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

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.

Overexpression of the sucrose transporter gene NtSUT1 alleviates aluminum-induced inhibition of root elongation in tobacco (Nicotiana tabacum L.)

“…The NtSUT1 gene (designated NtSUT1-L) cloned previously (Sameeullah et al 2013) from the non-chlorophyllous cultured tobacco cell line SL derived from Nicotiana tabacum L. cv. Samsun (Nakamura et al 1988) was used to construct the OX and RNAi transformants of tobacco (cv.…”

“…The sequence of NtSUT1-L is available at the DNA Data Bank of Japan (DDBJ) under accession number AB823663. The plasmid constructs of OX and RNAi prepared previously (Sameeullah et al 2013) were used to transform leaf disks of hydroponically grown tobacco seedlings (4-6 weeks old) using Agrobacterium tumefaciens strain EHA101 (Hood et al 1986) and LBA4404 (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, to prepare the OX and RNAi constructs, the full-length open reading frame (ORF) of NtSUT1-L (for the OX construct) and the fragment from 1 to 724 bp (for the RNAi construct) were cloned into the Gateway entry vector, and were then recombined into the destination vector, pGWB2, under the control of the CaMV35 promoter for overexpression or pBI-sense-antisense-GW for suppression.…”

“…To determine the expression level of NtSUT1, root apices (5mm tip segment) were harvested and ground in liquid nitrogen. Total RNA was extracted and used for real-time reverse transcription polymerase chain reaction (RT-PCR), as described previously (Sameeullah et al 2013). To quantify NtSUT1 transcript levels in WT, OX and RNAi lines, an NtSUT1-L genespecific primer set was used for WT and OX lines, and primers designed from the 3ʹUTR region of NtSUT1 conserved in both NtSUT1-L and NtSUT1-S were used for RNAi lines to evaluate the transcriptional suppression of both NtSUT1 genes.…”

“…Full details of these amplifications are provided in our previous report (Sameeullah et al 2013). Under the same conditions, the transcript levels of NtSUT3 (AAD34610) and NtSUT4 (BAI60050) were also determined using the primer sets described previously (Sameeullah et al 2013). Sequence information was not available for NtSUT2, so its transcript level was not analyzed.…”

“…Based on our previous findings of the biphasic Al action (Abdel-Basset et al 2010) (see above), we explored the role of the plasma membrane-localized SUT (NtSUT1) in Al-induced inhibition of cell elongation using cultured tobacco cells (line BY-2). We constructed over-expression (OX) and suppression (RNAi) lines in which NtSUT1 was controlled by the cauliflower mosaic virus (CaMV) 35S promoter (Sameeullah et al 2013). The expression of NtSUT1 depended on the presence of 2, 4-D under its native promoter or the CaMV 35S promoter.…”