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

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

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

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

Subcellular localization.

<p>(A) Total microsomes from super roots were fractionated on a discontinuous sucrose gradient consisting of 20%, 30%, 40%, and 50% (w/v) sucrose. Membrane fractions were collected from the interfaces between different sucrose concentrations and analyzed via SDS-PAGE and western blotting. Blots were probed with antisera to <i>LjABCG1</i>, PM H⁺-ATPase (W1C), tonoplast and endomembrane H⁺-PPase (AVP1), and endoplasmic reticulum BiP (BiP). (B) Two-phase separation of plasma membrane. Microsomal membranes (M) from super roots were fractionated by the aqueous two-phase portioning method, with plasma membranes enriched in the upper phase (U) and other intracellular membranes remaining in the lower phase (L). Proteins from each fraction were blotted and probed with antisera to <i>LjABCG1</i>, PM H⁺-ATPase (W1C), tonoplast and endomembrane H⁺-PPase (AVP1), and endoplasmic reticulum BiP (BiP).</p

Phylogenetic analysis of plant full-size ABCG transporters.

<p>A phylogenetic tree was generated using MEGA 5.0 software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139127#pone.0139127.ref053" target="_blank">53</a>]. The amino acid sequences of <i>LjABCG1</i>, Arabidopsis full-size ABCG transporters and reported full-size ABCG transporters were aligned using the ClustalW program. The Neighbor-joining method was then used to construct a phylogenetic tree with 1,000 bootstrap replications. Boot strap values (max 100) are shown at nodes generating clades. Accession numbers and AGI codes are: ABCG29, At3g16340.1; ABCG30, At4g15230.1; ABCG31, At2g29940.1; ABCG32, At2g26910.1; ABCG33, At2g37280.1; ABCG34, At2g36380.1; ABCG35, At1g15210.1; ABCG36, At1g59870.1; ABCG37, At3g53480.1; ABCG38, At3g30842.1; ABCG39, At1g66950.1; ABCG40, At1g15520.1; GmPDR12, NP_001237697; LR34, ADK62371; MtABCG10, TC184736; OsPDR9, AAQ02685; PhPDR1, AFA43816.1; ScPDR5, CAA99359; StPDR1, AEO22187; StPDR2, AEB65936; StPDR3, AEO22188; StPDR4, AEO22189. At, <i>Arabidopsis thaliana</i>; Gm, <i>Glycine max</i>; Lj, <i>Lotus japonicus</i>; Mt, <i>Medicago truncatula</i>; Nt, <i>Nicotiana tabacum</i>; Np, <i>Nicotiana plumbaginifolia</i>; Os, <i>Oriza sativa</i>; Ph, <i>Petunia hybrida</i>; Sc, <i>Saccharomyces cerevisiae</i>; Sp, <i>Spirodela polyrhiza</i>; St, <i>Solanum tuberosum</i>.</p

Expression profile of <i>LjABCG1</i> in <i>L</i>. <i>japonicus</i>.

<p>(A) Expression of <i>LjABCG</i>1 in various tissues. Leaves, stems and inoculated roots were obtained from plants inoculated with <i>M</i>. <i>loti</i>. (B) Response of <i>LjABCG1</i> expression to various treatments. Two-week-old seedlings were treated for 24 h with 10 μM indole-3-acetic acid (IAA), 10 μM 1-naphthaleneacetic acid (NAA), 10 μM abscisic acid (ABA), 10 μM gibberellic acid (GA₃), 100 μM salicylic acid (SA), 100 μM methyl jasmonate (JA), or 100 μM sclareol (Scl). There chemicals were applied into the medium at the final concentration described above. Significant difference was based on one-way ANOVA with Tukey's HSD test. Asterisks indicate significant differences from control (p<0.05). (C) Time course of expression of <i>LjABCG1</i> after treatment with 100 μM MeJA. (D) Response of <i>LjABCG1</i> expression to MeJA applied to shoots. A cotton ball containing MeJA was placed onto the wall of an air-tight plant box. After 24 hours, shoots and roots were frozen in liquid nitrogen until RNA preparation. Data represent the means ± S.D. of three replicates.</p

GUS staining of <i>ProLjABCG1</i>:<i>GUS</i> transformants infected with <i>M</i>. <i>loti</i>.

<p>The transgenic hairy roots were stained with X-Gluc for 24 hours. (A, B) GUS activity in (A) roots and (B) lateral roots. (C) Longitudinal section of a root. (D) Cross-section of a root. (E) Root with a nodule and lateral root primordia. (F-H) Nodules of different stages from F (young) to H (mature). (I) Section of a nodule. Bars = 500 μm (A, B, E), 100 μm (C, F-I), and 50 μm (D).</p

Arabidopsis plants inoculated with <i>P</i>. <i>infestans</i>.

<p>Leaves stained with trypan blue were observed under a microscope 24 h after inoculation with <i>P</i>. <i>infestans</i>; (A) Col-0, (B) <i>pdr8-1</i>, (C) <i>LjABCG1/pdr8-1</i>. (D) Beneath image of C. (A-D) SP, spore; AP, appressorium; IH, infection hyphae. Bars = 20 μm. (E) Frequency of penetration of <i>P</i>. <i>infestans</i> into leaf epidermal cells. Different letters indicate significant differences (p<0.05) in one-way ANOVA with Tukey's HSD test.</p