title: Involvement of the leaf-specific multidrug and toxic compound extrusion (MATE) transporter Nt-JAT2 in vacuolar sequestration of nicotine in Nicotiana tabacum.
abstract: Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues.
authors.affiliation: Department of Natural Medicinal Chemistry, Kobe Pharmaceutical University, Kobe, Hyogo, Japan; Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Kyoto, Japan.
authors.forename: Nobukazu
authors.initials: N
authors.lastname: Shitan
authors.affiliation: Department of Natural Medicinal Chemistry, Kobe Pharmaceutical University, Kobe, Hyogo, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Minami
authors.affiliation: Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Kyoto, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Morita
authors.affiliation: Department of Natural Medicinal Chemistry, Kobe Pharmaceutical University, Kobe, Hyogo, Japan.
authors.forename: Minaho
authors.initials: M
authors.lastname: Hayashida
authors.affiliation: Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Kyoto, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Ito
authors.affiliation: Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Kyoto, Japan.
authors.forename: Kojiro
authors.initials: K
authors.lastname: Takanashi
authors.affiliation: Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Omote
authors.affiliation: Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Moriyama
authors.affiliation: Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Kyoto, Japan.
authors.forename: Akifumi
authors.initials: A
authors.lastname: Sugiyama
authors.affiliation: Department of Plant Systems Biology, VIB, Gent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent University, Gent, Belgium.
authors.forename: Alain
authors.initials: A
authors.lastname: Goossens
authors.affiliation: Department of Natural Medicinal Chemistry, Kobe Pharmaceutical University, Kobe, Hyogo, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Moriyasu
authors.affiliation: Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Kyoto, Japan.
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Yazaki
mesh_terms: D000085:Acetates / Q000494:pharmacology; D000470:Alkaloids / Q000378:metabolism; D000691:Anabasine / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003517:Cyclopentanes / Q000494:pharmacology; D005419:Flavonoids / Q000378:metabolism; D020869:Gene Expression Profiling; D009538:Nicotine / Q000378:metabolism* / Q000494:pharmacology; D054883:Oxylipins / Q000494:pharmacology; D010802:Phylogeny; D059828:Plant Cells / Q000187:drug effects / Q000382:microbiology; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects; D014617:Vacuoles / Q000378:metabolism
chemical_list: D000085:Acetates; D000470:Alkaloids; D003517:Cyclopentanes; D005419:Flavonoids; D054883:Oxylipins; D010940:Plant Proteins; D011725:Pyridines; D012333:RNA, Messenger; C012737:anatabine; D009538:Nicotine; C072239:methyl jasmonate; D000691:Anabasine