A Soybean Seed Urease-Null Produces Urease in Cell Culture

Abstract

Itachi, a soybean (Glycine max [L.] Merr.) variety with 0.2% normal seed urease activity, was recovered from a screen of 6,000 entries in the United States Department of Agriculture soybean germplasm collection. No urease antigen in Itachi seed extracts was detected by double diffusion or by rocket immunoelectrophoresis. Native gels stained for protein or ureolytic activity revealed no detectable urease holoenzyme. An anti-urease antibody affinity column was used to remove all detectable urease activity and antigen from `wild type' (cv. Prize) seed extracts. Affinity column effluent and nonchromatographed Itachi extracts both lack a species which comigrates with purified urease subunits in sodium dodecylsulfate polyacrylamide gels. Inability to detect urease antigen or urease protein suggests that during development of Itachi seeds there is no synthesis of urease protein or that, at most, its synthesis is 0.2% of wild type (Prize). No urease activity or only traces of urease activity were detected in cotyledons of developing or germinating Itachi seeds. In contrast, callus cultures induced from cotyledon, shoot tip, root, or root tip tissues of Itachi seedlings exhibited ureolytic activity equivalent to that of Prize cultures. Shoot tip cultures of both Prize and Itachi grew with urea as sole nitrogen source. Most or all of the ureolytic activity in crude extracts of Prize and Itachi suspension culture cells is seed-like urease in thermal stability, recognition by antibodies to the seed enzyme, hydroxyurea sensitivity, and nickel requirement for synthesis. It has been reported previously (Polacco, Havir 1979 J Biol Chem 254: 1707-1715; Polacco, Sparks, Jr, Havir 1979 Genet Eng 1: 241-259) that partially purified cell culture urease is identical to seed urease by immunological and electrophoretic criteria. These results suggest that urease is under different developmental controls in the seed and in cell culture. In both Prize and Itachi cultures, utilization of the ureide allantoin, unlike that of urea, is not dependent on nickel. This suggests that ureide catabolism does not require urease.