**3. Discussion**

#### **3.1 Role of the NtNRAMP1 transporter following excess iron application**

The NRAMP family transporters function as general metal ion transporters (Nevo and Nelson 2006), and we have shown in this study that NtNRAMP1 overexpression in tobacco BY-2 cells suppressed cell cycle arrest and cell death upon excess iron application (Fig. 5). Plasma membrane localization of NtNRAMP1 (Fig. 6) and the decreased rate of iron uptake in the NtNRAMP1 overexpressing cells (Fig. 7B) implies the role of NtNRAMP1 as a modulator of iron uptake or an iron exporter.

Concerning the latter hypothes, the metal ion efflux activity of the NRAMP family members are somewhat uncommon. The TcNRAMP3 transporter was found to exclude Ni when expressed in yeast but transported iron and cadmium into cells in yeast and plants (Wei et al. 2009). Our iron uptake experiments in yeast implies the iron uptake of NtNRAMP1 rather than the export since the amout of iron uptake was higher than that in the cells expressing the effulux pump of AtHMA4 (Fig. 4).

In this context, the increaed iron resistance upon NtNRAMP1 overexpression might be explaned by the role of AtNRAMP1 as a physiological manganese (Mn) transporter

NR1 cells proliferated about 70 times per week (Fig. 8A). When 1.0 or 10 M CdSO4 was added to the medium, the growth rate of controls cells decreased to 40 or 20 times per week, respectively (Fig. 8A). In contrast, the NR1 cell growth rates in 1.0 M CdSO4 were comparable to those without cadmium treatment, and were still about 55 times per week in 10 M CdSO4 (Fig 8A). After 10 M CdSO4 application, the amount of cadmium taken up into the control BY-2 cells increased in 24 h but decreased thereafter (Fig. 8B). In the NR1 cells, the amount of cadmium was smaller than that in the control BY-2 cells in 24 h (Fig. 8B).

Fig. 8. Effect of NtNRAMP1 overexpression on cell growth following cadmium application. (A) Growth rate of control BY-2 cells and NtNRAMP1 overexpressing cells during culture for 7 days in control conditions (BY, NR), or with 1.0 M (BY + Cd 1.0, NR + Cd 1.0) or 10 M CdSO4 (BY + Cd 10, NR + Cd 10). (B) Changes in the amount of cadmium taken up into

The NRAMP family transporters function as general metal ion transporters (Nevo and Nelson 2006), and we have shown in this study that NtNRAMP1 overexpression in tobacco BY-2 cells suppressed cell cycle arrest and cell death upon excess iron application (Fig. 5). Plasma membrane localization of NtNRAMP1 (Fig. 6) and the decreased rate of iron uptake in the NtNRAMP1 overexpressing cells (Fig. 7B) implies the role of NtNRAMP1 as a

Concerning the latter hypothes, the metal ion efflux activity of the NRAMP family members are somewhat uncommon. The TcNRAMP3 transporter was found to exclude Ni when expressed in yeast but transported iron and cadmium into cells in yeast and plants (Wei et al. 2009). Our iron uptake experiments in yeast implies the iron uptake of NtNRAMP1 rather than the export since the amout of iron uptake was higher than that in the cells

In this context, the increaed iron resistance upon NtNRAMP1 overexpression might be explaned by the role of AtNRAMP1 as a physiological manganese (Mn) transporter

cells cultured with 10 M CdSO4. Data show the means ± SE of four independent

**3.1 Role of the NtNRAMP1 transporter following excess iron application** 

experiments.

**3. Discussion** 

modulator of iron uptake or an iron exporter.

expressing the effulux pump of AtHMA4 (Fig. 4).

(Cailliatte et al. 2010). Although the AtNRAMP1 was capable of transporting both iron and Mn in yeast cells (Curie et al. 2000, Thomine et al. 2000), Cailliatte et al. (2010) discussed the competence of iron uptake by Mn uptake increased the resistance to iron toxicity. Similar competence of iron uptake might be occured in the NtNRAMP1 overexpressing cells (Fig. 9). In this model, the supposed metal transporters other than NtNRAMP1 that actively mediate iron uptake are remained to be determined.

Fig. 9. A model for the increased resistance to iron in NtNRAMP1 overexpressing cells. In the control cells (left), excess iron application increases the rate of iron uptake by metal transporters (M) with high iron uptake activity other than NtNRAMP1 (N). In contrast in the NtNRAMP1 overexpressing cells (right), iron uptake is competed by manganese uptake through the increased number of the NtNRAMP1 proteins.

In graminaceous plants, the enhanced tolerance upon excess iron application was achieved by overproduction of a metal chelator, nicotianamine (Lee et al. 2009). The chelated iron was discussed to be an inactive form for reactive-hydroxyl radical generation as well as to be easily transported from roots to aerial organs (Curie et al. 2009). The increased translocation of iron to rice seeds was expected to provide iron-fortified plants and improve human health (Lee et al. 2009, Wirth et al. 2009, Zheng et al. 2010). Recently, transporters involved in iron translocation was identified in which iron-nocotianamine complex was transported to the rice shoots and phytosiderophore for iron acquisition was secreted to the soil (Ishimaru et al. 2010, Nozoye et al. 2011). In dicot plants, loss of nicotianamine synthase genes did not to fully supply iron to flowers and seeds (Klatte et al. 2009) whereas overaccumulation of nicotianamine did not affect iron translocation (Cassin et al. 2009). The role of metal chelator in dicot plants on iron translocation and resistance against iron application has still been controversial. The combination of the iron uptake moduration and the enhanced iron translocation could enhance the iron fortification and torelance in dicot plants.

#### **3.2 Increased resistance of NtNRAMP1 overexpressing cells to cadmium**

In plants, cadmium has various effects, such as the inhibition of photosynthesis, respiration and metabolism, and may finally lead to plant growth inhibition (Deckert 2005). NRAMP family members can potentially transport toxic heavy metals, including cadmium, and further characterization of the NtNRAMP1 overexpressing cells in this study revealed their

Metal Ion Homeostasis Mediated by NRAMP Transporters in

**5.3 Quantification of iron and cadmium concentrations** 

**5.4 Molecular cloning of tobacco iron transporter genes** 

vector (Invitrogen Corp., Carlsbad, CA, USA).

**5.5 Gene expression analysis by quantitative RT-PCR** 

**5.2 Cell cycle and cell death analyses** 

Kadota et al. (2004).

Plant Cells – Focused on Increased Resistance to Iron and Cadmium Ion 223

aphidicolin treatment, the cell culture was washed with 1 L of LSD medium on a glass filter and then incubated further in this medium. The cell culture was divided into two to four portions, and several different FeSO4 concentrations were applied as described in Results

The mitotic index (MI) was determined by fluorescence microscopy after the nuclei were stained with 1 M of SYTOX (Molecular Probes Inc., Eugene, OR, USA). For flow cytometry, cells were fixed with 100 % ethanol, then rehydrated in Galbraith's buffer (45 mM MgCl2, 30 mM Na-Citrate, 20 mM MOPS and 1 g l-1l Triton X-100, pH 7.0, Galbraith et al. 1983), and finally treated with 20 g l-1 RNase A (Sigma) and 10 g l-1 propidium iodide (Sigma) for 1 h at room temperature. Cytometric analysis was performed on 5 x 103 cells with a laser scanning cytometer (LSC101, Olympus, Tokyo, Japan) as described by Sano et al. (2006). Cell death was determined after staining the cells with 0.05 % Evans Blue (Sigma) as described in

Intracellular iron and cadmium concentrations were measured by atomic absorption spectrograph. Cells were sedimented by centrifugation to determine their packed cell volumes, and were then washed with 3 % sucrose on a glass filter before being resuspended in distilled water. For iron or cadmium extraction, cells were disrupted by a bead cell disrupter (MS-100, Tomy Seiko Co. Tokyo, Japan) and the iron or cadmium concentrations determined by atomic absorption spectrograph (AA-6800, Shimadzu Co., Kyoto, Japan).

Tobacco total RNA was isolated with the E.Z.N.A. Plant RNA Kit (Omega Bio-tek, Inc. Doraville, GA, USA), and cDNA synthesized using M-MLV reverse transcriptase (Promega, Heidelberg, Germany) with oligo-dT primers. Tobacco BY-2 *NRAMP* cDNA fragments were amplified with degenerate primers of 5'-CCNCAYAAYCTNTTYCTNCAYTSNGC-3' and 5'- TGNCCNGCRTANGTNCCNGTDATNGT-3' designed from homologous regions of known plant NRAMP proteins. *NtZIP1* cDNA was obtained based on the sequence information with high homology to *AtZIP* gene families deposited in the tobacco BY-2 EST database (TAB, Transcriptome Analysis of BY-2, http://mrg.psc.riken.jp/strc/). Amplification of the 5' and 3' cDNA ends was performed by RACE (SMART RACE cDNA Amplification kit, Clontech, Palo Alto, CA, USA), and the amplified fragments then subcloned into the pCR2.1

Real-time quantitative PCR was performed in a Smart Cycler II System (Takara Bio Inc., Shiga, Japan) using the SYBR Green Real time PCR Master Mix (Toyobo Co., LTD., Osaka, Japan). *NtNRAMP1* and *NtZIP1* fragments from nucleotides 635 to 833 and 313 to 487 were amplified with primers 5'-TCTTCAAGGGATTCCCAGGA-3' (NRAMP1 FW) and 5'- TGTTATCCCACGGCATGCAAC-3' (NRAMP1 RV) or 5'- TCGCCATGTTTG AAAGAGAATCC-3' (ZIP1 FW) and 5'- CCAGACTGAGCCACCAATCCA-3' (ZIP1 RW),

before cell cycle and cell death analyses were conducted as described below.

enhanced resistance to cadmium application (Fig. 8A). Changes in plant cadmium sensitivity as a consequence of NRAMP transporter activity have also been reported in which overexpression of AtNRAMP3 or AtNRAMP6 resulted in cadmium hypersensitivity of *Arabidopsis* growth (Thomine et al. 2000, Cailliatte et al. 2009). These proteins were considered to remobilize cadmium to cytoplasm from a detoxifying compartment such as a vacuole and an endomembrane compartment (Thomine et al. 2003, Cailliatte et al. 2009). The plasmamembrane localization of NtNRAMP1 may explain the increased resistance of NtNRAMP1 overexpressing cells to cadmium by the moderation of Cd2+ uptake similar to iron uptake.

The basis of cadmium toxicity is not completely understood, but it appears to affect cellular metabolism through its high affinity for sulfydryl compounds that therefore leads to the misfolding of enzymes, while its chemical similarity to other divalent cations reduces the activity of enzymes similar to the divalent trace metals described above (DalCorso et al. 2008, Verbruggen et al. 2009). In addition, cadmium is thought to be related to ROS generation and subsequent oxidative stress, although primarily through reduced antioxidative capacities rather than a direct effect on ROS generation (Schützendübel and Polle 2002, Deckert 2005, Heyno et al. 2008). In tobacco BY-2 cells, H2O2 production and subsequent cell death was reported upon application of 3 or 5 mM CdCl2 (Olmos et al. 2003, Garnier et al. 2006). Upon application of 50 M CdSO4, cell cycle phase-specific death was also observed in these cells (Kuthanova et al. 2008). In our observation, as cell death was not clearly observed 24 h after application of 10 M CdSO4, effect of cadmium application was monitored by measurement of cell growth (Fig. 8A). Although we can not exclude the possible effects of cadmium on ROS generation, the reduced cellular growth rates shown in this study may have resulted from the reduced enzymatic activities of metabolic pathways since the amounts of cadmium accumulated in the NtNRAMP1 overexpressing cells decreased (Fig. 8B).
