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

Arabidopsis ABCB21 is a Facultative Auxin Importer/Exporter Regulated by Cytoplasmic Auxin Concentration

The phytohormone auxin is critical for plant growth and many developmental processes. Members of the P-glycoprotein (PGP/ABCB) subfamily of ATP-binding cassette (ABC) transporters have been shown to function in the polar movement of auxin by transporting auxin over the plasma membrane in both monocots and dicots. Here, we characterize a new Arabidopsis member of the ABCB subfamily, ABCB21/PGP21, a close homolog of ABCB4, for which conflicting transport directionalities have been reported. ABCB21 is strongly expressed in the abaxial side of cotyledons and in junctions of lateral organs in the aerial part, whereas in roots it is specifically expressed in pericycle cells. Membrane fractionation by sucrose density gradient centrifugation followed by Western blot showed that ABCB21 is a plasma membrane-localized ABC transporter. A transport assay with Arabidopsis protoplasts suggested that ABCB21 was involved in IAA transport in an outward direction, while naphthalene acetic acid (NAA) was a less preferable substrate for ABCB21. Further functional analysis of ABCB21 using yeast import and export assays showed that ABCB21 mediates the 1-N-naphthylphthalamic acid (NPA)-sensitive translocation of auxin in an inward direction when the cytoplasmic IAA concentration is low, whereas this transporter mediates outward transport under high internal IAA. An increase in the cytoplasmic IAA concentration by pre-loading of IAA into yeast cells abolished the IAA uptake activity by ABCB21 as well as ABCB4. These findings suggest that ABCB21 functions as a facultative importer/exporter controlling auxin concentrations in plant cell

Involvement of auxin distribution in root nodule development of Lotus japonicus.

The symbiosis between legume plants and rhizobia causes the development of new organs, nodules which function as an apparatus for nitrogen fixation. In this study, the roles of auxin in nodule development in Lotus japonicus have been demonstrated using molecular genetic tools and auxin inhibitors. The expression of an auxin-reporter GH3 fused to β-glucuronidase (GUS) was analyzed in L. japonicus roots, and showed a strong signal in the central cylinder of the root, whereas upon rhizobium infection, generation of GUS signal was observed at the dividing outer cortical cells during the first nodule cell divisions. When nodules were developed to maturity, strong GUS staining was detected in vascular tissues of nodules, suggesting distinct auxin involvement in the determinate nodule development. Numbers and the development of nodules were affected by auxin transport inhibitors (1-naphthylphthalamic acid, NPA and triindobenzoic acid, TIBA), and by a newly synthesized auxin antagonist, α-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA). The common phenotypical alteration by these auxin inhibitors was the inhibition in forming lenticel which is normally developed on the nodule surface from the root outer cortex. The inhibition of lenticel formation was correlated with the inhibition of nodule vascular bundle development. These results indicate that auxin is required for the normal development of determinate nodules in a multidirectional manner