Purification and Characterization of Vegetative Storage Proteins from Alfalfa (Medicago sativa L.) Taproots

Abstract

Summary Alfalfa ( Medicago sativa L.) accumulates C and N reserves in taproots and utilizes these reserves for shoot growth in spring and for shoot regrowth after defoliation. Three proteins are very abundant in taproots and undergo a cyclic pattern of utilization during early shoot growth followed by reaccumulation during late shoot development. Our objectives were to purify and characterize these putative vegetative storage proteins from alfalfa taproots. The proteins were purified using organic-solvent and ionic-precipitation techniques, gel filtration, and affinity chromatography. Polyclonal antibodies were raised against the purified proteins, and electrophoresis and immunoblotting were utilized to determine protein distribution and relative abundance. These proteins are present in high concentrations in alfalfa taproots, but were not found in seeds, nodules, leaves, or stems of alfalfa. Taproots of all perennial Medicago species examined contained these proteins, whereas roots of annual Medicago species had very low to undetectable amounts of these proteins. Taproots of other forage legume species ( Lotus, Melilotus, and Trifolium ) did not contain proteins that cross-reacted with antibodies raised against the three alfalfa taproot proteins. The three proteins have molecular masses of 15, 19, and 32 ku, are glycosylated, and have epitopes in common. The amino acids asparagine and aspartate make up 15 mole percent of the three alfalfa taproot proteins. These proteins possess features consistent with their role being vegetative storage proteins.