**Metal Ion Homeostasis Mediated by NRAMP Transporters in Plant Cells – Focused on Increased Resistance to Iron and Cadmium Ion**

Toshio Sano1, Toshihiro Yoshihara1, Koichi Handa2, Masa H. Sato3, Toshiyuki Nagata1 and Seiichiro Hasezawa2,4 *1Faculty of Bioscience and Applied Chemistry, Hosei University 2Graduate School of Frontier Sciences, The University of Tokyo 3Graduate School of Life and Environmental Sciences, Kyoto Prefectural University 4Advanced Measurement and Analysis, Japan Science and Technology Agency (JST) Japan* 

#### **1. Introduction**

212 Crosstalk and Integration of Membrane Trafficking Pathways

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Plants have developed several adaptive systems to control the cellular concentrations of essential metals in which the ion transporters play significant roles. At the cell surface, transporters localized on plasmamembrane controlled metal ion uptake and release whereas inside of the plant cells, those localized on endomembrane sequestered and remobilized metal ions in organella, such as vacuoles and plastids (Pilon et al. 2009, Puig and Peñarrubia, 2009).

Iron is one of several essential nutrients but a problematic one for living organisms (Conte and Walker 2011). At the cellular level, iron is used as a cofactor in enzymatic activities based on the reversible reaction between Fe2+ (ferrous) and Fe3+ (ferric) ions (Hell and Stephan 2003). In plants, it is essential for chlorophyll synthesis and hence iron deficiency results in chlorosis and pale-yellow or white leaves (Wiedenhoeft 2006). Usually, iron is chelated to organic matter in insoluble forms in soils that causes iron deficiency whereas in anaerobic and acidic conditions, iron toxicity occurs because of the increase of iron solubility (Ricachenevsky et al. 2010). The basis of iron toxicity was usually discussed to be oxidative stress by generation of reactive-hydroxyl radicals (Neyens and Baeyens 2003). Iron homeostasis in plant cells is partly achieved through the control of iron transport across membranous structures, and several families of putative iron transporters, including ZIP (ZRT, IRT-like proteins) and NRAMP (natural resistance associated macrophage protein), have been described (Guerinot 2000, Curie and Briat 2003, Kim and Guerinot 2007). Among the ZIP transporters, the *Arabidopsis AtIRT1* was the first iron transport molecule identified in plants (Eide et al. 1996) and was shown to be the major transporter mediating iron uptake into roots (Vert et al. 2002). Recently, IRT2, a close homolog of IRT1 in *Arabidopsis*, was suggested to compartmentalize iron into vesicles to prevent toxicity from excess free iron in the cytosol (Vert et al. 2009). On the other hand, among the ubiquitous NRAMP family of

Metal Ion Homeostasis Mediated by NRAMP Transporters in

Plant Cells – Focused on Increased Resistance to Iron and Cadmium Ion 215

Fig. 1. Effect of excess iron application on tobacco BY-2 cell growth. (A) Amount of iron taken up into cells. After transfer of 7-day-old cells to medium containing 0.1, 1.0, 2.0 or 5.0 mM FeSO4, the amount of iron taken up into the cells in 6 h was measured. Values shown are those after subtraction of measurements taken just after transfer to the medium as background. Data show the means ± SE of three independent experiments. (B) In the culture conditions in (A), the population undergoing cell death was measured. Data represent means ± SE of three independent experiments with more than 400 cells in each experiment.

2A, B). When the cells were additionally treated with 1.0 mM FeSO4 after aphidicolin release, with the cell cycle restarting from S phase, cell cycle progression was delayed and the percentage of cells entering mitosis decreased (Fig. 2A). Further flow cytometric analysis

Fig. 2. Effects of excess iron application on cell cycle progression. Cell cycle progression of cells in control conditions (open diamonds) and those cultured with 1.0 mM FeSO4 after aphidicolon treatment (open squares). Cell cycle progression was monitored by the mitotic index (A) and by flow cytometry (B). The data show representatives of three independent

experiments.

demonstrated the cell cycle arrest of these cells in the S to G2 phase (Fig. 2B).

metal transporters, it was the mouse *Nramp1* that was first cloned as the gene responsible for resistance to mycobacterial infection (Nevo and Nelson 2006). In *Arabidopsis*, six NRAMP transporters, AtNRAMP1-6, have been identified and categorized by phylogenic analysis into two subfamilies: AtNRAMP1 and 6 forming the first group and AtNRAMP2 through 5 comprising the second group (Mäser et al. 2001). Of these, *AtNRAMP1*, *3*, *4* and *6* have been shown to encode functional plant metal transporters (Krämer et al. 2007, Cailliatte et al. 2009). *AtNRAMP1* can complement the *fet3fet4* yeast mutant that is defective in both lowand high-affinity iron transporters, while overexpression of *AtNRAMP1* in *Arabidopsis* increases plant resistance to toxic iron concentrations (Curie et al. 2000). AtNRAMP3 and AtNRAMP4 mediate the remobilization of iron from the vacuolar store and are essential for seed germination under low iron conditions (Thomine et al. 2003, Lanquar et al. 2005).

In addition to iron transport activities, these transporters can mediate the transport of a wide range of metal cations because of their similar chemical characteristics (Hall and Williams 2003, Krämer et al. 2007). AtNRAMP1 can functionally complement a manganeseuptake defective mutant and confer cadmium sensitivity to yeast (Thomine et al. 2000). This transporter was recently demonstrated to act as a physiological manganese transporter in *Arabidopsis* (Cailliatte et al. 2010). Similarly, TcNRAMP3 and TcNRAMP4 from the metal hyperaccumulator, *Thlaspi caerulescens*, can transport various metal cations, including Fe2+, Mn2+, Cd2+, Ni2+ and Zn2+ when expressed in yeast, and MbNRAMP1 from apple trees of *Malus baccata* was found to mediate Mn2+ uptake in addition to Fe2+ (Oomen et al. 2008, Xiao et al. 2008, Wei et al. 2009). Recently, rice Nrat1 that belongs to the NRAMP family has been reported to transport trivalent aluminum ion, but not other divalent ions such as manganese, iron and cadmium (Xia et al. 2010).

In the present chapter, we demonstrate that tobacco NtNRAMP1 is a plasma membrane transporter, and that overexpression of this protein in tobacco BY-2 cells increases the resistance of the cells to both iron and cadmium ions. We propose that NtNRAMP1 moderates metal ion-uptake and prevents toxicity resulting from excess ironor cadmium application.
