No abstract

No abstract

Rapidly growing tomato (Lycopersicon esculentum) cell suspension cultures contain transiently high levels of cell surface, salt-elutable, monomeric precursors to the covalently cross-linked extensin network of the primary cell wall. Thus, we purified a highly soluble monomeric extensin substrate from rapidly growing cells, and devised a soluble in vitro cross-linking assay based on Superose-6 fast protein liquid chromatography separation, which resolved extensin monomers from the newly formed oligomers within 25 minutes. Salt elution of slowly growing (early stationary phase) cells yielded little or no extensin monomers but did give a highly active enzymic preparation that specifically cross-linked extensin monomers in the presence of hydrogen peroxide, judging from: (a) a decrease in the extensin monomer peak on fast protein liquid chromatography gel filtration, (b) appearance of oligomeric peaks, and (c) direct electron microscopical observation of the cross-linked oligomers. The cross-linking reaction had a broad pH optimum between 5.5 and 6.5. An approach to substrate saturation of the enzyme required extensin monomer concentrations of 20 to 40 milligrams per milliliter. Preincubation with catalase completely inhibited the cross-linking reaction, which was highly dependent on hydrogen peroxide and optimal at 15 to 50 micromolar. We therefore identified the cross-linking activity as extensin peroxidase.the identification of IDT2 in cell wall hydrolysates (15), and hence a new protein cross-linking amino acid.Demonstration of IDT as a short intramolecular cross-link in two extensin tryptic peptides (13) made the suggested involvement of peroxidase in extensin cross-linkage (9, 29) more plausible and led to the discovery of a monomeric extensin pool in muro readily eluted from intact cells of rapidly growing suspension cultures (40). Pool turnover kinetics were consistent with the status of these monomers (P1 and P2) as soluble precursors to an insoluble network (40). Moreover, peptide mapping and amino acid sequencing identified the hexapeptide Val-Lys-ProTyr-His-Pro of P1 as a putative cross-link domain, occurring about five times in the monomer (41).Our extensin precursor preparations routinely yield substrate (mg) amounts, which have enabled us to develop a quantitative assay for in vitro cross-linking. We describe here methods for the isolation and assay of extensin precursor cross-linking activity. This involved CaCl2 elution of extensin cross-linking activity from intact cells and assay of the enzymic activity as the conversion rate ofextensin monomers to oligomers, measured by FPLC on Superose-6. We confirmed cross-linking by direct TEM visualization of the oligomeric products.The essential enzymic characterization of the cross-linking activity included: pH optimum, substrate saturation, inhibitors, and dependence on exogenous hydrogen peroxide. We therefore propose the trivial name 'extensin peroxidase' for the isolated cross-linking activity, which we have previously described briefly in progr...

No abstract

Fruit ripening is associated with cell wall modifications. The present review focuses on cell wall components and the nature of noncovalent and covalent interactions in the primary cell wall. The role of structural protein cross-links are evaluated within the context of cell wall-mediated changes in texture during fruit ripening. The article discusses molecular approaches in fruit cell wall interactions to regulate processes in fruit ripening in order to improve post-harvest textural characteristics.