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

Nobukazu Shitan,Masataka Moriyasu,Hiroshi Omote,Kazufumi Yazaki,Yoshinori Moriyama,Akifumi Sugiyama,Shota Minami,Minaho Hayashida,Kojiro Takanashi,Masahiko Morita,Shingo Ito,Alain Goossens

doi:10.1371/journal.pone.0108789

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.

Highlights

As sessile organisms, higher plants have evolved various strategies to adapt to their environment
Phylogenetic analysis of the relationships between Nicotiana tabacum jasmonateinducible alkaloid transporter 2 (Nt-JAT2) and other plant multidrug and toxic compound extrusion (MATE) transporters showed that Nt-JAT2 belongs to clade I, with most proteins of the proteins in this clade being transporters of secondary metabolites as substrate (Figure 1)
Nt-JAT2 showed 69% amino acid similarity with the protein encoded by tomato MTP77, a gene regulated by MYB transcription factor its function has not yet been characterized [19]

Summary

Introduction

Higher plants have evolved various strategies to adapt to their environment. A group of secondary metabolites involved in defending plants against pathogens and herbivores [1], have various biological activities, with some showing strong cytotoxicity. Some of these alkaloids are used as medicines, for example as anticancer drugs and analgesics. Alkaloids usually accumulate in a particular organelle of a specific organ or are excreted from cells, with some alkaloids transported from a source to a sink organ [2] These findings suggest that plants have various transport systems, likely involving transporter proteins, which play important roles in the efficient production and accumulation of secondary metabolites

Methods

Results

Discussion

Conclusion