Cloning of nicotianamine synthase genes from Arabidopsis thaliana

Nicotianamine synthase (NAS) catalyzes the trimerization of S-adenosylmethi onine 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 meta l homeostasis in plants. Moreover, in graminaceous monocotyledonous plants, NA is an essential intermediate in the biosynthesis of mugineic acid famil y phytosiderophores (MAs), In order to identify the gene encoding NAS in di cotyledonous plants, Arabidopsis thaliana databases were searched using the nucleotide sequence of the NAS gene from barley (HvNAS), which we have rec ently isolated. We found several ESTs and three genomic sequences highly ho mologous to HvNAS in the databases. Based on these nucleotide sequences and that of HvNAS, we designed 2 sets of primers to isolate the NAS orthologue s in Arabidopsis and succeeded in obtaining three DNA clones encoding AtNAS (AtNAS1, 2, and 3), These clones were expressed in Escherichia coli and th eir protein products displayed the NAS activity. The expression of AtNAS1 w as detected in both shoots and roots of A. thaliana by RT-PCR; AtNAS3 expre ssion was only detected in the shoots. In contrast, AtNAS2 expression was n ot detected in any organs.