**5. Material and methods**

#### **5.1 Plant materials and culture conditions**

A tobacco BY-2 cell line (*Nicotiana tabacum* L. cv. Bright Yellow 2) was maintained by weekly subculture in a modified Linsmaier and Skoog medium supplemented with 2,4-D (LSD medium), in which KH2PO4 and thiamine HCl were increased to 370 and 1 mg l-1, respectively. To this basal medium, sucrose and 2,4-D were supplemented to 3 % and 0.2 mg l-1, respectively, and the pH was adjusted to 5.8 before autoclaving (Nagata et al. 1992). The cell suspension was cultured on a rotary shaker at 130 rpm and 27C in the dark.

Cell synchrony was established by treatment with 5 g l-1 aphidicolin (Sigma Chemical Co., St. Louis, MO, USA) essentially as described by Kumagai-Sano et al. (2006). After 24 h of 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 before cell cycle and cell death analyses were conducted as described below.

## **5.2 Cell cycle and cell death analyses**

222 Crosstalk and Integration of Membrane Trafficking Pathways

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

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

Upon excess metal ion application, plant cells modulated transporter activities. The activation of the plasma membrane transporters upon excess metal application might be inconsistent to keep the cellular metal homeostasis. Our findings suggest that activation of transporters with low affinity to metal ions could be involved in avoiding metal ion toxicity.

A tobacco BY-2 cell line (*Nicotiana tabacum* L. cv. Bright Yellow 2) was maintained by weekly subculture in a modified Linsmaier and Skoog medium supplemented with 2,4-D (LSD medium), in which KH2PO4 and thiamine HCl were increased to 370 and 1 mg l-1, respectively. To this basal medium, sucrose and 2,4-D were supplemented to 3 % and 0.2 mg l-1, respectively, and the pH was adjusted to 5.8 before autoclaving (Nagata et al. 1992). The

Cell synchrony was established by treatment with 5 g l-1 aphidicolin (Sigma Chemical Co., St. Louis, MO, USA) essentially as described by Kumagai-Sano et al. (2006). After 24 h of

cell suspension was cultured on a rotary shaker at 130 rpm and 27C in the dark.

iron uptake.

decreased (Fig. 8B).

**5. Material and methods** 

**5.1 Plant materials and culture conditions** 

**4. Conclusion** 

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 Kadota et al. (2004).

#### **5.3 Quantification of iron and cadmium concentrations**

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).

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

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

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

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),

Metal Ion Homeostasis Mediated by NRAMP Transporters in

**7. References** 

1249-1259.

54:183-206.

706.

50: 280-289.

220: 1049-1051.

*Plant* 4: 464-476.

Plant Cells – Focused on Increased Resistance to Iron and Cadmium Ion 225

The nucleotide sequences reported in this paper have been submitted to GenBank as

This work was financially supported in part by a Grant-in-Aid for Scientific Research on Priority Areas to S.H. (No. 23012009), a Grant-in-Aid for Scientific Research on Innovative Areas to S.H. (No. 22114505) from the Japanese Ministry of Education, Science, Culture, Sports and Technology, an Advanced Measurement and Analysis grant from the Japan Science and Technology Agency (JST) to S.H. and Wada Kunkokai Foundation, Japan to T.S.

Cailliatte, R., Lapeyre, B., Briat, J.F., Mari, S. & Curie, C. (2009) The NRAMP6 metal transporter contributes to cadmium toxicity. *Biochem. J.* 422:217-228. Cailliatte, R., Schikora, A., Briat, J.F., Mari, S. & Curie, C. (2010) High-affinity manganese

Cassin, G., Mari, S., Curie, C., Briat, J.F. & Czernic, P. (2009) Increased sensitivity to iron

Conte, S.S. & Walker, E.L. (2011) Transporters contributing to iron trafficking in plants. *Mol.* 

Curie, C., Alonso, J.M., Le, J.M., Ecker, R. & Briat, J.F. (2000) Involvement of NRAMP1 from

Curie, C. & Briat, J.F. (2003) Iron transport and signaling in plants. *Annu. Rev. Plant Biol.* 

Curie, C., Cassin, G., Couch, D., Divol, F., Higuchi, K., Le Jean, M., Misson, J., Schikora, A.,

Deckert, J. (2005) Cadmium toxicity in plants: is there any analogy to its carcinogenic effect

Eide, D., Broderius, M., Fett, J. & Guerinot, M.L. (1996) A novel iron regulated metal

Enami, K., Ichikawa, M., Uemura, T., Kutsuna, N., Hasezawa, S., Nakagawa, T., Nakano, A.

Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma, D.P. & Firoozabady, E.

nicotianamine and yellow stripe 1-like transporters. *Ann. Bot.* 103:1-11. DalCorso, G., Farinati, S., Maistri, S. & Furini, A. (2008) How plants cope with cadmium: staking all on metabolism and gene expression. *J. Integr. Plant Biol*. 50:1268-1280. Dambrauskas, G., Aves, S.J., Bryant, J.A., Francis, D. & Rogers, H.J. (2003) Genes encoding

Czernic, P. & Mari, S. (2009) Metal movement within the plant: contribution of

two essential DNA replication activation proteins, Cdc6 and Mcm3, exhibit very different patterns of expression in the tobacco BY-2 cell cycle. *J. Exp. Bot.* 54: 699-

transporter from plants identified by functional expression in yeast. *Proc. Natl.* 

& Sato, M.H. (2009) Differential Expression Control and Polarized Distribution of Plasma Membrane-Resident SYP1 SNAREs in *Arabidopsis thaliana*. *Plant Cell Physiol.* 

(1983) Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues. *Science* 

*Arabidopsis thaliana* in iron transport. *Biochem. J*. 347: 749–755.

uptake by the metal transporter NRAMP1 is essential for *Arabidopsis* growth in low

deficiency in Arabidopsis thaliana overaccumulating nicotianamine. *J. Exp. Bot.* 60:

accession numbers AB505625 for NtNRAMP1 and AB505626 for NtZIP1.

manganese conditions. *Plant Cell* 22: 904-917.

in mammalian cells? *Biometals.* 18:475-481.

*Acad. Sci. USA*. 93: 5624–5628.

respectively. As internal standards of the cDNA amounts, GAPdH fragments were amplified with primers 5'-CCGGACAAGGCTGCTGCTAC-3' (GAP FW) and 5'- GACCCTCCACAATGCCAAACC-3' (GAP RW), designed on the basis of the tobacco GAPdH (cytosolic glyceraldehyde-3-phosphate dehydrogenase) gene (Accession number: M14419, Dambrauskas et al., 2003) and the relative transcript values then calculated.

### **5.6 Transformation of tobacco BY-2 cells**

The coding region of *NtNRAMP1* was amplified by PCR using gene specific primers of 5'- CACCATGGCGGCGAACTCGTCCCC-3' and 5'-ATTAGTGGTCCTCTGCTGAGGCAA-3', then cloned into the pENTR/D-TOPO vector (Invitrogen) and finally introduced into the pGWB502 binary vector (Nakagawa et al. 2007) by the Gateway cloning system using LR clonase (Invitrogen). The pGWB502 vector gave the cauliflower 35S promoter sequence to the PCR products. *Agrobacterium*-mediated transformation of the tobacco BY-2 cells was performed as described by Mayo et al. (2006). Transformants were selected with 50 mg l-1 hygromycin.

Transient gene expression was carried out by particle bombardment. A cell suspension of 2 d-old BY-2 cells was filtrated onto filter paper, and the cells bombarded with gold particles (1.0 m) coated with the appropriate vector constructs using a particle delivery system (PDS-1000/He, Bio-Rad, Hercules, CA, USA) according to the manufacturer's recommendations. Filtrated BY-2 cells were placed at a distance of 6 cm under the stopping screen and were bombarded in a vacuum of 28 inches Hg at a helium pressure of 1100 psi. Following bombardment, the cells were diluted in LSD medium and kept in the dark at 27 C for 6 to 12 h before observation. The GFP fluorescence was detected on the inverted platform of a fluorescence microscope equipped with a spinning disc confocal laser scanning system (CSU-X1, Yokogawa, Tokyo, Japan) and a cooled CCD camera (Cool-SNAP HQ, PhotoMetrics, Huntington Beach, Canada).

### **5.7 Yeast experiments**

Yeast cells INVSc1 (Invtrogen) were transformed by pYES2.1/V5-His-TOPO vectors (Invitrogen) containing an entire ORF region of the respective metal transporter cDNAs according to standard procedures (Invitrogen). The transformants were selected on synthesic complete medium omitted uracil (SC-uracil) containing 2 % glucose, 0.67 % yeast nitrogen base (without amino acids, Difco), amino acids omitting uracil (-Ura DO Supplement, Clontech Laboratories Inc.), 0.5 % ammmoniumu sulfate and 2 % agar. The transporter proteins were induced by application of 2 % galactose instead of glucose in the SC-uracil medium. For iron uptake measurements, yeast cells precultured in the SC-uracil medium were diluted to OD600 of 0.3 and cultured in the medium supplied with 2 % galactose and 0.2 mM FeCl3. After 18 h incubation, OD600 were measured and the yeast culture was washed with deionized water twice. For iron extraction, yeast cells were digested with 2N HCl and the iron concentrations were determined by atomic absorption spectrograph (AA-660, Shimadzu Co., Kyoto, Japan).

## **6. Acknowledgment**

We are grateful to Dr. T. Nakagawa (Shimane University) for the kind gift of the pGWB502 binary vector.

The nucleotide sequences reported in this paper have been submitted to GenBank as accession numbers AB505625 for NtNRAMP1 and AB505626 for NtZIP1.

This work was financially supported in part by a Grant-in-Aid for Scientific Research on Priority Areas to S.H. (No. 23012009), a Grant-in-Aid for Scientific Research on Innovative Areas to S.H. (No. 22114505) from the Japanese Ministry of Education, Science, Culture, Sports and Technology, an Advanced Measurement and Analysis grant from the Japan Science and Technology Agency (JST) to S.H. and Wada Kunkokai Foundation, Japan to T.S.
