Molecular cloning and characterization of MxNAS2, a gene encoding nicotianamine synthase in Malus xiaojinensis, with functions in tolerance to iron stress and misshapen flower in transgenic tobacco

Abstract Iron (Fe) is one of the essential micronutrients required by all plants. Nicotianamine (NA) is considered as the chelate substance in the transport of Fe. In the present study, a gene encoding putative nicotianamine synthase (NAS) is isolated from Malus xiaojinensis and designated as MxNAS2 . The MxNAS2 gene encodes a protein of 325 amino acid residues with a predicted molecular mass of 36.2 kDa and a theoretical isoelectric point of 5.18. Subcellular localization reveals that MxNAS2 is preferentially localized in vesicles and cytoplasmic membrane. The expression of MxNAS2 is more enriched in leaf, root, and phloem than xylem, which is highly affected by Fe stress and indoleacetic acid (IAA) treatment, whereas, weakly affected by abscisic acid (ABA) treatment in M. xiaojinensis seedlings. When MxNAS2 was introduced into tobacco, it promotes the synthesis of NAS and increases NA and chlorophyll contents. Overexpression of MxNAS2 improves the tolerance to Fe stress in transgenic tobacco, but leads to delayed flowering. Higher levels of MxNAS2 expression in transgenic tobacco contribute to misshapen flowers and increased levels of Fe, Mn, Cu and Zn in leaf and flower. In addition to its role in metal transport in plants, NA may be involved in the regulation of metal transfer within cells. These results suggest that NA excess influences the functions of metal-requiring proteins, including some of the transcription factors.