Identificação, purificação, caracterização e atividade biológica de proteínas do látex de Calotropis procera envolvidas na defesa da planta

Abstract

Latex is a fluid with milky aspect remarkably common in plants. Nearly 8 % of all plant species have canal systems from which latex is exuded upon damage. Nonetheless, few laticifer plants are studied in detail, mainly biochemical aspects of their latex. The role played by latex in plants is poorly understood. However, the most accepted hypothesis is its involvement in plant defense against insects and phytopathogens. In an attempt to investigate this hypothesis, a partial biochemical and proteolytic characterization of laticifer proteins were performed to obtain new information on the occurrence and biological activities of proteins from the latex of Calotropis procera. Besides, antifungal activity and purification of an osmotin are shown in this study. Laticifer proteins of C. procera exhibited strong proteolytic activity shared by at least four distinct proteinases with molecular masses ranging from 28 kDa to 66 kDa. Cysteine proteinase activities were predominant, whilst aspartic proteinase activities were barely visible. Serine and metaloproteinases were not detected. The optima pH and temperature for proteolytic activity were 5.0-6.0 and 37-60 °C, respectively, when azocasein or BANA were used as substrates. Polyclonal antibodies raised against papain cross-reacted weakly with laticifer proteins of C. procera showing that papain and cysteine proteinases of this latex were immunologically distinct. Proteomic approaches of 2-D electrophoresis and analysis by MALDI-TOF-TOF allowed identification of several Pathogenesis-Related proteins in latex: peroxidase (1), chitinases (2), cysteine proteinases (2), proteinase inhibitor (1), lipid-transfer proteins (3) and osmotins (3). In view of this high diversity of antifungal proteins, laticifer proteins were tested on different phytopathogen fungi. Laticifer proteins inhibited fungi growth of Fusarium solani, Neurospora sp. and Colletrotricum gloeosporioides with IC50 of 134.5 ± 8.1, 549.9 ± 14.3 and 455.0 ± 9.3 pg/ml, respectively. The inhibitory activity was always lost after heat treatment (98 °C for 30 min) or proteolysis, giving strong evidence for the protein nature of inhibitory molecules. Treatment of this latex with specific inhibitor of cysteine proteinases (E- 64) completely eliminated the inhibitory activity to F. solani but only partially for Neurospora sp. and C. gloerosporioides. Similar results were observed when spores were challenged to germinate in the presence of laticifer proteins. Papain, a cysteine protease from the latex of Carica papaya, but not trypsin and chymotrypsin, two serine proteases, repeated the results observed with C. procera, suggesting the involvement of cysteine proteinase as inhibitors of spore germination and fungal growth. Laticifer proteins were fractionated and a highly purified protein was obtained. The N-terminal sequence (40 AA) exhibited similarity to osmotin (87%) and thaumatin-like (82%) proteins. Its isolation procedures entailed two ion exchange chromatography steps. C. procera osmotin appeared as a single band (20.1 kDa) in SDS-PAGE and as two spots in 2-D electrophoresis (pl 8.9 and 9.2) identified by mass spectrometry as two osmotins. The two isoforms were glycoproteins as indicated by Schiff's reagent. Circular dichroism assays showed that osmotin was stable in different pH and temperatures. Secondary structure content was 35% ahelix, 33% 6-sheet, 19% turn and 28% unordered. Fluorescence analysis showed emission in the 300-420nm range upon excitation at 280 nm or 295 nm, with maximum at 340 nm. The osmotin caused membrane leakage in F. solani spores and artificial membranes, but did not disrupt rabbit erythrocytes. In presence of DTT, osmotin was fragmented in three peptides with molecular masses of 14, 11 and 7 kDa and lost antifungal activity, showing that the disulfide bonds are essential to its activity. These results reinforce the hypothesis of the involvement of different proteins of laticifer fluids in plant defense against phytopathogenic fungi.