Expression and functional characterization of plant pleiotropic drug resistance transporters in heterologous and homologous hosts

ABC transporters mediate ATP-dependent translocation of a wide range of structurally and functionally unrelated compounds across biological membranes. In our laboratory, we have identified plasma membrane ABC transporters belonging to the pleiotropic drug resistance (PDR) subfamily in the plant species Nicotiana plumbaginifolia and Nicotiana tabacum. Analysis of transgenic plants silenced for NpPDR1 and NtPDR3 expression indicated that they are involved in the plant response to fungal attack and to iron deficiency, respectively. However, identification of their substrates is required to fully understand their physiological roles. For that purpose, this work aimed at setting up a convenient expression system for plant PDR transporters. First, NpPDR1 and NtPDR3 were expressed in a heterologous system, the yeast Saccharomyces cerevisiae. Even though they did not properly localize to the plasma membrane, phenotypic characterization indicated that these plant PDR transporters are functional in yeast. Growth tests in the presence of toxic concentrations of putative substrates showed that NpPDR1 probably transports antifungal diterpenes while NtPDR3 seems to transport molecules structurally related to phenolic compounds that help to solubilize iron in the soil. Second, NtPDR3 expression in N. tabacum BY2 suspension cells resulted in its localization to the plasma membrane. Growth tests and direct transport assays confirmed phenolic compounds as substrates. Finally, expression of a 10-His tagged NtPDR3 led to its solubilization and partial purification by Ni-chromatography. Altogether, these data allowed us to identify NtPDR3 substrates and indicate that N. tabacum BY2 suspension cells are an interesting expression system for transport assays of plant PDR and possibly other ABC transporters.(AGRO 3) -- UCL, 201

Plant ABC transporters: time for biochemistry?

ATP-binding cassette (ABC) proteins form a large and ubiquitous family, most members of which are membrane-associated primary transporters. Plant genomes code for a particularly large number of these ABC proteins, with more than 120 genes present in both Arabidopsis thaliana and Oryza sativa (rice). Although plant ABC transporters were initially identified as detoxifiers, sequestering xenobitotics into the vacuole, they were later found to be involved in a wide range of essential physiological processes. Currently, the exact substrates transported by most of these transporters are still unknown and we therefore cannot exclude that a single substrate (e.g. a hormone) is responsible for the diversity of physiological roles. This gap in our knowledge is mainly due to the fact that only a few studies have used direct methods to identify the substrates of these membrane transporters. To address this issue, transport assays involving isolated cells, vesicular membranes or reconstituted liposomes are essential. In this review, we will highlight the importance of the direct biochemical characterization of plant ABC transporters and give some insights into the current status of the homologous and heterologous expression of such proteins

The Nicotiana tabacum ABC transporter NtPDR3 secretes O-methylated coumarins in response to iron deficiency

Although iron is present in large amounts in the soil, its poor solubility means that plants have to use various strategies to facilitate its uptake. In this study, we show that expression of NtPDR3/NtABCG3, a Nicotiana tabacum plasma-membrane ABC transporter in the pleiotropic drug resistance (PDR) subfamily, is strongly induced in the root epidermis under iron deficiency conditions. Prevention of NtPDR3 expression resulted in N. tabacum plants that were less tolerant to iron-deficient conditions, displaying stronger chlorosis and slower growth than those of the wildtype when not supplied with iron. Metabolic profiling of roots and root exudates revealed that, upon iron deficiency, secretion of catechol-bearing O-methylated coumarins such as fraxetin, hydroxyfraxetin, and methoxyfraxetin to the rhizosphere was compromised in NtPDR3-silenced plants. However, exudation of flavins such as riboflavin was not markedly affected by NtPDR3-silencing. Expression of NtPDR3 in N. tabacum Bright Yellow-2 (BY-2) cells resulted in altered intra- and extracellular coumarin pools, supporting coumarin transport by this transporter. The results demonstrate that N. tabacum secretes both coumarins and flavins in response to iron deficiency and that NtPDR3 plays an essential role in the plant response to iron deficiency by mediating secretion of O-methylated coumarins to the rhizosphere