Crude cell wall preparations from Cicer urietinurn L. cell suspension cultures show high activity for the hydrolysis of coniferyl alcohol P-D-glucoside (coniferin). Various 0-glucosidase activities could be solubilized from these preparations by 0.5 M NaCl treatment and one of these could be shown to possess a high activity for the hydrolysis of coniferin. The enzyme activities were purified to near homogeneity by Sephadex G-200 and CM-Sephadex chromatography. Isoelectric focussing indicated the occurrence of P-glucosidase isoenzymes with identical catalytic activity (PI 8.5 -10).Molecular weights were determined as 110000, with two subunits of 63000 and 43000. Maximum hydrolytic activity is at pH 5.The P-glucosidase isoenzymes catalyze the hydrolysis of various P-glucosides with aromatic aglycone structure and different sugar moieties. However, coniferin has been found to be one of the best substrates ( k , = 0.8 mM; V = 6 pmol . min-' . mg protein-') for these P-glucosidase isoenzymes. The data suggest that a P-glucosidase-catalyzed reaction might be involved in lignification of these plant cell cultures.

A combination of the phytohormones naphthalene acetic acid and benzylaminopurine (5gM each) allows Lignification in various plant cell cultures. This system has been used to investigate the relationship between the coniferin-hydrolyzing 13-glucosidase activity and lignification. In Petroselinum hortense and Triticum aestivum cell cultures the appearance of this enzymatic activity coincided with lignification. In parsley cell cultures it was moreover shown that this activity appears concomitantly with other lignin biosynthetic enzymes. The unique enzymes of the flavonoid pathway did not appear by this phytohormone treatment. In other cell cultures investigated the correlation between the coniferin-hydrolyzing activity and lignification was not as evident as in the above two cases. This was probably due to the high activity of coniferin glucosidase already present in the normally grown cultures. Coniferin t3-glucosidase was found in all lignified cell cultures.