Isolation and characterisation of a nicotianamine synthase gene MxNas1 in Malus xiaojinensis

Iron (Fe) is one of the essential micronutrients required by all living organisms. In order to isolate genes involved in Fe-uptake, a subtracted cDNA library from roots of Malus xiaojinensis 'Cheng et Jiang' (Accession No. DGB0458 in the National Fruit Germplasm Repository) was constructed using suppression subtractive hybridisation (SSH). MxNas1, a gene that encodes a putative nicotianamine (NA) synthase, was cloned by differential screening of the subtracted library. By complementation, we demonstrated that MxNas1 was able to rescue the phenotype of an NA synthesis-defective tomato (Solanum lycopersicum) mutant 'chloronerva', suggesting it was a functional NA synthase. Transgenic tobacco (Nicotiana tabacum) plants that over-expressed MxNas1 were more tolerant to Fe-deficiency than wild-type tobacco plants. In contrast, when MxNas1 was introduced into plants in an anti-sense orientation, to reduce endogenous transcript levels of NA synthase genes, the resulting transgenic tobacco plants exhibited symptoms of Fe-deficiency earlier than control plants. Increased expression of MxNas1 in transgenic tobacco plants also resulted in increased concentrations of Fe, Mn, Cu, and Zn in the leaves.