Cloning of nicotianamine synthase genes from Arabidopsis thaliana

Nicotianamine synthase (NAS) catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine (NA), NA is present in all the plants; it chelates metal cations, and is considered to play a role in metal homeostasis in plants. Moreover, in graminaceous monocotyledonous plants, NA is an essential intermediate in the biosynthesis of mugineic acid family phytosiderophores (MAs), In order to identify the gene encoding NAS in dicotyledonous plants, Arabidopsis thaliana databases were searched using the nucleotide sequence of the NAS gene from barley (HvNAS), which we have recently isolated. We found several ESTs and three genomic sequences highly homologous to HvNAS in the databases. Based on these nucleotide sequences and that of HvNAS, we designed 2 sets of primers to isolate the NAS orthologues in Arabidopsis and succeeded in obtaining three DNA clones encoding AtNAS (AtNAS1, 2, and 3), These clones were expressed in Escherichia coli and their protein products displayed the NAS activity. The expression of AtNAS1 was detected in both shoots and roots of A. thaliana by RT-PCR; AtNAS3 expression was only detected in the shoots. In contrast, AtNAS2 expression was not detected in any organs.