numerous IgE reactive proteins that are completely cross-reactive among orange, lemon, and mandarin. When peanut allergy coexists with citrus seed allergy, IgE cross-reactivity between peanut and citrus seed proteins can be demonstrated, suggesting a basis to this cosensitivity.

Generation of monoclonal antibody specific to (1-->5)-alpha-L-arabinan.

A neoglycoprotein (a heptasaccharide of (1-->5)-alpha-L-linked-arabinosyl residues linked to bovine serum albumin) has been used to generate a rat monoclonal antibody specific to a linear chain of (1-->5)-alpha-L-arabinan which is a structural feature of the side chains of pectins. The antibody, designated LM6, detected 100 ng of debranched sugar beet arabinan in an immunodot binding assay and 1 microgram of commercial citrus pectin in a similar assay. Hapten inhibition studies indicated that the antibody recognized 5-6 Ara residues and 50% inhibition of antibody binding in a competitive inhibition ELISA was achieved with ca. 2ng (21 nM) of (1-->5)-alpha-L-Arabinohexaose. The antibody will be useful for the localization of arabinans in plant tissue and will have uses in the analyses of pectin structure. We report here on the localization of the arabinan epitope in lemon fruits using tissue printing.

Induction of anti-orange antibodies in rabbits.

The capacity of certain foods to cause allergic reactions is well known. The four types of mechanisms that Gell and Coombs described in 1968, are involved in these reactions, although the reaginic antibodies retain the paramount attention of the immunologists. The physiochemical composition of the allergen molecule is the goal of investigators with the purpose to clarify the intrinsic kinetics of antibody synthesis. This paper contributes to the conflicting data about orange allergens especially those obtained from Citrus Aurantium Sinensis and Citrus Silension (CAS and CS, respectively). Glycoproteins were separated by gel filtration through a Sephadex G-50 column. A definite protein peak was obtained meanwhile several hexoses appeared throughout the fractionation procedure. These molecules have adequate physiochemical properties that make them able to trigger the immunological response (molecular weight, definite chemical composition and glycoprotein content). Although CAS and CS have a similar chemical composition a slight inverse proportion of proteins and hexoses was demonstrated between the two classes. Molecular weights were different for CAS (51.500) and for CS (37.000) in comparison with well established protein makers. Ouchterlony revealed two precipitin lines in the CAS-anti-CAS system but none in the CS-anti-CS one. The Boyden technique showed a titre of 1/256 in the first case and only of 1/64 in the second of specific anti-orange antibodies. All the eluted fractions gave negative results although they were concentrated ten times by pre-evaporation. This animal model reinforced the statement that after a long and continuous exposure to orange antigens it was possible to develop specific antibodies. It is assumed that this phenomenon happens in atopic children with it's diagnostic and therapeutic importance.