Active chitinases in the apoplastic fluids of healthy white lupin (Lupinusalbus L.) plants

Acidic exocellular class III chitinase (EC 3.2.1.14) was previously identif ied in healthy white lupin (Lupinus albus L.) plants and suspension-culture d cells by N-terminal microsequencing. In this study, the detection of chit inase activity with Remazol Brilliant Violet 5R (RBV)-labelled chitin deriv atives is described. Chitinase activity was observed in protein fractions o f cytoplasmic or exocellular origin from roots, hypocotyls, cotyledons, and leaves of healthy white lupin plants. Using isoelectrofocusing followed by a new overlay technique with carboxymethyl chitin-RBV conjugate-containing gel, up to six different chitinase isoforms were visualised. Their activit y was distributed fairly evenly within a plant with acidic isoforms predomi nating in cell walls and basic (or neutral) ones found intracellularly. Exo cellular location of some chitinase isoforms were also confirmed by detecti on of their activities in intercellular washing fluids from white lupin tis sues. Chitinase activity was demonstrated in culture filtrates and cell wal ls of suspension-cultured white lupin cells.