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. 2009 Feb;149(2):708–718. doi: 10.1104/pp.108.132811

Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots1,[C],[W]

Tsubasa Shoji 1,2, Koji Inai 1,2, Yoshiaki Yazaki 1, Yasutaka Sato 1, Hisabumi Takase 1,3, Nobukazu Shitan 1, Kazufumi Yazaki 1, Yumi Goto 1, Kiminori Toyooka 1, Ken Matsuoka 1,4, Takashi Hashimoto 1,*
PMCID: PMC2633862  PMID: 19098091

Abstract

Nicotine is a major alkaloid accumulating in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATE1 and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane. Reduced expression of the transporters rendered tobacco plants more sensitive to the application of nicotine. In contrast, overexpression of NtMATE1 in cultured tobacco cells induced strong acidification of the cytoplasm after jasmonate elicitation or after the addition of nicotine under nonelicited conditions. Expression of NtMATE1 in yeast (Saccharomyces cerevisiae) cells compromised the accumulation of exogenously supplied nicotine into the yeast cells. The results imply that these MATE-type proteins transport tobacco alkaloids from the cytosol into the vacuole in exchange for protons in alkaloid-synthesizing root cells.


Alkaloids are a chemically diverse group of low-molecular weight, nitrogen-containing secondary metabolites with characteristic toxicity and pharmacological activity and may function in the chemical defense of plants against herbivores and pathogens (Facchini, 2001; Steppuhn et al., 2004). Natural hydrophilic products, including alkaloids, are usually stored in the vacuole, which appears to be especially adapted to the bulk storage of chemicals for defensive functions. Due to its nitrogen atom(s), an alkaloid can be protonated and is a base. Because several weakly basic alkaloids, such as nicotine, are present in the lipophilic non-charged form in slightly alkaline solutions, a portion of these alkaloids in the cytoplasm may pass through the tonoplast by simple diffusion. An ion-trap mechanism has been proposed to drive an apparent uphill transport of weakly basic alkaloids against a concentration gradient, in which alkaloids are protonated in the acidic vacuole to become membrane-impermeable hydrophilic molecules (Wink and Roberts, 1998). This trapping mechanism removes transport-competent “free” molecules and thus enables the uphill transport process. As attractive as this model is, it is not known whether and how much the actual vacuolar transport of weakly basic alkaloids depends on the trapping mechanism. In contrast, other alkaloids, which are charged under cytosolic pH conditions, are thought to pass through the tonoplast via a carrier-mediated mechanism (Deus-Newmann and Zenk, 1986; Otani et al., 2005).

Nicotine is a major alkaloid synthesized in most commercial varieties of tobacco (Nicotiana tabacum). In tobacco, nicotine is synthesized exclusively in the root and distributed throughout the plant via the xylem, concentrating in the young tissues of aerial parts (Hashimoto and Yamada, 1995; Baldwin, 2001). As much as 60 mm of nicotine accumulates in the vacuoles of the leaf epidermal cells at the tip (Lochmann et al., 2001). Putrescine N-methyltransferase (PMT) catalyzes the first committed step in the nicotine-specific pathway, and a PIP-family reductase, called A622, was also suggested to function in a late step in nicotine biosynthesis (Hibi et al., 1994; Shoji et al., 2000a, 2000b; DeBoer et al., 2009; Kajikawa et al., 2009). PMT and A622 proteins are specifically expressed in the same cell types in the root (Shoji et al., 2000a, 2002). Both enzymes were abundant in the endodermis and cortex cells of the root tips, whereas in the differentiated region of the root, the outermost layer of the cortex and parenchyma cells surrounding the xylem in the vascular bundle contained these proteins. These localization patterns not only substantiated root-specific nicotine biosynthesis but also suggested nicotine synthesis to be intimately associated with the xylem-based transport.

Nicotine biosynthesis is positively regulated by the jasmonate-signaling cascade involving the COI1 F-box protein and JAZ repressors (Paschold et al., 2007; Shoji et al., 2008) and by the NIC regulatory loci that specifically control the gene expression of all enzymes known to be involved in the biosynthesis (Legg, 1984; Hibi et al., 1994; Reed and, Jelesko, 2004; Cane et al., 2005; Heim et al., 2007; Katoh et al., 2007). In flavonoid biosynthesis, regulatory genes coordinately regulate not only enzyme genes but also transporter genes responsible for intracellular transport of the metabolites (Koes et al., 2005). In this study, we identified two related tobacco transporters that are coordinately regulated by the NIC loci with nicotine biosynthetic enzymes. Our results suggest that these transporters promote the uptake of nicotine and related alkaloids into the vacuole by using a H+-gradient across the tonoplast in the alkaloid-synthesizing root cells.

RESULTS

Molecular Cloning of NtMATE1 and NtMATE2

A fluorescent differential display technique was used to comprehensively survey differences in the transcriptome between wild-type tobacco roots and nic1nic2 regulatory mutant roots. We detected more than 30,000 cDNA fragments from each genotype and obtained several cDNA clones whose transcripts were less abundant in the nic mutant roots (Supplemental Fig. S1). In addition to the PMT and A622 transcripts, we found that transcripts encoding quinolinate synthase and two closely related transporters were considerably less abundant in the mutant roots. Tobacco quinolinate synthase is the second enzyme in the de novo NAD biosynthetic pathway, which provides the pyridine moiety of nicotine (Katoh et al., 2006). The two transporters NtMATE1 and NtMATE2 (collectively called NtMATE1/2) share 96.4% amino acid sequence identity with each other and belong to the multidrug and toxin extrusion (MATE) family (Fig. 1A). Although their biochemical transporter functions are not well known, some MATE-type proteins, including NorM of Vibrio parahaemolyticus and human hMATE1, mediate the H+- or Na+-coupled export of cationic drugs in bacteria and mammalian cells (Omote et al., 2006). NtMATE1/2 is part of a MATE clade that includes Arabidopsis Transparent Testa12 (TT12; Debeaujon et al., 2001; Marinova et al., 2007) and is most closely related to an Arabidopsis (Arabidopsis thaliana) protein, At3g21690, of unknown function (Fig. 1B). Genomic DNA-blot analysis at high stringency conditions (Supplemental Fig. S2) indicated that there may be no closely related sequences other than NtMATE1 and NtMATE2 in the amphidiploid tobacco genome and that the NtMATE1/2 genes originated from the two presumed progenitor species (Nicotiana sylvestris and Nicotiana tomentosiformis) of tobacco (Fulnecek et al., 2002, and refs. therein).

Figure 1.

Figure 1.

Comparison of the NtMATE1 and NtMATE2 amino acid sequences with MATE protein sequences. A, Amino acid sequences were aligned using ClustalW. Residues identical to the NtMATE1 sequence are shaded. Twelve transmembrane domains (TM1–TM12) were predicted by the TMHMM program version 2.0 and are shown in solid lines. An N-terminal hydrophilic region (dotted line) was used as an antigen to produce NtMATE antibodies. B, Phylogenetic relationship of NtMATE1/2 with representative or closely related MATE family members. The nearest-joining method was used to create the phylogenetic tree, and TREEVIEW was used to visualize the resulting tree. The scale indicates the average number of substitutions per site for each cladogram, and the numbers give the bootstrap values of each node (1,000 bootstrap trials). AtDTX1, ALF5, TT12, EDS5, FRD3, TT12, At1g61890, and At3g21690 are from Arabidopsis, MTP77 is from tomato, NorM is from V. parahaemolyticus, and hMATE1 is from humans.

Expression Patterns

The expression of NtMATE1/2 was analyzed by RNA gel blotting, using a DNA probe that hybridized to both the NtMATE1 and NtMATE2 transcripts. The abundance of NtMATE1/2 mRNA in tobacco roots decreased in the following order: the wild type (NIC1NIC2), NIC1nic2, nic1NIC2, and nic1nic2 of the Burley 21 background (Fig. 2A). NtMATE1/2 transcripts were abundant in the root tissue, were detectable at low levels in the flowers, and were absent in the leaves and the stems in both wild-type and nic1nic2 plants (Fig. 2B). A similar suppression of NtMATE1/2 was observed in the roots of the nic1nic2 mutant with the NC95 background (data not shown). Mechanical damage to the tobacco leaves significantly increased the transcript levels of NtMATE1/2, as well as of PMT, within 4 h in the root but not in the leaf, while the general wound-responsive gene PI-II (Balandin et al., 1995) was expressed in both tissues with a distinct time course (Fig. 2C). The application of methyl jasmonate (MeJA) to tobacco plants led to similar expression patterns of these genes (Supplemental Fig. S3).

Figure 2.

Figure 2.

Expression patterns of NtMATE1/2. A to C, RNA gel-blot analysis. A, NtMATE1/2 expression in tobacco roots (cv Burley 21) with different NIC genotypes. B, Root-specific expression of NtMATE1/2 in the wild type and nic1nic2. C, Wound-induced gene expression. After tobacco leaves were wounded, expression levels of NtMATE1/2, PMT, and PI-II were monitored in the leaf and the root of wild-type plants. D to G, Histochemical GUS staining of PNtMATE1GUS transgenic tobacco seedlings. D, Five-day-old seedling. E, Root tip. F and G, Cross section (F) and longitudinal section (G) of the root in the differentiation zone.

To further characterize the cell type-specific expression, we fused the 1.1-kb 5′-flanking region of NtMATE1 to the GUS gene and introduced the transgene into tobacco plants. In transgenic seedlings, GUS activity was only detectable in the roots, with enhanced staining at the root tip (Fig. 2D). GUS staining was not observed in the root meristem, the epidermis, or the root cap (Fig. 2E). Longitudinal and cross sections showed that outer cortex cells were stained strongly (Fig. 2, F and G). MeJA treatment up-regulated the NtMATE1 promoter without affecting the spatial expression pattern (Supplemental Fig. S3). These expression patterns of NtMATE1/2 are very similar to those of nicotine biosynthetic genes (Hibi et al., 1994; Shoji et al., 2000a, 2002; Reed and, Jelesko, 2004; Cane et al., 2005; Heim et al., 2007; Katoh et al., 2007).

Subcellular Localization

The subcellular distribution of NtMATE1/2 was first examined using a GFP fused to the C terminus of NtMATE1. When NtMATE1-GFP was expressed under the control of the cauliflower mosaic virus 35S promoter in tobacco Bright Yellow-2 (BY-2) cells, GFP fluorescence was observed in endomembrane structures that were stained with the endocytic tracer dye FM4-64 (Fig. 3A), which in our conditions was mostly localized to the tonoplast (Kutsuna and Hasezawa, 2002). The plasma membrane was not stained with either NtMATE1-GFP or FM4-64. To analyze the localization of endogenous NtMATE1/2, microsomal membrane fractions from tobacco roots were separated by centrifugation on a Suc density gradient and probed with organelle-specific antibodies (Fig. 3B). Anti-NtMATE antibodies were produced against an N-terminal hydrophilic region of NtMATE1, which was mostly conserved in NtMATE2. The fractions of NtMATE1/2 coincided with those of the tonoplast-localized V-ATPase but did not match those of the plasma membrane-localized P-ATPase or the endoplasmic reticulum chaperone BiP.

Figure 3.

Figure 3.

Subcellular localization of NtMATE1/2. A, Confocal images of NtMATE1-GFP in transgenic BY-2 cells. Images for NtMATE1-GFP fluorescence (top left), FM4-64 fluorescence on the tonoplast (bottom left), and bright field (top right) are shown; a magnified image of an overlay between the fluorescence images (bottom right) corresponds to the boxed regions. A dotted line indicates the position of the plasma membrane. Note that NtMATE1-GFP and FM4-64 are not localized at the plasma membrane. B, Suc density gradient fractionation of endomembranes. A microsomal fraction from tobacco roots was separated on a Suc density gradient. Proteins were electrophoresed, blotted, and immunodetected using antibodies against NtMATE, P-ATPase (plasma membrane marker), BiP (endoplasmic reticulum marker), and V-ATPase (tonoplast marker). C and D, Immunoelectron microscopy. The presence of gold particles is indicated by arrows. Vac, Vacuole. C, Wild-type BY-2 cells treated with MeJA at 50 μm for 12 h. D, NtMATE1-overexpressing OX5 BY-2 cells.

Next, ultra-thin sections of tobacco BY-2 cells were examined by immunogold electron microscopy using purified NtMATE antibodies. In NtMATE1-overexpressing cells (see below) and MeJA-treated wild-type cells, 33% (n = 235) and 55% (n = 218) of all the gold particles were located on the tonoplast membranes (Fig. 3C), whereas only 4% (n = 136) were found on the tonoplast of untreated wild-type cells. On the tonoplast, the majority of the gold particles (171 out of 201 observed) were located on the cytoplasmic side (Fig. 3D), indicating that the N terminus of NtMATE1/2 is exposed to the cytosol.

Tobacco Roots with Reduced NtMATE1 Expression Are More Sensitive to the Application of Nicotine

To investigate the function of NtMATE1/2 in whole plants, we generated transgenic tobacco plants in which expression of NtMATE1/2 was suppressed by RNAi. RNA gel-blot analysis of the root samples showed that NtMATE1/2 transcript levels were much lower in the RNAi suppression lines (R18 and R24) than in the vector control line (Fig. 4A). Alkaloid contents (mostly nicotine and some nornicotine) of the leaf and the root were not significantly different among the vector control line and the two RNAi lines (Fig. 4B).

Figure 4.

Figure 4.

Down-regulation of NtMATE1/2 renders tobacco roots more sensitive to exogenous application of nicotine. A, RNA gel-blot analysis of tobacco plant roots. In the NtMATE1-RNAi lines (R18 and R24), transcript levels of NtMATE1 and NtMATE2 were highly reduced compared to the level in the vector-transformed control line (VC). Equal loading of total RNA was confirmed with ethidium bromide staining. B, Alkaloid contents in leaves and roots of the 8-week-old tobacco plants. The data shown are the mean values (±sd) for more than four individual plants. C, Exogenous supply of nicotine retards root growth of tobacco seedlings. Seven-day-old seedlings that had been grown in a standard culture medium were transferred to a medium containing nicotine at 2 mm and grown for an additional 7 d. Root growth after the transfer was measured. The nic1nic2 double mutants in two different genetic backgrounds (cv Burley 21 or NC95) had highly reduced levels of NtMATE1/2 (see Fig. 2) and were included in the assay. The presented data are representative of three independent experiments. The data shown are the mean values (±sd) for more than 20 seedlings. Significant differences (P < 0.01) among the control and RNAi lines were determined by one-way ANOVA followed by Tukey-Kramer test and are indicated by different letters, while those (P < 0.01) between the nic1nic2 mutant and corresponding wild type were determined by Student's t test and are indicated by asterisks. D, Tobacco seedlings grown in the nicotine-containing medium for 7 d. Arrows indicate the positions of the root tips at the time of the transfer.

To explore the possible contribution of NtMATE to alkaloid accumulation, we examined the inhibitory effect of exogenously supplied nicotine on root growth. In the presence of 2 mm nicotine, the growth of R18 and R24 roots was moderately but significantly more inhibited than the growth of the vector control roots (Fig. 4, C and D). Root growth on the nicotine-free agar medium was not significantly different among the three transgenic lines. We have been unable to generate transgenic tobacco plants in which NtMATE1 was overexpressed. These results suggest that NtMATE1/2 partly contribute to the growth of tobacco roots in the presence of high exogenous nicotine concentrations.

Overexpression of NtMATE1 Acidifies Cytoplasm upon Jasmonate Elicitation or Nicotine Addition

Because several MATE-type transporters mediate H+-coupled electroneutral organic cation exchange (Omote et al., 2006), we measured intracellular pH with the in vivo 31P-NMR spectroscopy (Blingny and Douce, 2001). We overexpressed NtMATE1 under the control of the cauliflower mosaic virus 35S promoter in cultured tobacco BY-2 cells. In other BY-2 cell lines, expression of NtMATE1/2 was suppressed by RNAi. RNA gel-blot analysis (Fig. 5A) showed that NtMATE1/2 expression was induced after treatment with MeJA for 48 h in wild-type tobacco cells. In contrast, NtMATE1 was constitutively overexpressed in three transgenic cell lines (OX1-3), whereas levels of NtMATE1/2 were consistently very low in two cell lines (RNAi1 and RNAi2) even after jasmonate elicitation.

Figure 5.

Figure 5.

MeJA elicitation or nicotine addition strongly acidifies the cytoplasm of NtMATE1-expressing tobacco cells. A, RNA gel-blot analysis of cultured tobacco BY-2 cells. When 4-d-old BY-2 cells were treated with MeJA at 100 μm in auxin-free medium for 1 d, expression of the endogenous NtMATE1/2 genes was induced. Compared to wild-type cells, NtMATE1-overexpressing cells (OX lines) had much higher levels of NtMATE1/2 transcripts, whereas NtMATE1-suppressed cells (RNAi lines) showed much lower expression levels. Gel staining with ethidium bromide confirmed equal loading of total RNA onto each lane. B, Cytoplasmic and vacuolar pH in cultured tobacco BY-2 cells. In vivo 31P-NMR spectroscopy was used to measure pH values of the cytoplasm and vacuole. Nicotine was supplied to the culture medium at 4 mm for 1 d. The data shown are the mean (±sd) of three NMR measurements of each sample. Significant differences among the lines at P < 0.05 were determined by one-way ANOVA followed by Tukey-Kramer test and are indicated by different letters. WT, Wild-type tobacco cells.

Figure 5B shows pH values in the cytoplasm and in the vacuole. In the control culture conditions, tobacco BY-2 cells of the vector control line, the three overexpression lines and the two RNAi lines all exhibited a cytosolic pH of approximately 7.6 and a vacuolar pH of approximately 5.1. Treatment with 100 μm MeJA for 1 d did not significantly affect the cytoplasmic and vacuolar pHs in the control line and the RNAi lines. However, upon jasmonate elicitation of the NtMATE1-overexpressing cells, the cytoplasmic pH decreased to 7.0 while the vacuolar pH remained the same. In the jasmonate-elicited control line, although expression of endogenous NtMATE1/2 genes was induced (at levels much lower than the NtMATE1-overexpressing lines), homeostasis of the cytoplasmic pH was maintained.

We next challenged the tobacco cells with 4 mm nicotine for 1 d and monitored the pH shifts. Like jasmonate treatment, the supply of nicotine did not significantly affect the intracellular pH values in the control and RNAi lines, whereas it markedly acidified the cytoplasm of the NtMATE1-overexpressing cells to pH 6.6 to 7.1. The vacuolar pH in the overexpressing cells remained the same as under the control conditions. These results suggest that nicotine, either synthesized de novo or supplied exogenously, triggers acidification of the cytoplasm when NtMATE1 is overexpressed.

NtMATE1 Affects Nicotine Accumulation in Yeast Cells

To examine the transport activity of NtMATE1 biochemically, we expressed the full-length NtMATE1 cDNA, alone or as a C-terminal GFP fusion, in a yeast (Saccharomyces cerevisiae) strain in which eight ATP-binding cassette transporter-related genes, including PDR5, were deleted (Decottignies et al., 1998). A previous study by Goossens et al. (2003) showed that Pdr5p is required for growth of wild-type yeast strains in the media containing high concentrations of nicotine, or tropane alkaloids (hyoscyamine or scopolamine). Fluorescence of NtMATE1-GFP was observed in not only the vacuolar membrane but also in other endomembranes and the plasma membrane (Fig. 6A). Because our nicotine uptake assay using isolated microsomal membrane vesicles gave a high background association of nicotine with the membranes (data not shown), we decided to conduct a nicotine uptake assay using intact yeast cells. In this whole cell assay, a population of NtMATE1 proteins residing in the plasma membrane is expected to contribute to the intracellular accumulation of the exogenously supplied nicotine.

Figure 6.

Figure 6.

Uptake of nicotine in NtMATE1-expressing yeast cells. A, Intracellular localization of the NtMATE1-GFP protein expressed in yeast. B, Time course of nicotine uptake into whole yeast cells. The culture medium contained nicotine at 1 mm. C, Reduction of nicotine uptake in NtMATE1- or NtMATE1-GFP-expressing yeast cells is blocked by ΔpH-disrupting drugs, NH4Cl (10 mm) and gramicidin D (5 μm). D, Uptake of hyocyamine or scopolamine (each supplied at 0.5 mm) in yeast cells and competitive inhibition of their uptake by 2.5 mm nicotine. The data shown are the mean values (±sd) for more than three separate samples. For each treatment, significant differences (P < 0.01) among empty vector, NtMATE1, and NtMATE1-GFP were determined by one-way ANOVA followed by Tukey-Kramer test and are indicated by different letters in C, while those between empty vector and NtMATE1 were determined by Student's t test and are indicated by asterisks (*P < 0.05, **P < 0.01) in D. [See online article for color version of this figure.]

When nicotine was supplied at 1 mm to a culture medium of pH 5.8, yeast cells accumulated nicotine in a time-dependent manner (Fig. 6B). The uptake of nicotine was significantly slower in the cells expressing NtMATE1 or NtMATE1-GFP by approximately 20% to 25% than in the control cells. This difference in accumulation suggests that NtMATE1 located in the plasma membrane transports the cytosolic nicotine out to the culture medium, thereby antagonizing the NtMATE1-independent uptake of nicotine into the yeast cells. In the presence of 10 mm NH4Cl or 5 μm gramicidin D, both of which disrupt pH gradients, the uptake of nicotine was not significantly different among the three yeast strains (Fig. 6C). To investigate the substrate specificity of NtMATE1-mediated transport, we tested two tropane alkaloids (hyoscyamine and scopolamine) at 0.5 mm in the absence or presence of 2.5 mm nicotine (Fig. 6D). These alkaloids, especially hyoscyamine, were taken up by the NtMATE1-expressing yeast cells more slowly than the control cells. Simultaneously supplied nicotine effectively competed against the NtMATE1-mediated transport of these alkaloids, indicating that NtMATE1 is capable of transporting nicotine, hyoscyamine, and scopolamine.

DISCUSSION

MATE Transporters in Plants

Evidence presented in this article implicates tonoplast-localized tobacco MATE transporters in the transport of tobacco alkaloids. The MATE transporter is the most recently defined member of the five multidrug transporter super families and possesses 12 putative transmembrane segments. The functional characterization of limited members of the MATE family showed that these proteins transport diverse low-molecular weight compounds, ranging from cationic dyes to aminoglycosides, and use drug/H+ or drug/Na+ antiport to energize drug efflux (Omote et al., 2006).

Plants are particularly abundant in the MATE transporters. Of 58 MATE family paralogs in Arabidopsis, five have been characterized based on mutant phenotypes. The tt12 mutant lacks proanthocyanidin deposition in vacuoles of the seed coat endothelium (Debeaujon et al., 2001). Transport assays using yeast vesicles suggest that TT12 is a vacuolar flavonoid/H+ antiporter (Marinova et al., 2007). The ion deficiency in the leaf cells of the frd3 mutant and transport studies using Xenopus oocytes support that FRD3 effluxes citrate, a potential iron chelator, into the root vasculature (Durrett et al., 2007). A T-DNA knockdown mutant of the FRD3-related AtMATE gene is deficient in aluminum-activated root citrate exudation (Liu et al., 2008). Lateral root formation in the alf5 mutant is highly sensitive to a soluble contaminant present in commercial Bacto agar, and expression of the ALF5 MATE efflux transporter in yeast confers resistance to the toxic cation tetramethylammonium (Diener et al., 2001). The eds5 mutant defective in a MATE transporter accumulates very little salicylic acid and is hyper-susceptible to pathogens (Nawrath et al., 2002). The nature of the substances transported by ALF5 and EDS5 remains unknown. Functional expression of an Arabidopsis MATE protein AtDTX1 in a bacterial mutant deficient in multidrug resistance indicates that AtDTX1 drives the efflux of positively charged plant alkaloids, antibiotics, Cd2+, and other toxic compounds from the cytoplasm, possibly as a proton-dependent antiporter (Li et al., 2002).

NtMATE1/2 belongs to a subgroup that includes Arabidopsis tonoplast-localized TT12 (Marinova et al., 2007) and a tomato (Solanum lycopersicum) MATE protein MTP77 (Fig. 1B). MTP77 is positively regulated by a transcriptional regulator of anthocyanin biosynthesis and thus is implicated in the transport of anthocyanins into the vacuole (Mathews et al., 2003). Proteome analysis of purified Arabidopsis vacuoles identified two uncharacterized MATE proteins, At1g61890 and At3g21690, which are included in this subgroup (Shimaoka et al., 2004). We speculate that this NtMATE1/2-containing clade may represent a tonoplast-localized MATE subgroup.

Possible Biochemical Function of NtMATE1/2

Immunoelectron microscopy indicated that the N-terminal portions of NtMATE1/2 face the cytoplasm. This topology of NtMATE1/2 is consistent with the suspected topology of other MATE proteins (Omote et al., 2006). As suggested for the tonoplast-localized TT12 (Marinova et al., 2007), NtMATE1/2 may use a transmembrane H+-gradient established between the vacuolar lumen and the cytoplasm to drive vacuolar uptake of their in vivo substrates. Because the PMT and NtMATE1/2 genes are expressed in a highly overlapping pattern in the tobacco root, are induced by exogenous jasmonates, and are positively regulated by the regulatory loci NIC, these tobacco transporters likely function in close association with nicotine biosynthesis. Although Kidd et al. (2006) reported that only a small subset of NIC-regulated genes are involved in nicotine biosynthesis, our extensive expression analyses of NIC-regulated genes in two independent genetic backgrounds (Burley 21 and NC95) support that NIC1 and NIC2 specifically regulate nicotine accumulation in tobacco (Hibi et al., 1994; Katoh et al., 2007; this study; M. Kajikawa and T. Hashimoto, unpublished data).

Uptake experiments performed with transformed yeasts expressing NtMATE1 demonstrated that NtMATE1 transports nicotine. The values of cytoplasmic pH in yeast vary depending on the growth conditions but are in the range of 6.4 to 7.1 (Shanks and Bailey, 1990). Because the pKa value between the monoprotonated form and nonionized form of nicotine is 7.84 (Nair et al., 1997), a majority of the nicotine molecules in the yeast cytoplasm should be in the monoprotonated form. The strong inhibitory effect of NH4Cl and gramicidin D indicates that the NtMATE1-catalyzed transport of nicotine is dependent on H+. In the NtMATE1-overexpressing tobacco cells, strong acidification of the cytoplasm was observed after jasmonate elicitation (which induced biosynthesis of anatabine and some pyridine alkaloids) and after the exogenous supply of nicotine. These in vivo results support that the tonoplast-localized NtMATE1 transports nicotine and other tobacco alkaloids into the vacuole in exchange for H+. At a cytoplasmic pH of around 7.5 in the tobacco cells, a significant proportion of the nicotine molecules should be monoprotonated, the form presumably transported by NtMATE1.

Proton-dependent antiport activity of MATE-family transporters has been shown for AtDTX1 (Li et al., 2002), FRD3 (Durrett et al., 2007), TT12 (Marinova et al., 2007), and hMATE1 (Otsuka et al., 2005). Nicotine and several other unrelated compounds inhibited a transport function of the human organic cation transporter hMATE1 in a dose-dependent manner, suggesting that nicotine can be transported by this MATE protein (Otsuka et al., 2005). Our assay using yeast cells also suggests that NtMATE1 transports two tropane alkaloids (hyoscyamine and scopolamine) besides nicotine. From our studies, it cannot be excluded that NtMATE1/2 might additionally transport nonalkaloid compounds, especially those induced by jasmonate treatment.

Possible Physiological Functions of NtMATE1/2

Our functional analysis of NtMATE1/2 is mostly based on overexpression in cultured tobacco cells and yeast cells. Although the NtMATE1/2-knockdown tobacco plants clearly exhibited increased sensitivity to exogenously supplied nicotine, they did not show obvious changes in the root-to-shoot translocation or synthesis of nicotine. It is possible that nicotine distribution between the cytoplasm and the vacuole is affected in the nicotine-synthesizing root cells in the absence of NtMATE1/2, but an in situ chemical analysis of tobacco alkaloids in subcellular compartments is technically very difficult. Alternatively, there might be other tobacco transporters that contribute to the vacuolar accumulation of nicotine in the root cells. Such transporters could be other members of the tobacco MATE family or might belong to other transporter families. Functional redundancy of nicotine transporters may underlie the observed subtle phenotype of NtMATE1/2-down-regulated plants.

What might be the physiological functions of NtMATE1/2 in the tobacco root cells? Because the expression of their genes is tightly coordinated with nicotine biosynthesis, these transporters are likely to function in the vacuolar sequestration of newly synthesized nicotine. A few millimolar concentrations of nicotine in the culture medium retard the root growth of tobacco seedlings, indicating that this alkaloid is toxic to root cells at relatively high concentrations. Tobacco roots actively synthesize nicotine as the plants mature, especially after the decapitation of flower heads and young leaves, a common practice among tobacco farmers, and the nicotine content under inductive conditions can reach 5% of leaf dry weight in some cultivated varieties (Tso, 1972). At a high rate of synthesis, nicotine may accumulate at a considerable concentration in the cytoplasm of the root cells before being transported to the aerial parts via the xylem. In the xylem sap of leaf-damaged N. sylvestris plants, as much as 1 mm nicotine was observed (Baldwin, 1989). Vacuolar sequestration of nicotine may then be necessary to protect the nicotine-synthesizing root cells from potential cytotoxicity. A transporter-independent ion-trap mechanism may not be sufficient to sequester nicotine efficiently into the vacuole. It has been reported that the incubation of tobacco roots with low millimolar concentrations of nicotine, at the time of decapitation, dramatically reduced the activities of PMT and N-methylputrescine oxidase (Mizusaki et al., 1973). Although the mechanisms by which nicotine inhibits the activities of these enzymes are not known, maintaining low cytoplasmic concentrations of nicotine may be necessary to ensure its active synthesis.

While NtMATE1/2 transporters likely function in association with nicotine synthesis in the root, there may be other transporters that facilitate sequestration of tobacco alkaloids into the vacuole in the aerial parts of tobacco plants. Tobacco transporters that may promote the xylem-loading of nicotine in the root cells for the root-to-shoot translocation are yet to be identified. The intra- and intercellular transport, as well as the long-distance translocation, of tobacco alkaloids should be an interesting area of alkaloid biology.

MATERIALS AND METHODS

Plant Materials

Sterile plants of tobacco (Nicotiana tabacum) were grown for 4 weeks and treated with MeJA (Shoji et al., 2000a) or wounded by mechanical damage to a leaf (Cane et al., 2005). The cv Burley 21 was mostly used for analyses of nontransgenic plants unless otherwise indicated, whereas the cv Petit Havana line SR1 was used for transformation. The tobacco BY-2 cell suspension was cultured as described (Nagata et al., 1992). To induce alkaloid biosynthesis, 4-d-old BY-2 cells were first rinsed to remove 2,4-dichlorophenoxyacetic acid and then transferred to fresh auxin-free medium supplemented with MeJA at specific concentrations. All samples were immediately frozen with liquid nitrogen and kept at −80°C prior to use.

Cloning of NtMATE1/2 cDNAs

Fluorescent differential display was performed basically as described (Hayama et al., 2002) using total RNA from tobacco roots (cv Burley 21) of the wild type and nic1nic2. Amplified cDNA fragments were detected with FMBIO II Multi-View (Takara). Candidate fragments were excised from the gel, reamplified by PCR, separated on agarose gels containing HA-yellow or HA-Red (Takara), and then subcloned into pGEM-T (Promega) for subsequent sequencing. Full-length cDNA clones were isolated by using a SMART RACE kit (Clontech) and a tobacco root cDNA library (Hibi et al., 1994).

Analysis of Gene Expression

The RNA gel-blot analysis was done as described (Shoji et al., 2000a). The coding region of NtMATE1 cDNA was produced by PCR and used as an NtMATE1/2 probe. cDNAs for tobacco PI-II (Balandin et al., 1995) and tobacco PMT (Hibi et al., 1994) were also used as hybridization probes.

Vector Construction and Transformation

The 5′-upstream sequence of NtMATE1 was cloned from tobacco cv Burley 21 genomic DNA by a TAIL-PCR method (Liu et al., 1995). A promoter region from −1,100 bp to +3 bp (with the first adenine of the translational start Met as +1) of NtMATE1 was flanked with SalI and NcoI sites by PCR and fused in-frame with the GUS coding sequence in pLM9 (Mlynarova et al., 1995). Histochemical analysis of GUS activity was done as described (Shoji et al., 2000b).

For expression of NtMATE1-GFP, SalI and NcoI sites were added to the NtMATE1 coding region by PCR and cloned into pAVA393 (von Arnim et al., 1998). To overexpress NtMATE1, SalI and XbaI sites were added by PCR, and the GFP sequence was replaced in pAVA319 (von Arnim et al., 1998). The expression cassettes in the pAVA vectors were then transferred to pBIN19 (Frisch et al., 1995). The gene-silencing construct was made by subcloning a NtMATE1 cDNA fragment (+1,434 bp to +1,739 bp) into pHANNIBAL (Wesley et al., 2001) and then replacing the GUS fragment of pBI121 (Clontech) with the insert.

Transgenic tobacco plants were produced by using Agrobacterium tumefaciens strain LBA4404 and a leaf disc protocol (Horsch et al., 1985). Transgenic plants of the T1 generation were analyzed and the non-transgenic T1 progeny were excluded from the analysis. Tobacco BY-2 cells were transformed as described (An, 1985) by using A. tumefaciens strain EHA105.

Subcellular Localization Analysis and Antibodies

To label the tonoplast, NtMATE1-GFP-expressing tobacco BY-2 cells were incubated with FM4-64 (Molecular Probes) at 32 μm for 12 h and examined with a Zeiss LSM510 confocal microscope as described (Kutsuna and Hasezawa, 2002).

Suc density gradient fractionation of endomembranes was done essentially as described (Matsuoka et al., 1997) using the root of tobacco cv Petit Havana line SR1. Immunoblot analysis was performed with Hybond-P membrane and the ECL-plus kit (Amersham Biosciences) according to the manufacturer's instructions.

A glutathione S-transferase (GST) fused to the N-terminal 40 amino acid residues of NtMATE1 was produced in Escherichia coli by using pGEX6P-1 (Amersham Biosciences), purified, and used as an antigen. The IgG fraction was obtained from crude rabbit antiserum by using protein A Sepharose beads, passed through the GST-Sepharose column, and affinity-purified by binding to recombinant GST-NtMATE1 protein blotted on a Hybond P membrane. Standard procedures (Lieberherr et al., 2005) and the manufacturer's protocol (Amersham Biosciences) were used for purification. Antisera against P-ATPase (Kobae et al., 2004), V-ATPase (Kobae et al., 2004), and BiP (Höfte and Chrispeels, 1992) were used, respectively, as markers for the plasma membrane, tonoplast, and endoplasmic reticulum.

Alkaloid Analysis

A dry sample of 10 mg was homogenized and soaked in 1 mL of 0.1 n H2SO4. After sonication and centrifugation, 1 mL of the supernatant was mixed with 0.1 mL of 25% NH4OH, loaded onto an Extrelut-1 column (Merck), and eluted with 6 mL of chloroform. The extract was dried at 37°C and dissolved in ethanol containing 0.1% (v/v) dodecane. Tobacco alkaloids were separated and quantified by gas-liquid chromatography (GC-14A, Shimadzu) with a Rtx-5 Amine capillary column (Restek) by using a thermal gradient: 100°C for 10 min; 25°C/min to 150°C; 1°C/min to 170°C; and 30°C/min to 300°C.

Immunoelectron Microscopy

Tobacco BY-2 cells were frozen in an EM Pact high-pressure freezer (Leica) and fixed with anhydrous acetone containing 1% glutaraldehyde and 1% OsO4 at −80°C. The frozen samples were embedded in LR White resin as described (Toyooka et al., 2006). Ultra-thin sections were treated as described (Follet-Gueye et al., 2003) and were first labeled with an affinity-purified NtMATE1 antibody (1:50) in Tris-buffered saline and then with 18-nm colloidal gold particles coupled to goat anti-rabbit IgG. The sections were stained with uranyl acetate and examined with a 1010EX transmission electron microscope (JEOL) at 80 kV as described (Toyooka et al., 2006).

Measurement of Intracellular pH

Cytosolic and vacuolar pH values were determined in suspension-cultured tobacco BY-2 cells by an in vivo 31P-NMR method, as described previously (Qiao et al., 2002). Four-day-old tobacco BY-2 cells were transferred to a fresh medium and cultured for 1 d before measurement. Nicotine was added to a final concentration of 5 mm, while MeJA was added at 100 μm to a medium lacking 2,4-dichlorophenoxyacetic acid.

In Vitro Transport Experiments in Yeast

The NtMATE1-coding sequence and its C-terminal fusion with GFP were subcloned into the multicloning site of a yeast (Saccharomyces cerevisiae) expression vector pDR196 (Rentsch et al., 1995) to give pDR-NtMATE1 and pDR-NtMATE1-GFP, respectively. The resulting plasmids were introduced into a yeast strain AD12345678 (yor1Δ, snq2Δ, pdr5Δ, pdr10Δ, ycf1Δ, pdr3Δ, pdr15Δ; Decottignies et al., 1998) by the lithium acetate method (Ito et al., 1983). Yeasts were grown in synthetic defined medium (lacking uracil) with vigorous shaking at 30°C. When the optical density at 600 nm of the culture reached 1.0, yeasts were collected by centrifugation, suspended in the same volume of half-strength synthetic defined medium (lacking uracil) supplemented with test chemicals, and cultured for specific periods. Nicotine, hyoscyamine, scopolamine, NH4Cl, and gramicidin D were added at final concentrations of 1 mm, 0.5 mm, 0.5 mm, 10 mm, and 5 μm, respectively. The cells were collected by centrifugation and washed with ice-cold water twice to carefully remove extracellular alkaloids. Alkaloids in the yeast cells were extracted into a two-fold volume of 50% ethanol, 49.5% methanol, and 0.5% acetate (v/v/v) by vigorous vortexing with acid-washed glass beads (Sigma). High accumulation of exogenously supplied alkaloids in yeast cells enabled us to use this simplified purification method for alkaloid quantification. Nicotine, hyoscyamine, and scopolamine were measured with gas-liquid chromatography as described above. To localize NtMATE-GFP in yeast cells, fluorescence images were captured with a Zeiss LCM5 PASCAL confocal microscope.

Sequence data from this article can be found in the GenBank database under accession numbers AB286961 to AB286963.

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure S1. cDNA differential display analysis of tobacco roots.

  • Supplemental Figure S2. Genomic DNA-blot analysis of NtMATE1/2-related sequences in N. tabacum and its progenitor species.

  • Supplemental Figure S3. Induction of NtMATE1/2 expression by MeJA.

Supplementary Material

[Supplemental Data]
pp.108.132811_index.html (1.1KB, html)

Acknowledgments

We thank Masayoshi Maeshima (Nagoya University) and Maarten Chrispeels (University of California, San Diego) for antibodies; John Hamill (Monash University) for the nic double mutant with the NC95 background; Wolfgang Frommer (Carnegie Institution, Stanford) for pDR196; M.A. DeWaard (Wageningen University) for the yeast strain AD12345678; and Kumi Yoshida (Nagoya University), Tetsuro Mimura (Kobe University), and Shinobu Satoh (Tsukuba University) for helpful discussions.

1

This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (RFTF program 00L01605 and Grant-in-Aid for Scientific Research on Priority Areas 17051022 to T.H. and Grant-in-Aid for Scientific Research on Priority Areas 17078009 to K.M.).

The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Takashi Hashimoto (hasimoto@bs.naist.jp).

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Some figures in this article are displayed in color online but in black and white in the print edition.

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The online version of this article contains Web-only data.

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