Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2005
Publication Date: 5/1/2005
Citation: Liang, X.Q., Holbrook, C.C., Lynch, R.E., Guo, B.Z. 2005. B-1,3-Glucanase activity in peanut seed (Arachis hypogaea) is induced by inoculation with Aspergillus flavus and copurifies with a conglutin-like protein. Phytopathology. 95(5):506-511.
Interpretive Summary: Infection of peanut (Arachis hypogaea L.) seeds by Aspergillus flavus and A. parasiticus is a serious problem. This infection can result in the contamination of the seeds with aflatoxins, which are toxic fungal metabolites. These fungi are ubiquitous, being found virtually everywhere in the world. Breeding the resistant cultivars is considered the most effective approach to eliminate aflatoxin accumulation in peanut. Most plants respond to pathogen attacks by synthesizing an assortment of PR (pathogen-related) proteins. Both chitinases and ß-1-3-glucanases are well studied PR proteins as result of plant responding against diseases. Peanut has been reported to have chitinases, but there are no reports of endogenous ß-1-3-glucanase gene in peanut. In this study, we present evidences that peanut seeds have ß-1-3-glucanase activities and produce the enzyme protein and protein isoforms differentially in the resistant and susceptible peanut cultivars, in which the ß-1-3-glucanase activities were increased significantly in the resistant cultivars in comparison with the susceptible cultivars. Eight different isoforms were detected in an in-gel (native PAGE) assay and isoelectric focusing gel. Conglutin is a peanut allergen, Ara h2, and has trypsin inhibitor function (Maleki et al., 2003). Our results also suggest that conglutin, a peanut storage protein, may have ß-1-3-glucanase activity and may be associated with the resistance to A. flavus infection in peanut seeds.
Technical Abstract: Peanut ß-1-3-glucanase was detected and the activities and isoform patterns were measured. Peanut cultivars, GT-YY9 and GT-YY20 (resistant to Aspergillus flavus infection), and Georgia Green and A100 (susceptible to A. flavus infection), were used in this study. The activities of ß-1-3-glucanase were similar in the uninfected seeds of all cultivars, but increased significantly in the resistant cultivars after inoculation in comparison with the susceptible cultivars. An in-gel (native PAGE) enzymatic activity assay of ß-1-3-glucanase revealed that there were earlier detection and more protein bands corresponding to ß-1-3-glucanase isoforms in the infected seeds of resistant cultivars than in the infected seeds of susceptible cultivars. Both acidic and basic ß-1-3-glucanase isoforms were detected in the IEF gel. Thin layer chromatography (TLC) analysis of the hydrolytic products from the reaction mixtures of total protein extract and individual band of native PAGE revealed the presence of the enzymatic hydrolytic oligomer products. Two bands were revealed on the SDS-PAGE with molecular weight of 10-kDa and 13-kDa, respectively, which are of the individual band corresponding to the bands of ß-1-3-glucanase isoforms from native PAGE. The 13-kDa protein was the majority. The sequences of the 13-kDa protein showed a high degree of homology to conglutin, a storage protein in peanut seeds. Conglutin is reported as a peanut allergen, Ara h2, and has trypsin inhibitor function. Our results suggest this 13-kDa conglutin-like protein has ß-1-3-glucanase activity and may be associated with the resistance to A. flavus infection in peanut seeds.