Isolation and lectin-binding properties of barley epidermal and mesophyll protoplasts

Abstract

Protoplasts from primary leaves of barley (Hordeum vulgare L.) were obtained by enzymatic digestion and fractionated by discontinuous density gradient centrifugation to yield highly enriched fractions of mesophyll and epidermal protoplasts. A characterization of both protoplast types resulted in a clear differentiation of the outer protoplast surfaces. The protoplasts were examined for affinity to various lectins by agglutination tests and by labeling with lectin – fluorescein isothiocyanate conjugates. Both types of protoplasts agglutinated with soybean lectin. Fluorescein isothiocyanate-labeled soybean lectin was uniformly distributed on the protoplast surface. Mesophyll protoplasts, but not protoplasts from the epidermis, were agglutinated by Concanavalin A. Both types of protoplasts exhibited fluorescence labeling with Concanavalin A – fluorescein isothiocyanate conjugate. This label often showed a patchy distribution on the protoplast surface. Tetragonolobus lectin and β-D-glucosyl Yariv artificial antigen agglutinated mesophyll but not epidermal protoplasts. One of three tested monoclonal antibodies with specificity for arabinogalactans had affinity to the surface of mesophyll and epidermal protoplasts. Key words: agglutination, arabinogalactan protein, cell surface, epidermal protoplasts, fluorescence labeling, lectin.