Isolation and Characterization of a
ßAmylase Gene from AlfalfaJ.A. Gana, S.M. Cunningham, and J.J. Volenec
Dept. of Agronomy, Purdue University, West Lafayette, IN 47907-1150
Alfalfa (Medicago sativa L.) roots contain large quantities of ß -amylase, but the role of this enzyme in planta is poorly understood. We have speculated that ß-amylase may be a storage protein providing regrowing shoots with N. This suggestion is based on several line of reasoning: 1) preferential accumulation of ß -amylase in storage taproots to between 5 and 8% of root soluble protein; 2) its disappearance from roots when shoot growth resumes in spring and after defoliation; and 3) no association between increases in ß -amylase activity and enhanced starch utilization. We studied the role of ß -amylase by isolating a ß -amylase cDNA and examining factors that affect expression of its gene. The ß -amylase cDNA encoded a 55.95 kD polypeptide with deduced amino acid sequence showing high similarity to other ß -amylases, especially those from soybean seeds. Starch concentrations, ß -amylase activity, and ß -amylase mRNA levels were measured in roots of alfalfa after defoliation and during cold-acclimation. Starch levels, ß -amylase activity, and ß -amylase transcripts were reduced significantly in roots of defoliated plants. The ß -amylase transcript was high in roots of undefoliated plants, but disappeared from roots within 2 d after defoliation and remained very low until 12 d after defoliation. ß -amylase transcript levels increased in roots of germplasms between September and October, then declined markedly in November and December after shoots were killed by frost (Fig. 1).
Figure 1. Cold acclimation effects on ß -amylase transcript levels in six alfalfa germplasms. The cultivars CUF 101 (O) and Norseman (O) were selected for three cycles for more (L) or less (H) fall dormancy. Roots of field-grown plants were sampled in the months indicated.
Expression of ß -amylase in alfalfa is tissue-specific, with very high expression in roots, moderate expression in stems, and very low expression in leaves. Alfalfa roots contain high ß -amylase transcript levels when compared to roots of sweetclover and red clover, with transcript levels below our detection limit in birdsfoot trefoil. Southern analysis indicates that ß -amylase is present in alfalfa as a multi-gene family. Our evidence indicates that ß -amylase is not involved in starch hydrolysis in alfalfa roots but may function as a storage protein.