Amylase was found in high activty (193 international units per milliam protein) in the tap root of alfalfa (Medcago satia L. cv. Sonra). The activity was separated by gel filtration chromatography into two fractions with molecular weights of 65,700 (beavy amylase) and 41,700 (light amylase). Activty staining of electropboretic gels indicated the presence of one isozyme in the heavy amylase fraction and two in the liht amylase fraction. Three amylase isozymes with electropboretic mobilties identical to those in the heavy and the light amylase fractions were the only amylases identified in crude root preparations. Both heavy and lht amylases hydrolyzed amylopectin, soluble starch, and amylose but did not hydrolyze puflulan or p-limit dextrin. The ratio of viscosity change to reducing power production during starch hydrolysis was identical for both alfalfa amylase fractions and sweet potato P-amylase, while that of bacterial a-amylase was considerably higher. The identification of maltose and -limit dextrin as hydrolytic end-products confirmed that these alfalfa root amylases are ail .8-amylases. Beta-amylase (a-1,4-glucan maltohydrolase, EC 3.2.1.2) is an exoamylase that attacks the nonreducing ends of starch molecules, producing ,B-maltose and fl-limit dextrin as products (22). Betaamylases are strictly plant enzymes that have been reported in ungerminated wheat and soybean seeds (11, 23); germinating barley, rice, sorghum, and wheat seeds (3,6,7,15); sweet potato roots (1); broad bean leaves (5); and pea seedling roots (21).Amylases are of particular interest in the perennial legume alfalfa (Medicago sativa L.), which stores large quantities of starch in its fleshy tap root. This starch serves as a carbohydrate reserve that is used for winter survival and shoot regeneration following harvest of the shoot as a forage (17,19). Amylase has been reported in high activity in the tap root of several alfalfa cultivars (8), and activity staining of electrophoretically separated proteins on polyacrylamide gels has shown the presence of two or three isozymes of amylase in alfalfa tap roots (13). In this study, we describe the separation, identification, and characterization of isozymes of 8-amylase from alfalfa root tissues. MATERIALS AND METHODS Plant Material. Alfalfa (Medicago sativa L. cv. Sonora) was planted in the spring as described previously (8). In the autumn of the seeding year, plants were collected, shoots clipped to within 2.5 cm of crowns, and all secondary roots removed from tap roots. Trimmed plants were either potted and maintained in a greenhouse or washed, wrapped in wet cheesecloth and plastic, and stored at -2°C until use. Greenhouse-grown plants were inoculated with a mixture of Rhizobium meliloti strains (Nitragen Co., Milwaukee, WI) and irrigated with a nitrogen-free nutrient solution (9).Separation of f8-Amylase Isozymes. Whole tap roots were washed, chopped into small pieces with a razor blade, and then mechanically homogenized with a 'homogeniser' (Measuring and Scientific Equipment,...