CELL-WALL-BOUND OXIDASES FROM TOBACCO (NICOTIANA-TABACUM) XYLEM PARTICIPATE IN LIGNIN FORMATION

A band of cells closest to the cambium in the xylem of tobacco (Nicoti ana tabacum L. cv. Samsun) stems oxidized ,2-azinobis-(3-ethylbenzo-th iazoline-6-sulphonate) (ABTS), o-dianisidine and syringaldazine in the absence of exogenously added hydrogen peroxide. The oxidation was not prevented by catalase which suggests that the oxidation is not depend ent on the production and utilisation of endogenous hydrogen peroxide by cell-wall peroxidases. Cell walls, isolated from tobacco xylem, als o oxidized these substrates in the absence of added hydrogen peroxide. The cell walls consumed molecular oxygen whilst oxidizing a range of compounds including coniferyl alcohol. The substrate preference and se nsitivity to inhibitors suggest the presence of laccase-type polypheno l oxidases (p-diphenol:O-2 oxidoreductase EC 1.14.18.1) which are cova lently bound to the wall. The oxidation of coniferyl alcohol by the xy lem cell walls was confirmed by assays based on the disappearance of c oniferyl alcohol and was not affected by the presence of 500 units.ml( -1) catalase or superoxide dismutase. Prolonged incubation of cell wal ls with coniferyl alcohol led to the production of a yellow-orange wat er-insoluble material that precipitated with the cell walls. Although a proportion of this material was soluble in methanol, the majority wa s tightly associated with the cell walls. These coloured cell walls ha d elevated lignin contents when assayed by the acetyl-bromide method. Fourier transform-infrared spectroscopic analysis of the coloured cell walls indicated that the increased lignin content is due to the depos ition of guaiacyl-type lignin. Digestion of the xylem cell walls with Driselase, a mixture of fungal glycases, produced a wall residue that had a dramatically reduced ability to oxidize ABTS in the absence of a dded H2O2. However, oxidase activity could not be detected in the Dris elase-solubilized extract, although small amounts of oxidase activity could be recovered from the Driselase-resistant wall residue by extrac tion in 3 M CaCl2.