CHANGES IN ALPHA-AMYLASE AND BETA-AMYLASE ACTIVITIES DURING GERMINATION OF SEEDS OF ALFALFA (MEDICAGO-SATIVA L)

We examined the methods available for the assay of alpha-amylase in alfalfa (Medicago sativa) and found the Phadebas test most suitable. The Phadebas assay and activity staining on ampholine gels after isoelectrofocusing revealed that an amylase is present in the dry seeds of alfalfa and that its activity decreases rapidly after the second day of seed germination. An amylase was purified by affinity chromatography and gel filtration. The kinds of sugar generated from soluble starch by the purified amylase resembled those generated by other alpha-amylases from plants, in particular those from mung bean (Vigna radiata). These results indicate that amylase in alfalfa seeds belongs to the family of alpha-amylases. The molecular weight and isoelectric point of the alpha-amylase were determined to be 43 kDa and 4.92, respectively. The Pantrac assay and activity staining on immobiline gels after isoelectrofocusing revealed that the activities of beta-amylases increased during the initial 4 to 5 days of germination. Furthermore, treatment of whole seedlings with cycloheximide or actinomycin D inhibited the increase in activity of beta-amylases but did not affect the reduction in activity of alpha-amylase. During germination of alfalfa seeds, alpha-amylase activity decreases while, in contrast, beta-amylase activity increases (in the cotyledons of germinating seeds), changes that are specific to the germinating seeds of alfalfa.