Targeted Approaches for Detecting Changes in Protein Expression with Mycorrhiza Development: Hydrolytic Isoenzyme Analyses and Immunological Detection of Known Proteins in Root Extracts

Abstract

The first proteins investigated in arbuscular mycorrhizal root extracts were various enzymes thought to be implicated in the functional symbiosis, such as enzymes of phosphorus and nitrogen metabolism (GianinazziPearson and Gianinazzi 1978; Smith et al. 1985). Besides classical methods for measuring in vitro enzyme activities of root extracts, gel electrophoresis of total proteins followed by specific enzymatic detection constitutes a powerful and useful tool for the study of plant proteins with known activities. Activity gel strategy has been adapted for the detection and assay of several plant enzymes including those of primary and secondary metabolism (Gabriel and Gersten 1992), and more recently those involved in defense mechanisms (Asselin 1993; Michaud and Asselin 1995). Two of the major interests of this technique lie in its great versatility and specificity, since most of the enzyme activities can be analyzed after protein separation by either the Davis electrophoretic system for acidic and neutral proteins (Davis 1964) or the Reisfeld system for basic proteins (Reisfeld et al. 1962), by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli 1970) or finally by twodimensional gel electrophoresis (2D-PAGE; Fig. 19.1). The specificity of the technique depends on the enzyme substrate, and many different substrates can be used ranging from chemicals, or modified chemicals, to bacterial or fungal wall components (Michaud and Asselin 1995). Since the general strategy is very similar for different hydrolytic enzymes, we describe first the protocol for analyzing chitinase activities in detail, and indicate only variations for others.