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Comparative Study
. 2001 Mar;13(3):695-705.
doi: 10.1105/tpc.13.3.695.

Identification of a mannitol transporter, AgMaT1, in celery phloem

N Noiraud  1 , L Maurousset, R Lemoine
Affiliations
Comparative Study

Identification of a mannitol transporter, AgMaT1, in celery phloem

N Noiraud et al. Plant Cell. 2001 Mar.

Abstract

A celery petiole phloem cDNA library was constructed and used to identify a cDNA that gives Saccharomyces cerevisiae cells the ability to grow on mannitol and transport radiolabeled mannitol in a manner consistent with a proton symport mechanism. This cDNA was named AgMaT1 (Apium graveolens mannitol transporter 1). The expression profile in source leaves and phloem was in agreement with a role for mannitol in phloem loading in celery. The identification in eukaryotes of a mannitol transporter is important because mannitol is not only a primary photosynthetic product in species such as celery but is also considered a compatible solute and antioxidant implicated in resistance to biotic and abiotic stress.

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Figures

Figure 1.
Figure 1.
Nucleotide and Deduced Amino Acid Sequences of the AgMaT1 cDNA. Putative transmembrane helices are underlined. Transmembrane regions and protein orientation were predicted using HMMTOP (Tusnady and Simon, 1998). The sequence (VAGIGVG) corresponding to LLGFGVG (used to design the initial degenerate primer for amplification) is boxed. The italicized residues correspond to the sequences conserved in the sugar transporter subfamily of the MFS.
Figure 2.
Figure 2.
Growth Curves of Transgenic Saccharomyces MaDH4 Cells in Minimal Medium Supplemented with Either 2% Glucose or 2% Mannitol. AgMaT1 was subcloned into the PstI-XhoI sites of the yeast expression vector YEP112A1XE (YEP; Riesmeier et al., 1992). Competent MaDH4 cells were transformed with this construct (Dohmen et al., 1991), and YEP112A1XE was used as a control. YEP, MaDH4 containing the expression plasmid YEP112A1XE; AgMaT1, MaDH4 containing the AgMaT1 cDNA cloned in the PstI-XhoI sites of YEP112A1XE. SC, synthetic complete.
Figure 3.
Figure 3.
Uptake of Mannitol by Transgenic Saccharomyces Cells. Yeast cells were grown to the early logarithmic phase in minimal medium supplemented with either 2% glucose or 2% mannitol. During uptake, the mannitol concentration was 500 μM and the external pH was 4.5. Squares represent uptake by cells transformed with AgMaT1, and circles represent mannitol uptake by control cells transformed with the plasmid YEP112A1XE. The results are means ±sd of three independent experiments (four replicates per experiment).
Figure 4.
Figure 4.
pH Dependence of Mannitol Transport in Transgenic Yeast Cells Expressing AgMaT1. Measurements were performed as described for Figure 3 except that the incubation time was 2 min and the incubation medium was buffered at the indicated pH values with 25 mM Mes. The results are means ±sd of two independent experiments (four replicates per experiment).
Figure 5.
Figure 5.
Concentration Dependence of Mannitol Transport in Transgenic Yeast Cells Expressing AgMaT1. Culture conditions were as described in Figure 3. Mannitol uptake rates of MaDH4 control cells (with the YEP112A1XE plasmid) were subtracted from mannitol uptake rates of AgMaT1-expressing cells to determine the AgMaT1-dependent mannitol uptake rates at different mannitol concentrations. Uptake duration was 2 min. The results are means ±sd of one typical experiment (four replicates). The inset shows a Lineweaver-Burk plot of the uptake data.
Figure 6.
Figure 6.
Expression Pattern of AgMaT1 in Saccharomyces Cells. Total RNA was extracted, and the RNA gel blot analysis was as described by Noiraud et al. (2000). Hybridization was conducted with AgMaT1 (A) or a riboprobe (B) for calibration. Level of hybridization was analyzed though radioactivity counting with an Instant Imager (Packard Instrument Co.). Saccharomyces cells were transformed either with the empty plasmid YEP and grown on glucose or with the plasmid containing AgMaT1 and grown on either glucose or mannitol.
Figure 7.
Figure 7.
Comparison of Glucose and Mannitol Uptake by Transgenic Saccharomyces Cells. RS453 cells were grown to the early logarithmic phase in SC medium supplemented with 3% glycerol and 0.05% glucose. Uptake of glucose (squares) and mannitol (circles) was measured as described in Figure 3. Closed symbols represent uptake by cells transformed with AgMaT1, and open symbols represent mannitol uptake by control cells transformed with the plasmid pDR196. The results are from one typical experiment (four replicates per experiment). The standard deviation is presented when larger than symbols.
Figure 8.
Figure 8.
Expression Pattern of AgMaT1 in Selected Organs from Celery. (A) Data are given as percentage of maximal expression in mature leaves and are means of four independent determinations ±se. Phloem and storage parenchyma were extracted from the petioles of mature leaves. (B) Typical scan of the image obtained after radioactivity counted by the Instant Imager (AgMaT1 probe). (C) Scan of the image obtained after calibration with a riboprobe (same RNA gel blot analysis as in [B]). Total RNA was extracted, and the RNA gel blot analysis was as described by Noiraud et al. (2000). After hybridization with labeled AgMaT1 probe, the radioactivity associated with the probe bound to AgMaT1 mRNA was counted with an Instant Imager (Packard Instrument Co.). Calibration was made with a riboprobe.
Figure 9.
Figure 9.
Phylogenetic Tree for AgMaT1. Homologous sequences were obtained through the National Center for Biotechnology Information via the BLAST site (Altschul et al., 1997), alignment was performed with DNASTAR-MegAlign (Genetics Computer Group, Madison, WI), and the tree was designed with PAUP 4.0 (Sinauer Associates, Sunderland, MA). The upper left cluster includes genes from bacteria, and the cluster at right includes several glucose and myo-inositol transporters from plants, yeast, and animals. GenBank accession numbers are as follows: probable sugar transporters from sugar beet, BvPST1, U64903 and BvPST2, U64902; putative sugar transporters from Arabidopsis, AtPST1, ACOO713412; AtPST2, AC00713413; AtPST3, Z99708; AtPST4, AC006135; AtPST5, AC006234 and AtPST6, AC002335; probable metabolite transporter from Bacillus subtilis CSBC, AB005554; metabolite transport protein homolog from B. subtilis yxcC, Z99124; metabolite transport protein homolog from B. subtilis ywtG, Z92954; d-xylose proton symporter from L. brevis XYLT, AF045552; l-arabinose transporter from B. subtilis araE, Z99121H+; monosaccharide transporter from tobacco MST1, X66856; plastidic glucose transporter from Arabidopsis AtpGlcT, Af215855; plastidic glucose transporter from maize ZmpGlcT, AF251854; myo-inositol transporter 2 from S. pombe ITR2, Z95334; sodium myo-inositol transporter from rat RnSMIT, CAA04650; and Arabidopsis putative sugar transporter ERD6, D89051.
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