Nobukazu Shitan

Involvement of CjMDR1, a plant multidrug-resistance-type ATP-binding cassette protein, in alkaloid transport in Coptis japonica

Alkaloids comprise one of the largest groups of plant secondary metabolites. Berberine, a benzylisoquinoline alkaloid, is preferentially accumulated in the rhizome of Coptis japonica, a ranunculaceous plant, whereas gene expression for berberine biosynthetic enzymes has been observed specifically in root tissues, which suggests that berberine synthesized in the root is transported to the rhizome, where there is high accumulation. We recently isolated a cDNA encoding a multidrug-resistance protein (MDR)-type ATP-binding cassette (ABC) transporter (Cjmdr1) from berberine-producing cultured C. japonica cells, which is highly expressed in the rhizome. Functional analysis of Cjmdr1 by using a Xenopus oocyte expression system showed that CjMDR1 transported berberine in an inward direction, resulting in a higher accumulation of berberine in Cjmdr1-injected oocytes than in the control. Typical inhibitors of ABC proteins, such as vanadate, nifedipine, and glibenclamide, as well as ATP depletion, clearly inhibited this CjMDR1-dependent berberine uptake, suggesting that CjMDR1 functioned as an ABC transporter. Conventional membrane separation methods showed that CjMDR1 was localized in the plasma membrane of C. japonica cells. In situ hybridization indicated that Cjmdr1 mRNA was expressed preferentially in xylem tissues of the rhizome. These findings strongly suggest that CjMDR1 is involved in the translocation of berberine from the root to the rhizome.

Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum.

Alkaloids play a key role in plant defense mechanisms against pathogens and herbivores, but the plants themselves need to cope with their toxicity as well. The major alkaloid of the Nicotiana species, nicotine, is translocated via xylem transport from the root tissues where it is biosynthesized to the accumulation sites, the vacuoles of leaves. To unravel the molecular mechanisms behind this membrane transport, we characterized one transporter, the tobacco (Nicotiana tabacum) jasmonate-inducible alkaloid transporter 1 (Nt-JAT1), whose expression was coregulated with that of nicotine biosynthetic genes in methyl jasmonate-treated tobacco cells. Nt-JAT1, belonging to the family of multidrug and toxic compound extrusion transporters, was expressed in roots, stems, and leaves, and localized in the tonoplast of leaf cells. When produced in yeast cells, Nt-JAT1 occurred mainly in the plasma membrane and showed nicotine efflux activity. Biochemical analysis with proteoliposomes reconstituted with purified Nt-JAT1 and bacterial F0F1-ATPase revealed that Nt-JAT1 functioned as a proton antiporter and recognized endogenous tobacco alkaloids, such as nicotine and anabasine, and other alkaloids, such as hyoscyamine and berberine, but not flavonoids. These findings strongly suggest that Nt-JAT1 plays an important role in the nicotine translocation by acting as a secondary transporter responsible for unloading of alkaloids in the aerial parts and deposition in the vacuoles.

Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots

Nicotine is a major alkaloid accumulating in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATE1 and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane. Reduced expression of the transporters rendered tobacco plants more sensitive to the application of nicotine. In contrast, overexpression of NtMATE1 in cultured tobacco cells induced strong acidification of the cytoplasm after jasmonate elicitation or after the addition of nicotine under nonelicited conditions. Expression of NtMATE1 in yeast (Saccharomyces cerevisiae) cells compromised the accumulation of exogenously supplied nicotine into the yeast cells. The results imply that these MATE-type proteins transport tobacco alkaloids from the cytosol into the vacuole in exchange for protons in alkaloid-synthesizing root cells.

Involvement of a Soybean ATP-Binding Cassette-Type Transporter in the Secretion of Genistein, a Signal Flavonoid in Legume-Rhizobium Symbiosis

Legume plants have an ability to fix atmospheric nitrogen into nutrients via symbiosis with soil microbes. As the initial event of the symbiosis, legume plants secrete flavonoids into the rhizosphere to attract rhizobia. Secretion of flavonoids is indispensable for the establishment of symbiotic nitrogen fixation, but almost nothing is known about the membrane transport mechanism of flavonoid secretion from legume root cells. In this study, we performed biochemical analyses to characterize the transport mechanism of flavonoid secretion using soybean (Glycine max) in which genistein is a signal flavonoid. Plasma membrane vesicles prepared from soybean roots showed clear transport activity of genistein in an ATP-dependent manner. This transport activity was inhibited by sodium orthovanadate, a typical inhibitor of ATP-binding cassette (ABC) transporters, but was hardly affected by various ionophores, such as gramicidin D, nigericin, or valinomycin, suggesting involvement of an ABC transporter in the secretion of flavonoids from soybean roots. The Km and Vmax values of this transport were calculated to be 158 μm and 322 pmol mg protein−1 min−1, respectively. Competition experiments using various flavonoids of both aglycone and glucoside varieties suggested that this ABC-type transporter recognizes genistein and daidzein, another signaling compound in soybean root exudates, as well as other isoflavonoid aglycones as its substrates. Transport activity was constitutive regardless of the availability of nitrogen nutrition. This is, to our knowledge, the first biochemical characterization of the membrane transport of flavonoid secretion from roots.

Characterization of Vacuolar Transport of the Endogenous Alkaloid Berberine in Coptis japonica

Alkaloids comprise one of the largest groups of plant secondary metabolites. Many of them exhibit strong biological activities, and, in most cases, they are accumulated in the central vacuole of alkaloid-producing plants after synthesis. However, the mechanisms involved in alkaloid transport across the tonoplast are only poorly understood. In this study, we analyzed the vacuolar transport mechanism of an isoquinoline alkaloid, berberine, which is produced and accumulated in the vacuole of cultured cells of Coptis japonica. The characterization of berberine transport using intact vacuoles and a tonoplast vesicle system showed that berberine uptake was stimulated by Mg/ATP, as well as GTP, CTP, UTP, and Mg/pyrophosphate. Berberine uptake was strongly inhibited by NH4+ and bafilomycin A1, while vanadate, which is commonly used to inhibit ATP-binding cassette transporters, had only a slight effect, which suggests the presence of a typical secondary transport mechanism. This is contrary to the situation in the plasma membrane of this plant cell, where the ATP-binding cassette transporter is involved in berberine transport. Model experiments with liposomes demonstrated that an ion-trap mechanism was hardly implicated in berberine transport. Further studies suggested that berberine was transported across the tonoplast via an H+/berberine antiporter, which has a Km value of 43.7 μm for berberine. Competition experiments using various berberine analogs, as well as other classes of alkaloids, revealed that this transporter is fairly specific, but not exclusive, for berberine.

Secondary transport as an efficient membrane transport mechanism for plant secondary metabolites

Plants produce a large number of secondary metabolites, such as alkaloids, terpenoids, and phenolic compounds. Secondary metabolites have various functions including protection against pathogens and UV light in plants, and have been used as natural medicines for humans utilizing their diverse biological activities. Many of these natural compounds are accumulated in a particular compartment such as vacuoles, and some are even translocated from source cells to sink organs via long distance transport. Both primary and secondary transporters are involved in such compartmentation and translocation, and many transporter genes, especially genes belonging to the multidrug and toxin extrusion type transporter family, which consists of 56 members in Arabidopsis, have been identified as responsible for the membrane transport of secondary metabolites. Better understandings of these transporters as well as the biosynthetic genes of secondary metabolites will be important for metabolic engineering aiming to increase the production of commercially valuable secondary metabolites in plant cells.

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 cells.

Cell and molecular biology of ATP-binding cassette proteins in plants.

ATP-binding cassette (ABC) proteins constitute a large and diverse superfamily of membrane-bound and soluble proteins, which are involved in a wide range of biological processes in all organisms from prokaryotes to eukaryotes. Genome analyses of model plants, for example, Arabidopsis and rice, have revealed that plants have more than double numbers of this family member in their genomes compared to animals and insects. In recent years, various biochemical and physiological functions of ABC proteins in plants have been reported. Some are relevant for the defense mechanisms to biotic and abiotic stresses, whereas others are involved in the basic functions necessary for maintaining the plant life. Here, we provide an updated inventory of plant ABC proteins and summarize their tissue specificities, membrane localizations, and physiological functions.

RCN1/OsABCG5, an ATP-binding cassette (ABC) transporter, is required for hypodermal suberization of roots in rice (Oryza sativa)

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C(28) and C(30) fatty acids or ω-OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier.

Heterologous expression of a mammalian ABC transporter in plant and its application to phytoremediation

Mammalian ATP-binding cassette (ABC) transporters involved in the multidrug-resistance of cancer cells can efflux cytotoxic compounds that show a wide variety of chemical structures and biological activities. Human multidrug resistance-associated protein (hMRP1) is one of the most intensively studied ABC transporters and many substrates have been identified, including both organic and inorganic compounds. In an attempt at novel ‘transport engineering’ using hMRP1 as a molecular pump, we established transgenic tobacco plants that showed clear resistance to cadmium and daunorubicin, although they were not resistant to etoposide, another known substrate of hMRP1. When expressed in tobacco cells, hMRP1 protein was localized at vacuolar membrane, while members of the MRP family are localized at plasma membrane in mammalian cells to reduce the cellular accumulation of various drugs. Thus, the hMRP1-expressing tobacco cells were able to take up these substrates across the tonoplast and sequestrate them in the vacuolar matrix. These results suggest that it may be possible to use the transgenic tobacco in phytoremediation, where a single transformation with an ABC transporter with broad substrate specificity should be effective for extracting various environmental pollutants including both organic and inorganic compounds, and accumulate them in the plant body. This should be advantageous for the remediation of a complex polluted environment, which is commonly found in the real world.