Map-based cloning of chloronerva, a gene involved in iron uptake of higherplants encoding nicotianamine synthase

The uptake of iron in plants is a highly regulated process that is induced on iron starvation. In tomato, the mutant chloronerva exhibits constitutive expression of iron uptake responses and intercostal chlorosis, Biochemical ly, chloronerva is an auxotroph for nicotianamine, a key polyamine in plant iron uptake metabolism. The chloronerva gene has been fine-mapped onto the long arm of chromosome 1 in a large segregating tomato population and yeas t artificial chromosome clones encompassing the region were isolated by usi ng flanking markers. A cosmid contig containing the chloronerva gene was es tablished, and complementing cosmids were identified by transformation into the mutant. The chloronerva transcript was identified by cDNA isolation us ing the complementing cosmids, The gene encodes a unique protein of 35 kDa. The mutant harbors a single base change compared with the wild type. Based on enzyme activity and sequence similarity to the coding DNA sequence of t he purified barley enzyme the chloronerva gene encodes the enzyme nicotiana mine synthase.