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

Effects of anesthesia and surgery on U (crit) performance and MO2 in chum salmon, Oncorhynchus keta

Telemetry is a useful technique for elucidating salmon behavior, but the recovery periods before fish can be safely released after the attachment of telemetry devices have not yet been established. Reported recovery times vary widely, from 2 h to 13 days. We examined how anesthesia and surgery to attach external electromyogram (EMG) transmitters affected chum salmon (Oncorhynchus keta) recovery based on three physiological parameters. Fish subjected to anesthesia plus EMG transmitter attachment (EMG group), anesthesia only (AO group), and no handling (control) were placed in a swim tunnel. Critical swimming speed (U (crit)), oxygen consumption (MO2), and muscle activity (EMG values) were assessed 0, 1, 6, 12, 24, and 30 h after treatment. The MO2 in the EMG and AO groups was higher than in the control group 1 h after treatment, but did not differ significantly from the control in all subsequent trials (from 6 to 30 h after treatment). Values for U (crit) and EMG were not significantly different from the control group in any of the trials conducted 1-30 h after treatment. We concluded that chum salmon had regained their normal swimming ability by 6 h after treatment and could be safely released into the natural environment

Comparison of the swimming ability and upstream-migration behavior between chum salmon and masu salmon

The spawning ground of chum salmon (Oncorhynchus keta) is usually located farther downriver than that of masu salmon (Oncorhynchus masou) in Hokkaido, Japan. To compare the swimming abilities of these two species, the relationship between swimming speed and oxygen consumption was compared using a swim tunnel in the laboratory. Then, the upstream-migration behaviors of chum salmon and masu salmon were compared using electromyogram telemetry at fish passages in the Toyohira River, Hokkaido. In the laboratory study, the standard metabolic rate of masu salmon was lower and the critical swimming speed (U-crit) was faster than those of chum salmon. In the field study, the holding time needed to recover the swimming performance exceeding U-crit at the fish passages and the trial number needed to pass the fish passages were significantly lower for masu salmon than chum salmon. These results revealed that masu salmon are more adaptable to extended swimming in high water velocity conditions than chum salmon and that masu salmon are better equipped for a long distance upstream migration to their spawning ground than chum salmon

Interaction between patch area and shape: lakes with different formation processes have contrasting area and shape effects on macrophyte diversity

Although both patch area and shape are key factors driving biodiversity in fragmented terrestrial landscapes, researchers have had limited and mixed success in documenting the effects of these two factors on aquatic ecosystems. Here we examined the effects of lake area and shape on macrophyte species richness in a lowland floodplain by considering the differences in lake types (i.e. marsh, oxbow, man-made lakes). We surveyed species richness of native macrophytes in 35 lakes including 11 marshes, 11 oxbows and 13 man-made lakes with various complex shapes covering from 0.25 to 46.3 ha. Model selection clearly supported the existence of interaction between area and shape effects: large-circular and small-complex lakes supported a higher macrophyte species richness, while it was lower in large-complex and small-circular lakes. Among the three lake types, marsh lakes were more circular and man-made lakes had more complex shapes, while oxbow lakes were intermediate between these two. Also, marsh lakes had positive species-area relationships, while man-made lakes had negative relationships. Our results suggest the opposing shape complexity and species-area relationships of these two contrasting lake types are the result of the interactions between lake area and shape. These results indicate that different lake types result in variations in their conservation value for preserving macrophyte diversity. We suggest that small complex-shaped patches (especially oxbow lakes), which are often given the lowest conservation priority in terrestrial ecosystems, cannot be disregarded when conserving macrophyte biodiversity in aquatic ecosystems

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

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