**2. Results**

#### **2.1 Excess iron application induces cell death and arrests cell cycle progression**

To examine the effect of excess iron application to plant cells, we monitored the growth of tobacco BY-2 cells in medium containing high amounts of iron. The cells took up about 50 to 90 g iron g cells-1 6 h after transfer to a medium containing 1.0, 2.0 or 5.0 mM FeSO4 and lacking other divalent cations (Mg2+, Ca2+, Mn2+, Zn2+ and Co2+), but only about 5 g iron g cells-1 in standard medium with 0.1 mM FeSO4 (Fig. 1A).

Under these conditions, about 50 % or more than 80 % of the cells died 24 h after transfer to medium containing 1.0 and 2.0 or 5.0 mM FeSO4, respectively, whereas only a few percent of the cells died in the standard medium (Fig. 1B).

As cell death is known to relate to the arrest of cell cycle progression (Kadota et al. 2004, Sano et al. 2006), we monitored the latter by flow cytometric and mitotic index (MI) analyses upon excess iron application.

In the control condition containing 0.1 mM FeSO4, the cell cycle progressed from the S to G2 phase 2 h after aphidicolin release, and entered mitosis at 8 h and the G1 phase at 10 h (Fig.

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

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.

**2.1 Excess iron application induces cell death and arrests cell cycle progression** 

To examine the effect of excess iron application to plant cells, we monitored the growth of tobacco BY-2 cells in medium containing high amounts of iron. The cells took up about 50 to 90 g iron g cells-1 6 h after transfer to a medium containing 1.0, 2.0 or 5.0 mM FeSO4 and lacking other divalent cations (Mg2+, Ca2+, Mn2+, Zn2+ and Co2+), but only about 5 g iron g

Under these conditions, about 50 % or more than 80 % of the cells died 24 h after transfer to medium containing 1.0 and 2.0 or 5.0 mM FeSO4, respectively, whereas only a few percent of

As cell death is known to relate to the arrest of cell cycle progression (Kadota et al. 2004, Sano et al. 2006), we monitored the latter by flow cytometric and mitotic index (MI) analyses

In the control condition containing 0.1 mM FeSO4, the cell cycle progressed from the S to G2 phase 2 h after aphidicolin release, and entered mitosis at 8 h and the G1 phase at 10 h (Fig.

manganese, iron and cadmium (Xia et al. 2010).

cells-1 in standard medium with 0.1 mM FeSO4 (Fig. 1A).

the cells died in the standard medium (Fig. 1B).

upon excess iron application.

**2. Results** 

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

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.

Metal Ion Homeostasis Mediated by NRAMP Transporters in

show the means ± SE of four independent experiments.

only showed cytoplasmic-localized fluorescence (Fig. 6D).

comparable in the two cell lines (Fig. 7B).

conditions (Fig. 5D).

Plant Cells – Focused on Increased Resistance to Iron and Cadmium Ion 217

expressing LacZ (Fig. 4). Therefore, both NtNRAMP1 and NtZIP1 could have the iron

Fig. 4. Iron uptake activity of metal transporters in yeast cells. Iron accumulation in yeast cells transformed with the vector containing LacZ, NtNRAMP1 (NR1), NtZIP1 (ZIP), AtNRAMP1 (AR1), AtNRAMP3 (AR3) and AtHMA4 (HMA) was measured by atomic absorption spectrograph after incubation for 18 h in a medium with 0.2 mM FeCl3. The data

In the NtNRAMP1-overexpressing tobacco line (NR1), cell cycle progression was similar to that of the non-transformed BY-2 cells under control conditions in which 0.1 mM FeSO4 was included. When 0.3 mM FeSO4 was applied at the S phase, cell cycle progression of the non-transformed cells was delayed and the value of the peak MI reduced (Fig. 5A). In contrast, in the NR1 cells, the tendency of cell cycle progression was comparable to that in the control condition even though these cells took up as much as amounts of iron compared to the non-transformed cells (Fig. 5A, B). Furthermore, the proportion of NR1 cells undergoing cell death was reduced in comparison with the non-transformed cells following 1.0 mM FeSO4 application (Fig. 5C), even though the amount of iron taken up by the NR1 cells was comparable to that in the non-transformed cells under these

To investigate the role of NtNRAMP1 on the suppression of cell cycle arrest and cell death upon excess iron application, we examined the subcellular localization of NtNRAMP1 by transient expression of NtNRAMP1-GFP fusion proteins. The GFP fluorescence was localized primarily on the plasma membrane, and confirmed by the plasma membrane marker, SYP132 (Enami et al. 2009, Fig. 6A-C). In contrast, cells transiently expressing GFP

As the above results suggested that NtNRAMP1 is a plasma membrane transporter, we examined the effect of *NtNRAMP1* overexpression on iron uptake. The total amount of iron taken up into cells 24 h after 1.0 mM FeSO4 application was comparable in the NR1 and nontransformed cells (Fig. 7A). However, when calculated on the basis of the rate of iron uptake, the non-transformed BY-2 cells had about 3-fold higher rates than the NR1 cells in the initial 1 hour after iron application (Fig. 7B). In subsequent periods, uptake rates were

uptake activity comparable to *Arabidopsis* AtNRAMP1 and AtNRAMP3.

#### **2.2 Overexpression of** *NtNRAMP1* **decreases sensitivity to excess iron application**

To investigate the molecular mechanisms of iron uptake and cell death of tobacco BY-2 cells, we identified and characterized several tobacco iron transporter genes. As the ZIP and NRAMP family proteins are known as iron/metal transporters in plants, we identified two tobacco cDNA clones that encoded proteins with high sequence similarity to ZIP or NRAMP and named them *NtZIP1* and *NtNRAMP1*, respectively. The amino acid sequence of NtZIP1 was 61 % identical to the MtZIP3 of *Medicago truncatula* (Lopez-Millan et al. 2004) and 53 % to AtZIP5 of *Arabidopsis thaliana*, whereas NtNRAMP1 was 71 % identical to *Arabidopsis* AtNRAMP1 and AtNRAMP6. Gene expression analysis revealed that 1.0 mM FeSO4 application increased the relative transcript levels of *NtNRAMP1* but decreased those of *NtZIP1* (Fig. 3A, B).

As the increased level of *NtNRAMP1* gene expression upon iron application implied an involvement of this transporter under these culture conditions, we prepared transgenic tobacco BY-2 cell lines that overexpressed *NtNRAMP1* by placing the gene under control of the cauliflower mosaic virus 35S promoter. In one (NR1) of the four transgenic lines obtained, *NtNRAMP1* transcript levels were about 2-fold those of the non-transformed BY-2 cells whereas the *NtZIP1* transcript levels were reduced (Fig. 3A, B). Similar increases in *NtNRAMP1* transcript levels were also observed in the other three transgenic lines obtained (data not shown).

Fig. 3. Gene expression of the *NtNRAMP1* and *NtZIP1* iron transporters. *NtNRAMP1* (A) and *NtZIP1* (B) gene expression in non-transformed BY-2 cells cultured in control conditions for 24 h (BY-2) or with 1.0 mM FeSO4 for 24 h (BY-2 + Fe), and in *NtNRAMP1* overexpressing cells cultured in control conditions (NR1) or with 1.0 mM FeSO4 for 24 h (NR1 + Fe). Gene expression was monitored by real-time quantitative PCR and the data show relative transcripts normalized with GAPdH gene expression. The data show the means ± SE of three independent experiments.

When the iron uptake activities of NtNRAMP1 and NtZIP1 were measured in yeast cells, the amount of iron accumulated in the yeast cells expressing NtNRAMP1 or NtZIP1 was about 1.5 times high compared to control cells expressing LacZ. The amout was comparable to those expressing *Arabidopsis* AtNRAMP1 or AtNRAMP3 whereas that expressing an effulux pump AtHMA4 (Verret et al. 2004, Mills et al. 2005) was comparable to that

To investigate the molecular mechanisms of iron uptake and cell death of tobacco BY-2 cells, we identified and characterized several tobacco iron transporter genes. As the ZIP and NRAMP family proteins are known as iron/metal transporters in plants, we identified two tobacco cDNA clones that encoded proteins with high sequence similarity to ZIP or NRAMP and named them *NtZIP1* and *NtNRAMP1*, respectively. The amino acid sequence of NtZIP1 was 61 % identical to the MtZIP3 of *Medicago truncatula* (Lopez-Millan et al. 2004) and 53 % to AtZIP5 of *Arabidopsis thaliana*, whereas NtNRAMP1 was 71 % identical to *Arabidopsis* AtNRAMP1 and AtNRAMP6. Gene expression analysis revealed that 1.0 mM FeSO4 application increased the relative transcript levels of *NtNRAMP1* but decreased those of

As the increased level of *NtNRAMP1* gene expression upon iron application implied an involvement of this transporter under these culture conditions, we prepared transgenic tobacco BY-2 cell lines that overexpressed *NtNRAMP1* by placing the gene under control of the cauliflower mosaic virus 35S promoter. In one (NR1) of the four transgenic lines obtained, *NtNRAMP1* transcript levels were about 2-fold those of the non-transformed BY-2 cells whereas the *NtZIP1* transcript levels were reduced (Fig. 3A, B). Similar increases in *NtNRAMP1* transcript levels were also observed in the other three transgenic lines obtained

Fig. 3. Gene expression of the *NtNRAMP1* and *NtZIP1* iron transporters. *NtNRAMP1* (A) and *NtZIP1* (B) gene expression in non-transformed BY-2 cells cultured in control conditions

overexpressing cells cultured in control conditions (NR1) or with 1.0 mM FeSO4 for 24 h (NR1 + Fe). Gene expression was monitored by real-time quantitative PCR and the data show relative transcripts normalized with GAPdH gene expression. The data show the

When the iron uptake activities of NtNRAMP1 and NtZIP1 were measured in yeast cells, the amount of iron accumulated in the yeast cells expressing NtNRAMP1 or NtZIP1 was about 1.5 times high compared to control cells expressing LacZ. The amout was comparable to those expressing *Arabidopsis* AtNRAMP1 or AtNRAMP3 whereas that expressing an effulux pump AtHMA4 (Verret et al. 2004, Mills et al. 2005) was comparable to that

for 24 h (BY-2) or with 1.0 mM FeSO4 for 24 h (BY-2 + Fe), and in *NtNRAMP1*

means ± SE of three independent experiments.

**2.2 Overexpression of** *NtNRAMP1* **decreases sensitivity to excess iron application** 

*NtZIP1* (Fig. 3A, B).

(data not shown).

expressing LacZ (Fig. 4). Therefore, both NtNRAMP1 and NtZIP1 could have the iron uptake activity comparable to *Arabidopsis* AtNRAMP1 and AtNRAMP3.

Fig. 4. Iron uptake activity of metal transporters in yeast cells. Iron accumulation in yeast cells transformed with the vector containing LacZ, NtNRAMP1 (NR1), NtZIP1 (ZIP), AtNRAMP1 (AR1), AtNRAMP3 (AR3) and AtHMA4 (HMA) was measured by atomic absorption spectrograph after incubation for 18 h in a medium with 0.2 mM FeCl3. The data show the means ± SE of four independent experiments.

In the NtNRAMP1-overexpressing tobacco line (NR1), cell cycle progression was similar to that of the non-transformed BY-2 cells under control conditions in which 0.1 mM FeSO4 was included. When 0.3 mM FeSO4 was applied at the S phase, cell cycle progression of the non-transformed cells was delayed and the value of the peak MI reduced (Fig. 5A). In contrast, in the NR1 cells, the tendency of cell cycle progression was comparable to that in the control condition even though these cells took up as much as amounts of iron compared to the non-transformed cells (Fig. 5A, B). Furthermore, the proportion of NR1 cells undergoing cell death was reduced in comparison with the non-transformed cells following 1.0 mM FeSO4 application (Fig. 5C), even though the amount of iron taken up by the NR1 cells was comparable to that in the non-transformed cells under these conditions (Fig. 5D).

To investigate the role of NtNRAMP1 on the suppression of cell cycle arrest and cell death upon excess iron application, we examined the subcellular localization of NtNRAMP1 by transient expression of NtNRAMP1-GFP fusion proteins. The GFP fluorescence was localized primarily on the plasma membrane, and confirmed by the plasma membrane marker, SYP132 (Enami et al. 2009, Fig. 6A-C). In contrast, cells transiently expressing GFP only showed cytoplasmic-localized fluorescence (Fig. 6D).

As the above results suggested that NtNRAMP1 is a plasma membrane transporter, we examined the effect of *NtNRAMP1* overexpression on iron uptake. The total amount of iron taken up into cells 24 h after 1.0 mM FeSO4 application was comparable in the NR1 and nontransformed cells (Fig. 7A). However, when calculated on the basis of the rate of iron uptake, the non-transformed BY-2 cells had about 3-fold higher rates than the NR1 cells in the initial 1 hour after iron application (Fig. 7B). In subsequent periods, uptake rates were comparable in the two cell lines (Fig. 7B).

Metal Ion Homeostasis Mediated by NRAMP Transporters in

cell transiently expressing GFP. Scale bar represents 20 m.

four independent experiments.

Plant Cells – Focused on Increased Resistance to Iron and Cadmium Ion 219

Fig. 6. Subcellular localization of NtNRAMP1. (A) NtNRAMP1 localization was monitored in a tobacco BY-2 cell transiently expressing NtNRAMP1-GFP. (B) Plasma membrane localization of the syntaxin, SYP132, monitored in cells transiently expressing tagRFP-SYP132. (C) Merged image of images (A) and (B). (D) GFP fluorescence in a tobacco BY-2

Fig. 7. Effect of NtNRAMP1 overexpression on iron uptake. (A) Changes in the amount of iron taken up into control BY-2 cells (BY-2) and NtNRAMP1 overexpressing cells (NR1). (B) Changes in iron uptake rate calculated from the data in (A). Data show the means ± SE of

To further characterize NtNRAMP1, we examined the effects of cadmium on cell growth since NRAMP transporters are known to transport a variety of metal ions (Nevo and Nelson 2006, Krämer et al. 2007). In control medium without cadmium, both non-transformed and

Fig. 5. Cell cycle progression and the population of NtNRAMP1 overexpressing cells undergoing cell death. (A) Cell cycle progression of non-transformed BY-2 cells (BY-2 + Fe 0.1 mM) and NtNRAMP1 overexpressing cells (NR1 + Fe 0.1 mM) cultured in control conditions or with 0.3 mM FeSO4, respectively (BY-2 + Fe 0.3 mM, NR1 + Fe 0.3 mM). The data show a representative sample of three independent experiments. (B) Amount of iron taken up into cells cultured for 6 h in the culture conditions shown in (A). (C) Population of cells undergoing cell death in non-transformed BY-2 cells (BY-2) and NtNRAMP1 overexpressing cells cultured in control conditions (NR1) for 24 h, or those cultured with 1.0 mM FeSO4 for 24 h, respectively (BY-2 + Fe, NR1 + Fe). (D) Amount of iron taken up into cells cultured for 24 h in culture conditions in (C). In (B), (C) and (D), the data show the means ± SE of three independent experiments.

Fig. 5. Cell cycle progression and the population of NtNRAMP1 overexpressing cells undergoing cell death. (A) Cell cycle progression of non-transformed BY-2 cells (BY-2 + Fe 0.1 mM) and NtNRAMP1 overexpressing cells (NR1 + Fe 0.1 mM) cultured in control conditions or with 0.3 mM FeSO4, respectively (BY-2 + Fe 0.3 mM, NR1 + Fe 0.3 mM). The data show a representative sample of three independent experiments. (B) Amount of iron taken up into cells cultured for 6 h in the culture conditions shown in (A). (C) Population of

cells undergoing cell death in non-transformed BY-2 cells (BY-2) and NtNRAMP1

means ± SE of three independent experiments.

overexpressing cells cultured in control conditions (NR1) for 24 h, or those cultured with 1.0 mM FeSO4 for 24 h, respectively (BY-2 + Fe, NR1 + Fe). (D) Amount of iron taken up into cells cultured for 24 h in culture conditions in (C). In (B), (C) and (D), the data show the

Fig. 6. Subcellular localization of NtNRAMP1. (A) NtNRAMP1 localization was monitored in a tobacco BY-2 cell transiently expressing NtNRAMP1-GFP. (B) Plasma membrane localization of the syntaxin, SYP132, monitored in cells transiently expressing tagRFP-SYP132. (C) Merged image of images (A) and (B). (D) GFP fluorescence in a tobacco BY-2 cell transiently expressing GFP. Scale bar represents 20 m.

Fig. 7. Effect of NtNRAMP1 overexpression on iron uptake. (A) Changes in the amount of iron taken up into control BY-2 cells (BY-2) and NtNRAMP1 overexpressing cells (NR1). (B) Changes in iron uptake rate calculated from the data in (A). Data show the means ± SE of four independent experiments.

To further characterize NtNRAMP1, we examined the effects of cadmium on cell growth since NRAMP transporters are known to transport a variety of metal ions (Nevo and Nelson 2006, Krämer et al. 2007). In control medium without cadmium, both non-transformed and

Metal Ion Homeostasis Mediated by NRAMP Transporters in

through the increased number of the NtNRAMP1 proteins.

plants.

iron uptake are remained to be determined.

Plant Cells – Focused on Increased Resistance to Iron and Cadmium Ion 221

(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

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

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

**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

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