Purification, characterization and DNA sequencing of nicotianamine aminotransferase (NAAT-III) expressed in Fe-deficient barley roots

Abstract

To study the mechanism of Fe-response in plants and to breed the Fe-deficiency tolerant cultivars, we purified Nicotianamine aminotransferase (NAAT), the key enzyme induced by Fe-deficiency in the biosynthetic pathway of mugineic-acid family phytosiderophores (MAs) in barley roots. Previously, two isozymes of NAAT (NAAT-I and NAAT-II) have been eluted by a linear KCl gradient on DEAE Sephacel column chromatography. Recently the third isozyme activity passing through DEAE Sephacel column has been found (NAAT-III). NAAT-III activity was detected only in Fe-deficient roots, and its specific activity was increased by the next nicotianamine (NA)-affinity chromatography. Among a few candidate spots of NAAT-III on 2D-PAGE, one peptide was concentrated and found to be specific to Fe-deficiency. Amino acid sequences of this peptide were determined. The probe was made by PCR method using degenerated primers, and cDNA encoding this peptide was isolated by this PCR product from a cDNA library constructed from Fe-deficient barley roots. The sequence of the cDNA had 51.7% identity to rat mRNA for tyrosine aminotransferase. NAAT activity was detected, after its introduction into yeast cells.