Proteínas antifúngicas do látex de Marsdenia megalantha [GOYDER & MORILLO, 1994] – Caracterização Bioquímica e mecanismos de ação

Abstract

Masdenia megalantha (Apocynaceae ) is a species which grows on the hinterland in Northeast, Brazil. Upon injuries, each organ of the plant exudates a latex which quickly coagulates on exposure to air, preventing losses of water and the penetrat ion of plant pathogens. This present work had the aim to describe the antifungal properties of M. megalantha latex and to identify the related proteins in plant defense against phytopathogenous. So, latex was harvested from trees in Quix adá, Ceará, Brazil, and diluted with 0.05 M Tris - HCl, pH 8.0, containing 0.15 M NaCl (10:1, v/v) , to prevent its coagulation. The diluted latex was centrifuged (10,000 g , 10 min, 25 ºC), dialyzed extensively against water, and further centrifuged under the same above conditi ons. The supernatant, denominated Latex Proteins (LP), after exhaustive dialysis against 0.05 M sodium acetate, pH 5.2, was applied to a Resource - Q column equilibrated with this same buffer. Two different fractionswas obtained, one the non - retained protein s (FnRq), eluted with the equilibrating buffer, and the other, the retained proteins (FRq), eluted with 0.2 M NaCl in the above buffer. The FnRq (22 μfP/mL) was the only fraction able to inhibited the spore germination of the phytophatogenic fungi Fusarium solani . In addition, the evaluation of some enzymatic activities involved in plant defense mechanisms against phytopathogenous fungi such as chitinases, peroxidases and proteases were observed in both fractions from Resource - Q. FnRq, after performed in a Superose - 12 HR 10/30 column chromatography, originated another two different protein peaks (S1 and S2), showing different enzymatic activities, but both capable to inhibit the spore germination of the phytopathogenic fungi F. solani . S1 concentrated all th e peroxidase activity, whereas S2, which concentrate the chitinolityc activity. Under SDS - PAGE, S1 was presented as a purified protein band, instead of S2. Indeed, the NH2 - Terminal sequence under Edman degradation showed that S1 protein was blocked. The an tifungal action mechanisms evaluated in this work has showed that these protein presents in M. megalantha latex were able to interfere with the proton pump present in plasmatic membrane, and also to induce the ROS production, and promote alteration in the membrane permeability within the F. solani spores. These results show the latex from M. megalantha as a rich source of antifungal proteins, with potential to been used in the defense against the phytopathogenous F. solani.