Soybean genes involved in nickel insertion into urease

Abstract

In soybean, mutation in the Eu2 or in the Eu3 gene eliminates the activities, but not the proteins, of the embryo-specific and the ubiquitous ureases, encoded by Eu1 and Eu4, respectively. This pleiotropic urease-negative phenotype is consistent with accessory gene functions encoded by Eu2 and Eu3, i.e. correct insertion of Ni into the metallocentre of each urease. To test an accessory gene function an examination was made of segregation of alleles at Eu2 and Eu3 with segregation of an RFLP revealed by a plant homologue of the bacterial urease accessory gene, ureG. The eu3-e1/eu3-e1 mutant, which has a urease activity-null phenotype, lacked a 1.4 kb EcoRV genomic fragment found in progenitor cultivar Williams, cv Williams 82 and in two mutants at the Eu2 locus, eu2/eu2 and EN24, the latter described here for the first time. The lack of the 1.4 kb band segregated with eu3-e1 in a cross of eu3-e1/eu3-e1 × EN24. The second approach was to attempt partial correction of the urease-negative trait by Ni supplementation in vitro. First, a small, reproducible stimulation of activity in mixed extracts of mutants which complement genetically, namely {eu2/eu2 plus eu3-e1/eu3-e1) and (EN24 plus eu3-e1/eu3-e1) was observed. Activation proceeded for several hours in these extracts containing endogenous Ni. In mixed extracts from Ni-free embryos, activation was dependent on added Ni; Ni had no effect on individual mutant extracts. By genetic and biochemical criteria the ubiquitous urease was the sole or major species activated, an activation which approached 10% normal activity.