RESUMO
KEY MESSAGE: Expression of the TaMDC1 in transgenic tomato plants confer resistance to bacterial and fungal pathogens, as well as an insect pest and thus prove in planta function of the wheat cystatin. Cystatins are the polypeptides with cysteine proteinase inhibitory activities. Plant cystatins or phytocystatins are known to contribute to plant resistance against insect pests. Recently, increasing data proved that some of the phytocystatins also have antifungal activities in vitro. Here, we functionally characterized a wheat multidomain cystatin, TaMDC1, using in planta assays. Expression of TaMDC1 in wheat seedlings is up-regulated in response to methyl jasmonate and salicylic acid, indicating that TaMDC1 is involved in biotic stress responses mediated by these plant hormones. The TaMDC1 cDNA was integrated in tomato genome and expressed under cauliflower mosaic virus 35S promoter. Four transgenic plants that show high level of the transgene expression were selected by RNA gel blot and immunoblot analysis and utilized to assess biotic stress resistance against the bacterial pathogen Pseudomonas syringae, the fungal pathogens Botrytis cinerea and Alternaria alternata, and the insect pest Colorado potato beetle (CPB, Leptinotarsa decemlineata). Detached leaf inoculation assays revealed that the tomato plants expressing TaMDC1 showed high levels of resistance against P. syringae and A. alternata, and elevated tolerance against B. cinerea. Sustenance of L. decemlineata larvae to the transgenic plants demonstrated inhibition of CPB larvae growth. Inhibitory activity of TaMDC1 against selected pathogens was also demonstrated by in vitro assays with total protein extracted from transgenic tomato plants. Taken together, the presented data suggest that TaMDC1 is involved in a broad spectrum biotic stress resistance in planta.
Assuntos
Cistatinas/metabolismo , Resistência à Doença/genética , Doenças das Plantas/imunologia , Reguladores de Crescimento de Plantas/metabolismo , Triticum/genética , Acetatos/metabolismo , Animais , Antibacterianos/metabolismo , Antifúngicos/metabolismo , Botrytis/fisiologia , Besouros/fisiologia , Ciclopentanos/metabolismo , Cistatinas/genética , Expressão Gênica , Larva , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Solanum lycopersicum/parasitologia , Oxilipinas/metabolismo , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Folhas de Planta/parasitologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Pseudomonas syringae/fisiologia , Ácido Salicílico/metabolismo , Triticum/imunologia , Triticum/microbiologia , Triticum/parasitologiaRESUMO
The conformational stability of gamma-II crystallin at pH 7.0 was estimated by studying its urea denaturation at isothermal conditions. The conformational states were monitored by far UV-CD and fluorescence measurements. Gamma-II crystallin shows sigmoidal order-disorder transition curves by both methods. The presence of more than one intermediate was confirmed but at neutral pH. The experiment results were critically analyzed in terms of both linear extrapolation and Tanford's models. The Gibbs free energy of unfolding delta G u,H2O = -36 kcal mol-1 was obtained. This value corresponds to the high conformational stability of the protein predicted qualitatively by its crystal structure.
Assuntos
Cristalinas/química , Dobramento de Proteína , Ureia/farmacologia , Animais , Bovinos , Dicroísmo Circular , Cristalinas/efeitos dos fármacos , Cristalinas/metabolismo , Cristalino , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
The electron transfer between the excited triplet state of zinc-substituted sperm whale myoglobin and Cu2+ has been studied by following the decay rate of delayed fluorescence. The Cu2+ bound on the surface of the myoglobin molecule are efficient quenchers of the excited electron state of Zn-myoglobin. Two bimolecular rate constants of quenching (KQ) for every pH investigated have been calculated. The pH-dependence of KQ1 indicates that the protonation of one amino acid residue (His-GH1 (119] is important for the process. Our results support the idea of the common nature of the mechanism of quenching by Cu2+ and oxidation of oxymyoglobin by Cu2+.