Identification of in vivo nitrosylated phytochelatins in Arabidopsis thaliana cells by liquid chromatography-direct electrospray-linear ion trap-mass spectrometry.

Reversed-phase liquid chromatography (RPLC) and electrospray (ESI)-linear ion trap (LIT) mass spectrometry was applied to the direct characterization of in vivo S-nitrosylated (SNO) phytochelatins (PCs) expressed in cadmium-stressed Arabidopsis thaliana cells. Cys-nitrosylation is under discussion as in vivo redox-based post-translational modification of proteins and peptides in plants in which the -NO group is involved as signal molecule in different biological functions. The gas-phase ion chemistry of in vivo and in vitro generated SNO-PC(s) was compared with the aim of evaluating NO binding stability and improving MS knowledge about peptide nitrosation. Using RPLC separation and ESI-LIT-MS, mono-nitrosylated PCs were identified in in vivo cadmium treated A. thaliana cells without derivatization. The in vivo binding of the NO group to PC(2), PC(3) and PC(4) resulted to occur selectively on only one cystein residue. The fragmentation pathway energies of the in vitro GSNO-generated NO-PCs with respect to the in vivo NO-PCs were investigated, suggesting the presence of a different internal stability for these molecules. By carrying out MS(2) experiments on these quasi-symmetric peptides, the different stability degree of the NO group was demonstrated to be correlated with the PC chain length. In addition, the data obtained highlight a putative role of the adjacent Glu/Cys motif in the gas-phase stability of the NO-containing molecule.

Proteomic analysis in the lichen Physcia adscendens exposed to cadmium stress.

This work was undertaken to explore the potential of proteomics to dissect parallel and consecutive events of cadmium stress response in the lichen Physcia adscendens (Fr.) H. Olivier. Thalli were exposed to 0 (control) and 36 microM Cd for 6, 18, 24 and 48 h. Two-dimensional electrophoresis and mass spectrometry analyses showed an 80-85% spot identity between 6 and 18 h vs. 24 and 48 h of Cd exposure. Putative heat-shock proteins and glutathione S-transferase generally increased their expression all over the Cd treatments. By contrast, ABC transporters were underexpressed after 6-18 h, but in some cases induced after 24-48 h of Cd exposure. The cytochrome P450 appeared to have a variable expression pattern over time. Overall these data suggest that a considerable importance in the response of P. adscendens thalli to Cd stress can be assumed by differential expression of various protein families.

Antifungal activity of diketopiperazines extracted from Alternaria alternata against Plasmopara viticola: an ultrastructural study.

Three dipeptides, belonging to the family of diketopiperazines (DKPs), were extracted from broth culture of the grapevine endophyte Alternaria alternata, and were tested against Plasmopara viticola on leaves of grapevine plants grown in greenhouse. DKPs, used at different concentrations (10(-3), 10(-4), 10(-5) and 10(-6)M) both singularly and in mixtures, demonstrated real effectiveness in inhibiting P. viticola sporulation when applied 2 or 24h after pathogen inoculation. Moreover, no necrotic lesions or other phytotoxicity symptoms were observed on DKP-treated grapevine leaf tissues. Ultrastructural analysis performed on grapevine leaf tissues revealed that the DKPs used singularly and in mixture, at above reported concentrations, did not cause leaf tissue damages. By contrast, hyphae of P. viticola exhibited marked structural changes, similar to those induced by the endophyte A. alternata. This demonstrates the involvement of these metabolites in the relationship of P. viticola and the endophyte. Further experimental trials will be carried out in the next future in order to test the effectiveness of these molecules also under field conditions, and to better understand the mechanism of action involved in the pathogen inhibition.

Inhibition of Sporulation and Ultrastructural Alterations of Grapevine Downy Mildew by the Endophytic Fungus Alternaria alternata.

ABSTRACT One hundred twenty-six endophytic microorganisms isolated from grapevine leaves showing anomalous symptoms of downy mildew were tested on grapevine leaf disks as biocontrol agents against Plasmopara viticola. Among the 126 microorganisms, only five fungal isolates completely inhibited the sporulation of P. viticola; all of them were identified as Alternaria alternata. Ultrastructural analyses were carried out by transmission electron microscopy to observe cellular interactions between P. viticola and A. alternata in the grapevine leaf tissue. Cytological observations indicated that, even without close contact with A. alternata, the P. viticola mycelium showed severe ultrastructural alterations, such as the presence of enlarged vacuoles or vacuoles containing electron-dense precipitates. Haustoria appeared necrotic and irregularly shaped or were enclosed in callose-like substances. Therefore, a toxic action of A. alternata against P. viticola was hypothesized. To examine the production of toxic low-molecular-weight metabolites by A. alternata, we analyzed the fungal liquid culture by thin layer chromatography and proton magnetic resonance spectroscopy. The main low-molecular-weight metabolites produced by the endophyte were three diketopiperazines: cyclo(l-phenylalanine-trans-4-hydroxy-l-proline), cyclo(l-leucine-trans-4-hydroxy-l-proline), and cyclo(l-alanine-trans-4-hydroxy-l-proline). When applied at different concentrations to both grapevine leaf disks and greenhouse plants, a mixture of the three diketopiperazines was very efficacious in limiting P. viticola sporulation.

Recovery in apple trees infected with the apple proliferation phytoplasma: an ultrastructural and biochemical study.

ABSTRACT Localization of hydrogen peroxide (H(2)O(2)) and the roles of peroxidases, malondialdehyde, and reduced glutathione in three apple cultivars were compared in healthy trees, trees infected with apple proliferation phytoplasma (APP), and trees that had recovered from the infection. In recovered apple trees, symptoms of the disease and the pathogen had disappeared from the canopy, but phytoplasmas remained in the roots. H(2)O(2) was detected cytochemically by its reaction with cerium chloride to produce electron-dense deposits of cerium perhydroxides.H(2)O(2) occurred in the plasmalemma of the phloem of leaves of recovered apple trees, but not in healthy or APP-infected leaves. In all cultivars, the peroxidase activity detected in tissue from APP-diseased trees was greater than or equal to that of tissue from recovered trees, which equaled or exceeded that of tissue from healthy trees, at two sampling times (May and September). In contrast, the glutathione content of leaves decreased in the reverse order. More malondialdehyde was observed in leaves from recovered trees than in leaves from healthy or APP-infected trees in three of six cultivar-date combinations; in the other three combinations, the malondialdehyde contents of leaves from healthy, infected, and recovered trees were not significantly different from one another. The results suggest that some components of the oxidant-scavenging system in recovered leaves are not very active, leading to an overproduction of H(2)O(2) and, possibly, to a membrane lipid peroxidation.The production of H(2)O(2) appears to be involved in counteracting pathogen virulence.