Differential expression of two soybean apyrases, one of which is an early nodulin

Two cDNA clones were isolated from soybean (Glycine soja) by polymerase cha in reaction with primers designed to conserved motifs found in apyrases (nu cleotide phosphohydrolase). The two cDNAs are predicted to encode for two, distinct, apyrase proteins of approximately 50 kDa (i.e., GS50) and 52 kDa (i.e,, GS52). Phylogenetic analysis indicated that GS52 is orthologous to a family of apyrases recently suggested to play a role in legume nodulation. GS50 is paralogous to this family and, therefore, likely plays a different physiological role. Consistent with this analysis, GS50 mRNA was detected in root, hypocotyls, flowers, and stems, while GS52 mRNA was found in root and flowers, Neither gene was expressed in leaves or cotyledons. Inoculatio n of roots with Bradyrhizobium japonicum, nitrogen-fixing symbiont of soybe an, resulted in the rapid (<6 h) induction of GS52 mRNA expression. The lev el of GS50 mRNA expression was not affected by bacterial inoculation. Weste rn blot (immunoblot) analysis of GS50 expression mirrored the results obtai ned by mRNA analysis. However, in contrast to the mRNA results, GS52 protei n was found in stems. Interestingly, anti-GS52 antibody recognized a 50-kDa protein found only in nodule extracts. Treatment of roots with anti-GS52 a ntibody, but not anti-GS50 antibody or preimmune serum, blocked nodulation by B. japonicum, Fractionation of cellular membranes in sucrose density gra dients and subsequent Western analysis of the fractions revealed that GS50 colocalized with marker enzymes for the Golgi, while GS52 colocalized with marker enzymes for the plasma membrane. Restriction fragment length polymor phism (RFLP)-based mapping placed the gs52 gene on major linkage group J of the integrated genetic map of soybean These data suggest that GS50 is like ly an endo-apyrase involved in Golgi function, while GS52 is localized on t he root surface and appears to play an important role in nodulation.