Apparently homogeneous polygalacturonase-elicitor purified from the filtrates of Rhizopus stolonifer cultures stimulates germinating castor bean seedlngs to produce greatly increased levels ofcasbene synthetase activity.The purification procedure involved gel-filtration chromatography on Sephadex G-25 and G-75 columns followed by cation-exchange chromatography on a Sephadex CM C-50 column. Homogeneity of the purified preparation was indicated by the results of cationic polyacrylamide disc gel electrophoresis and isoelectric focusing (pI = 8.0). The identity of the casbene elicitor activity and polygalacturonase were indicated by the coincidence of the two activities at all stages of purification, the coincidence of both activities with the single protein-staining band detected on a cationic polyacrylamide disc gel and an isoelectric focusing gel, and the identical behavior of both activities on an agarose gel affinity column. The purified polygalacturonaseelicitor is a glycoprotein with approximately 20% carbohydrate content and an estimated molecular weight of 32,000 by polyacrylamide disc gel electrophoresis.
Some properties of the polygalacturonase-elicitor from the filtrates of Rhizopus stolonifer cultures have been examined in an attempt to understand its mode of action as an elicitor of casbene synthetase activity in castor bean seedlings. Both the polygalacturonase activity and the elicitor activity are heat-labile with similar heat-sensitivity profiles. AMso, the catalytic activity of the enzyme is lost on treatment with sodium periodate, as had been shown previously for the elicitor activity. The pH optimum of the enzyme activity with polygalacturonic acid as the substrate is 4.9. Exposures of germinating castor bean seedlings to the elicitor for shortterm periods of 1 to 10 minutes followed by washing and incubation in sterile, distilled water are partially effective in elicitation in comparison with the continuous exposure of the seedlings over 11 hours to the same amount of the elicitor. The initial rate of reaction catalyzed by the enzyme is about 3 times faster with polygalacturonic acid as a substrate than with partiay (S0%) methylated polygalacturonic acid (pectin). The Km value of the enzyme for polygalacturonic acid is about 4.2 mfllimolar in terms of monomeric units and about 0.07 millimolar in terms of polymer concentration. Examination of the types of products formed by the action of the enzyme suggests that It is an endo-hydrolase. The amino acid composition of this enzyme Is shimlar to those of other extracellular fungal proteins reported. The carbohydrate moiety of the glycoprotein polygalacturonaseelicitor Is composed of 92% mannose and 8% glucosamine by gas chromatography-mass spectrometry analysis. The linkage group analysis of the carbohydrate moiety showed that mannosyl residues which are 1,2-linked comprse about 70% of the total glycosyl residues and demonstrated the presence of some 1,3,6-and 1,2,6-linked branching mannosyl residues.(1) to suggest that elicitors may be recognized by plant cell surface receptors in a process involving carbohydrate-protein interactions. There is little direct evidence available to date to evaluate this proposal.Previous studies in this laboratory by Stekoll and West (23) showed that a partially purified elicitor from culture filtrates of Rhizopus stolonifer, which acts to elicit casbene synthetase activity in castor bean seedlings, possesses the characteristics expected of a glycoprotein requiring both protein and carbohydrate for its activity. The dependency of the elicitor activity on the native protein structure, and an evolutionary argument that the elicitor must have a role of the advantage of the fungus in addition to its seemingly deleterious role for the fungus of being recognized as a signal to initiate casbene biosynthesis, led us to search for an enzymic activity that might be associated with the elicitor. In the accompanying paper (15), the identification of the elicitor from R. stolonifer with a polygalacturonase is described. The apparently homogeneous polygalacturonase-elicitor catalyzes the hydrolysis of polygalacturonic acid, a subs...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.