Lectina de cotilédones de Luetzelburgia auriculata Ducke e seu possível papel na defesa da planta

Abstract

This work describes the purification, physico-chemical and structural :haracterisation of the lectin from cotyledons of Luetzelburgia auriculata. In addition, we discuss the envolvement of this lectin in plant defense against pathogens. This lectin, named LAL, was purified on agarose-N-acetylgalactosamine column. LAL is a potent agglutinin for rabbit erythrocytes, reacts against human A+, B+ and O+ red cells, but is ;reactive against cow, sheep and goat erythrocytes. Hemagglutination of rabbit red cells was inhibited by galactose, raffinose, mellibiose, lactose, and mainly, by N-acetyl-Dzalactosamine and N-acetylneuraminic acid. The apparent molecular mass determined by gel filtration in Sephadex G-100, at pH 7.6, was 123.5 kDa. However, the lectin assumed different conformation by varying the pH. By SDS-PAGE the lectin showed two bands of apparent molecular masses of 28 e 15 kDa, not associated by dissulfide bonds. The 28 kDa band corresponds to the monomer (a chain) and that of 15 kDa the two subunits 13 e y. LAL is a glycoprotein (3.2 % carbohydrates) and the a chain has the following N-terminal sequence SEVVSFSFTKFNPNQKDII. LAL is rich in acidic and hydroxy amino acids, which justifies the pl of 5.8. LAL is relatively heat stable. It requires about 104.75 kJ/mol of energy for denaturation to initiate. The absorption spectrum showed maximum at 280 nm, with El%rm, of 5.2 and an increase in the extinction coefficient was observed in the presence of the inhibiting sugar D-galactose. The secondary structure ofthe lectin showed a high content of (3-sheet. In the fluorescence spectroscopy, excitation of the lectin solution at 280 run gave an emission spectrum in the 285-445 nm range. The wavelength maximum emission was in 334.5 nm, which is typical for tryptophan residues buried inside the protein. LAL is located on the outer epidermis of L. auriculata cotyledon, what justifies-the ease of exsudation to the medium during seed germination. LAL slowed the growth of the filamentous fungi Colletothricum lindemuthianum, Fusarim solani, and Aspergilllus niger, and of the yeast Saccharomyces cerevisiae. This activity was inhibited by galactose, except for the inhibition of F. solani, suggesting the interaction of the lectin with another type of glycoconjugated receptor, which galactose can not counteract. The lectin inhibiteck the acidification of the yeast culture medium stimulated by glucose, suggesting an action upon ion transport mechanism across plasmatic membrane. The lectin was bound effectively to yeast surface, as shown by fluorescence and electron microscopy, interacting also with plasmatic membrane. The lectin detected in roots, hypocotyls, germinating cotyledons, epycotyls and leaves of L. auriculata was recognized by anti-LAL antibody, showing an homology between LAL and the lectin present in the tissues. It appears that LAL is under processing by enzymes activated during germination, since the proposed pattern for this lectin, a chain and 13 e y subunits, was observed in germinating cotyledons. The treatment of L. auriculata with salicilic acid and F. solani caused alterations in lectin content in roots, hypocotyls, germinating cotyledons, epycotyls and leaves, suggesting the envolvement of the lectin in plant defense against aggressors.