Molecular cloning of the cDNA encoding aspartate aminotransferase from bean root nodules and determination of its role in nodule nitrogen metabolism

Abstract

A cDNA clone encoding aspartate aminotransferase (PVAAT-2) (EC 2.6.1.1) was isolated from the common bean Phaseolus vulgaris nodule cDNA library. The nucleotide sequence analysis of the full-length cDNA allowed its identification by comparison with sequence databases. The amino acid sequence of the bean PvAAT-2 showed high similarity with the AAT-2 isoforms described in other leguminous plants. The amino-terminal region of the PvAAT-2 contains a sequence, which shares common features of plastid transit peptides. Southern blot analysis showed that the PvAAT-2 clone is encoded by a single gene in the P. vulgaris genome. Analysis of the PvAAT-2 mRNA levels suggests that the expression of this gene is nodule enhanced. The PvAAT-2 transcript is more abundant in nodules with increased synthesis of amides and is down-regulated in conditions where ureides accumulate. When plants were supplemented with ureides or with amides, PvAAT-2 expression was reduced, while it was not affected when plants were treated with allopurinol, an inhibitor of ureide synthesis. On the other hand, the expression of asparagine synthetase (another enzyme involved in the synthesis of amides) is not affected either by ureides or amides. These data suggest a role for AAT-2 in the mechanism involved in the synthesis of nitrogen compounds in bean nodules.