Involvement of the leaf-specific multidrug and toxic compound extrusion (MATE) transporter Nt-JAT2 in vacuolar sequestration of nicotine in Nicotiana tabacum

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

Nicotine transport activity of Nt-JAT2.

<p>(A) Time course analysis of nicotine transport in Nt-JAT2-expressing yeast. Control (dashed line) and Nt-JAT2-expressing (solid line) yeast cells were incubated in half-strength SD medium supplemented with 1 mM nicotine and sampled at the times indicated. Results are mean ± SD of triplicates. *<i>P</i><0.05; **<i>P</i><0.01 compared with control by Student’s t-test. (B) Localization of Nt-JAT2–GFP in yeast cells. Yeast cells expressing Nt-JAT2–GFP were grown at 30°C to logarithmic growth phase and observed by fluorescence microscopy. (i) Fluorescence of yeast cells transformed with Nt-JAT2–GFP; (ii) bright-field contrast (scale bar, 5 µm). (C) Nicotine content in control (white bar), Nt-JAT1-expressing (gray bar) and Nt-JAT2 -expressing (black bar) yeast cells incubated in half-strength SD medium containing 0.5 mM nicotine for 6 h. Results are mean ± SD of triplicates. *<i>P</i><0.01 compared with control by Student’s t-test.</p

Subcellular localization of Nt-JAT2 in cultured tobacco cells.

<p>(A) Confocal images of Nt-JAT2-GFP in transgenic BY-2 cells. (left) Nt-JAT2-GFP fluorescence images, (right) bright field images, (bottom left) merged images. (B) Nt-JAT2-GFP fluorescence (green, top left), FM4-64 fluorescence (red, bottom left), merged (bottom right), and bright field (top right) images after treatment for 24 h (scale bar, 2 µm.).</p

Expression of Nt-JAT2 in tobacco plants.

<p>(A) Organ-specific expression of <i>Nt-JAT2</i> mRNA in tobacco plants. Total RNA (7.5 µg) prepared from each tobacco organ was probed with ³²P-labeled <i>Nt-JAT2</i> fragment (0.5 kb) (top). The amount of total RNA applied to each lane is shown by EtBr-stained 18S rRNA (bottom). For comparison between NtJAT1 and NtJAT2 expression, analysis was performed using the same membrane as our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108789#pone.0108789-Morita1" target="_blank">[15]</a>. (B, C) Immunoblot analysis of Nt-JAT2 and Nt-JAT1 proteins in control (B) and MeJA treated (C) plants. Microsomes from tobacco leaves, stems and roots were subjected to electrophoresis, blotted, and incubated with antibody to Nt-JAT2 or Nt-JAT1. L, leaves (Leaves were numbered from top to bottom).</p

MeJA induction of <i>Nt-JAT2</i> mRNA expression in tobacco seedlings.

<p>(A) <i>Nt-JAT2</i> expression in response to various treatments. Fourteen-day-old seedlings were treated for 24 h with 10 µM 1-naphthaleneacetic acid (NAA), 10 µM IAA, 10 µM 6-benzyladenine (BA), 10 µM abscisic acid (ABA), 10 µM gibberellic acid (GA₃), 5 µM brassinolide (BL), 100 µM MeJA, 100 µM salicylic acid (SA), or 100 µM sclareol (SC), at 4°C (cold)/low light, dark (dark), and drought (dry) conditions. Cont., untreated control. Total RNA (7.5 µg) prepared from the aerial parts of seedlings was probed with a ³²P-labeled <i>Nt-JAT2</i> fragment (top). Loading controls are shown as EtBr-stained 18S rRNA (bottom). (B) RNA gel blot analysis of <i>Nt-JAT2</i> in tobacco seedlings. Seedlings were harvested 0 to 72 h after MeJA treatment. Total RNA (7.5 µg) was probed with a ³²P-labeled <i>Nt-JAT2</i> fragment (0.5 kb) (top). Loading control is shown as EtBr-stained 18S rRNA (bottom). For comparison between NtJAT1 and NtJAT2, expression analyses were performed using the same membrane as our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108789#pone.0108789-Morita1" target="_blank">[15]</a>.</p