Direct covalent linkage of fluorescent probes to the plant protoplast surface

Abstract

Methods for fluorescently labeling the surface of live plant protoplasts are presented and characterized. Two classes of chemical reactions were exploited to accomplish direct covalent attachment of reactive fluorophores to plasma membrane components. One class of reactions involved the derivatization of existing amino-groups on the protoplast surface. The other class of reactions involved a two-step procedure to derivatize surface carbohydrates. In the first step, aldehyde groups were generated by oxidation of surface carbohydrates with either periodate or galactose oxidase. The second step involved formation of a Schiff base linkage between the aldehydes and a hydrazide or aromatic amine on a fluorophore. Both classes of reactions were demonstrated with several fluorophores including fluorescein, rhodamine, and Lucifer yellow. The reactions were carried out under conditions that allowed good protoplast survival while producing a high ratio of surface fluorescence to cytoplasmic fluorescence. The reaction products were characterized to the extent of determining whether the fluorophores were incorporated into proteins or into lipids. These fluorescence-labeling procedures should prove useful in a variety of cell biological and biochemical applications.