Reconstitution in vitro of the components and conditions required for the oxidative cross-linking of extracellular proteins in French bean (Phaseolus vulgaris L.).

Oxidative cross linking of three (hydroxy)proline-rich cell wall proteins, known to be immobilised during the elicitor-induced oxidative burst in French bean cells, was modelled using peroxidases with cysteine or H2O2. Further reconstitution of the homologous system was achieved with a 46 kDa bean cell-wall peroxidase using conditions known to appertain in cell walls prior to the immobilisation. Thus, cell wall alkalinisation and secretion of a reductant have been reconstituted by addition of apoplastic fluid to the wall proteins and the 46 kDa peroxidase with resultant cross-linking. This is the first demonstration of the oxidative cross-linking of cell wall glycoproteins without the addition of external H2O2.

Specificity in the immobilisation of cell wall proteins in response to different elicitor molecules in suspension-cultured cells of French bean (Phaseolus vulgaris L.).

A characteristic of the defence response is the immobilisation of wall proteins possibly through the formation of covalent cross-links and the subsequent barrier formation against pathogens. A requirement for this is the generation of active oxygen species, particularly hydrogen peroxide. In the present work, we examine in depth the requirement for H2O2 and the specificity of the immobilisation with respect to particular wall proteins. Salt-extractable wall proteins were analysed for hydroxyproline content and the subset of proteins with this post-translational modification was found to be small. About 50 proteins were found to be easily salt-extractable and in response to elicitor treatment about 5 were found to be specifically immobilised. Immobilisation was very rapid and completed within 15 min after elicitation, and dependent upon the type of elicitor and the intensity of the production of active oxygen species. N-terminal sequencing and amino acid analysis revealed that, apart from one polypeptide, all immobilised proteins were (hydroxy)proline-containing glycoproteins with O-linked oligosaccharide side chains. In contrast, N-linked glycoproteins were not immobilised. N-terminal protein sequencing revealed the immobilised HRGPs to be novel, but both extensin and PRP-like. Implications of these findings for both pathogenic and symbiotic processes are also discussed.