Cloning and functional analysis of MxNRAMP1 and MxNRAMP3, two genes related to high metal tolerance of Malus xiaojinensis

• MxNRAMP1 is localized to the plasma membrane.
• MxNRAMP3 is localized to the vacuolar membrane.
• MxNRAMP1 and MxNRAMP3 were able to transport Cd in yeast.
• MxNRAMP1 and MxNRAMP3 response to Fe, Cd, Mn, and Zn stress

Metal stresses are common nutritional disorder in plants, especially in fruit trees grown in calcareous soil. The natural resistance-associated macrophage protein (NRAMP) family comprises integral membrane proteins that play an important role in regulating metal ion transport. Our research focused on an iron deficiency-resistant apple (Malus xiaojinensis) rootstock, as the study model. Based on conserved regions of NRAMP1 and NRAMP3 in other plants, we designed primers and cloned MxNRAMP1 and MxNRAMP3 genes from M. xiaojinensis, the functions of which were preliminarily characterized in yeast, a eukaryotic model organism. In this study, MxNRAMP1 is localized to the plasma membrane, whereas MxNRAMP3 is localized to the vacuolar membrane. In yeast, both proteins were able to transport Cd but not other metal ions. Upon Fe, Cd, Mn, and Zn stress treatments for M. xiaojinensis, MxNRAMP1 expression was specifically induced in roots. MxNRAMP3 was expressed in both roots and leaves, and was immediately expressed in roots at the onset of metal stress, whereas its expression was delayed in leaves. These results showed that MxNRAMP1 and MxNRAMP3 play an important role in metal absorption and transportation.