Sucrose transporter NtSUT1 confers aluminum tolerance on cultured cells of tobacco (Nicotiana tabacum L.)

Abstract

The role of plasma membrane-localized sucrose transporter (NtSUT1) was investigated using cultured tobacco cell (Nicotiana tabacum L.) line BY-2. The wild type (WT) cells were first transformed with the NtSUT1 gene or its fragments cloned from tobacco cell line SL to form the over-expression (OX) and suppression (RNAi) cell lines, respectively. Using OX and RNAi transgenics, the role of NtSUT1 in growth capacity of actively growing cells and in aluminum (Al)-treated cells was examined. During the logarithmic phase of growth in nutrient medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), both the rate of sucrose uptake measured with radio-tracer and the content of soluble sugars were higher in OX and lower in RNAi cell lines compared to WT. Overall, the content of soluble sugars negatively correlated with the time necessary for doubling mass (fresh weight). When cells were treated without (control) or with Al in a simple medium containing calcium, sucrose and 2-(N-morpholino)ethanesulfonic acid (MES; pH 5.0) for up to 18 h, the expression of NtSUT1 under its native promoter, or under the control of strong constitutive cauliflower mosaic virus (CaMV) 35S promoter, was strongly dependent on the presence of 2,4-D. Thereafter, the cells were preferentially treated in the presence of 2,4-D. During 6 h after a start of the control treatment, sucrose uptake rates were, compared to WT, slightly higher and lower in OX and RNAi lines respectively. The addition of Al reduced the sucrose uptake rates of OX and WT to the level of RNAi line, indicating that Al inhibits sucrose uptake via NtSUT1. During the post-Al culture of control and Al-treated cells in a nutrient medium, sucrose uptake rates were much higher in OX compared to WT and RNAi lines, which closely and positively correlated with the growth capacity of the cells. Judging from the growth capacity of Al-treated cells relative to that of control cells, OX cells were more tolerant to Al than WT and RNAi. In summary, we conclude that over-expression of NtSUT1 confers higher growth capacity in actively growing cells as well as in Al-treated cells.

Key words:

• aluminum tolerance
• cultured tobacco cells
• 2,4-dichlorophenoxyacetic acid
• NtSUT1
• sucrose transporter.

ACKNOWLEDGMENTS

We thank Prof. Toshiyuki Nagata (Hosei Univ.) for providing the BY-2 cell line and Prof. Tsuyoshi Nakagawa (Shimane Univ.) and Prof. Yasuo Niwa (Univ. Shizuoka) for providing the plasmid vectors. We thank Prof. Takuya Furuichi (Gifu Women’s Univ.), Prof. Ivan Galis (Okayama Univ.) and Mr. Yoshiyuki Tsuchiya (Okayama Univ.) for valuable discussions. This study was supported by a Grant-in-Aid for General Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 24380039), a special grant from MEXT for the Environmental and Life Scientific Studies on the Integration of Low-Carbon Society and Ensuring Food Safety and Security, and the Ohara Foundation for Agricultural Science.