Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots

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.