Isolation, characterization and cDNA cloning of nicotianamine synthase from barley - A key enzyme for iron homeostasis in plants

Basic cellular processes such as electron transport in photosynthesis and r espiration require the precise control of iron homeostasis. To mobilize iro n, plants have evolved at least two different strategies. The nonproteinoge nous amino acid nicotianamine which is synthesized from three molecules of S-adenosyl-L-methionine, is an essential component of both pathways, This c ompound is missing in the tomato mutant chloronerva, which exhibits severe defects in the regulation of iron metabolism. We report the purification an d partial characterization of the nicotianamine synthase from barley roots as well as the cloning of two corresponding gene sequences. The function of the gene sequence has been verified by overexpression in Escherichia coli. Further confirmation comes from reduction of the nicotianamine content and the exhibition of a chloronerva-like phenotype due to the expression of he terologous antisense constructs in transgenic tobacco plants. The native en zyme with an apparent M-r of approximate to 105 000 probably represents a t rimer of S-adenosyl-L-methionine-binding subunits. A comparison with the re cently cloned chloronerva gene of tomato reveals striking sequence homology , providing support for the suggestion that the destruction of the nicotian amine synthase encoding gene is the molecular basis of the tomato mutation.