Journal of Experimental Botany, Vol. 53, No. 376, pp. 1879-1886,
September 1, 2002
© 2002 Oxford University Press
Characterization of berberine transport into Coptis japonica cells and the involvement of ABC protein
Received 4 March 2002; Accepted 11 June 2002
1 Laboratory of Molecular and Cellular Biology of Totipotency, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Kyoto 606-8502, Japan
2 Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-8502, Japan
Abbreviations: ABC, ATP-binding cassette; DMSO, dimethylsulphoxide; DTT, dithiothreitol; EGTA, ethylene glycol bis(2-aminoethyl)-tetra-acetic acid; MDR, multi-drug resistance protein; MRP, multi-drug resistance-associated protein.
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Cultured Coptis japonica cells are able to take up berberine, a benzylisoquinoline alkaloid, from the medium and transport it exclusively into the vacuoles. Uptake activity depends on the growth phase of the cultured cells whereas the culture medium had no effect on uptake. Treatment with several inhibitors suggested that berberine uptake depended on the ATP level. Some inhibitors of P-glycoprotein, an ABC transporter involved in multiple drug resistance in cancer cells, strongly inhibited berberine uptake, whereas a specific inhibitor for glutathione biosynthesis and vacuolar ATPase, bafilomycin A1, had little effect. Vanadate-induced ATP trap experiments to detect ABC proteins expressed in C. japonica cells showed that three membrane proteins of between 120 and 150 kDa were photolabelled with 8-azido-[
-32P] ATP. Two revealed the same photoaffinity-labelling pattern as P-glycoprotein, and the interaction of these proteins with berberine was also demonstrated. These results suggest that ABC proteins of the MDR-type are involved in the uptake of berberine from the medium. Key words: Key words: ABC protein, berberine, Coptis japonica, MDR, transport.
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Introduction |
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Alkaloids are a large group of plant secondary metabolites having divergent chemical structures and biological activities. Some of them are used as medicines both in modern and traditional medicine. For instance, vincristine and taxol are prescribed as anticancer drugs, morphine and scopolamine are used as analgesics, and berberine, a yellow benzylisoquinoline alkaloid, is conventionally used as an antidiarrhetic, bitter stomachic, and antimaleria drug in many countries (Yamamoto et al., 1993). They often show strong cytotoxicity to prokaryotic and eukaryotic cells; for example, vincristine inhibits microtubule formation, and berberine inhibits DNA and protein synthesis (Ghosh et al., 1985). Because of these activities, alkaloids are presumed to play an important role as a biological barrier to protect the plant tissue from pathogens. Indeed, berberine shows strong antimicrobial activity to both Gram-positive and Gram-negative bacteria as well as other micro-organisms (Iwasa et al., 1998).
On the other hand, alkaloid-producing plant cells seem to be insensitive to their metabolites, probably because they have a detoxification mechanism to prevent their cytotoxicity. However, the mechanism of detoxification of secondary metabolites in plant cells is less well understood. One possible strategy is the compartmentation of alkaloids in the vacuole, by which the primary metabolic enzymes in the cytosol and DNA in the nucleus may not be negatively affected by the alkaloid.
In this study, the high tolerance of cultured Coptis japonica cells that produce a large amount of berberine is reported and compared with cells that do not produce berberine. While cultured C. japonica cells exclusively accumulate endogenous berberine in the vacuoles, they also take up berberine added to the culture medium and transport it to the vacuoles (Sato et al., 1992, 1994). It is reported here that the uptake of berberine by Coptis cells depends on the growth phase of the cells and requires intracellular ATP. Further analyses using specific inhibitors of ATP-binding cassette (ABC) proteins have suggested that an ABC transporter is involved in the transport of berberine in Coptis cells.
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Cultured cells
The high berberine-producing cultured Coptis cells, which were originally induced from rootlets of C. japonica Makino var. dissecta (Yamabe) Nakai, were maintained as described elsewhere (Sato and Yamada, 1984). Two-week-old cultured cells were used for the experiment of berberine uptake, unless otherwise stated. The cultured cells of Lithospermum erythrorhizon (line M18) and Nicotiana tabacum (cv. SamsunNN, line NII) were also continuously subcultured according to the methods of Yazaki et al. (1997) and Takeda et al. (1990), respectively.
Chemicals
Berberine and the other chemicals used in this study were purchased from Wako Pure Chemicals (Osaka Japan), or Nakalai Tesque (Kyoto Japan). The 8-azido-[-32P] ATP (specific activity 740 GBq mmol–1) was purchased from ICN Biochemicals.
Toxicity of berberine to various plant cells
In order to evaluate the sensitivity of various plant cells to berberine, cells (1.0 g FW) were inoculated into 30 ml of medium containing various concentrations of berberine and cultured on a rotary shaker for 14 d in darkness. Cells were harvested by filtration through Miracloth (Calbiochem Co.) and the fresh weight was measured.
Berberine uptake by cultured cells
Cultured C. japonica cells were harvested by filtration and 0.25 g fresh mass was inoculated into 3 ml of the culture medium in which the cells had grown (conditioned medium) in a test tube. Berberine chloride was added to the medium at a final concentration at 250 µM and the cells were incubated on a rotary shaker at 25 °C. Every 2 h, a 150 µl aliquot of the medium was mixed with 350 µl water and centrifuged at 15 000 g for 5 min. The concentration of berberine was determined by measurement of the absorbance of the supernatant at 420 nm (
=3330). Experiments were done in triplicate. For evaluating the effect of growth stage, C. japonica cells were filtered to separate the cells from the culture medium 3, 6, 9, 12, 16, 21, and 25 d after inoculation, and 0.25 g fresh cells were used for the experiment as mentioned above. Berberine uptake was measured after 10 h of incubation in this experiment.
Inhibitor experiments
Prior to the addition of berberine, C. japonica cells (0.25 g) were treated with inhibitors (vanadate, 1 mM; azide, 100 µM; nifedipine, 50 µM; cyclosporine A, 100 µM; bafilomycin A1, 1 µM; NH4+ 10 mM; glutathione 100 µM; buthionine sulphoximine, 1 mM) in 3 ml of conditioned medium for 1 h. These concentrations were chosen from the normal range used to inhibit the function of ABC proteins (Hu et al., 1996; Klein et al., 1996; Lu et al., 1998). After the addition of berberine (final concentration 250 µM) the uptake was measured at 25 °C. Sodium vanadate was depolymerized before use according to the method of Goodno (1979). Quinidine, nifedipine, and cyclosporin A were dissolved in DMSO solution, and 10 µl each was added to the medium. In the control, 10 µl of DMSO or H2O was added. DMSO did not affect berberine uptake or cell viability at this concentration. The viability of the cells was confirmed by staining with neutral red in the conventional assay. Alternatively, cell growth was measured after the treated cells were washed and recultivated.
Photoaffinity labelling of ABC proteins with 8-azido ATP
Fresh cells (1.0 g) were homogenized in 3 ml of 0.1 M potassium phosphate buffer (pH 6.5) containing 10 mM dithiothreitol (DTT) and 0.1 g polyvinylpolypyrrolidone using a mortar and pestle. After removal of cell debris by centrifugation at 3000 g, the supernatant was gel-filtered with PD-10 (Amersham-Pharmacia) to change the buffer to 0.1 M TRIS–HCl (pH 7.4) containing 10 mM DTT, and then the membrane fraction was recovered by centrifugation at 20 000 g for 30 min at 4 °C. Membrane proteins (20 µg) were incubated in a buffer mixture composed of 10 µM 8-azido-[-32P] ATP, 2 mM ouabain, 0.1 mM EGTA, 3 mM MgSO4, 40 mM TRIS–Cl (pH 7.5), and 0.2 M orthovanadate in a total volume of 8 µl for 10 min at 37 °C (Senior et al., 1995). The reactions were stopped by the addition of 400 µl of ice-cold TGM buffer (40 mM TRIS–Cl (pH 7.5), 0.1 mM EGTA, and 1 mM MgSO4), and the membrane proteins were sedimented by centrifugation (15 000 g, 10 min, 4 °C) to separate unbound ATP. Pellets were washed with fresh ice-cold TRIS–EGTA buffer, then irradiated with UV light at 254 nm (5.5 mW cm–2) in 8 µl of TGM buffer for 5 min on ice. Samples were electrophoresed on a 7% SDS-polyacrylamide gel, and autoradiographed. The radioactivity trapped by ABC protein was analysed with a radioimaging analyzer BAS 2000 (Fuji Film Co.). Experiments were repeated three times.
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Different sensitivity and uptake activity among plant cells
Cultured cells of C. japonica produce berberine and accumulate it in the vacuoles in large amounts, although this alkaloid generally shows strong cytotoxicity to many bacteria (Iwasa et al., 1998; Schmeller et al., 1997). The inhibitory effect of berberine on the cell growth of cultured C. japonica cells and on plant cells that do not produce berberine was examined (Fig. 1A–C). Berberine added to the medium strongly inhibited the growth of cultured cells of both Lithospermum erythrorhizon and tobacco, suggesting that berberine was toxic to plant cells as well as to bacterial cells. Conversely, C. japonica cells showed a clear tolerance to berberine, which was a native secondary metabolite of this plant. The tolerance of C. japonica to berberine was more than 10-fold higher than that of tobacco and Lithospermum cells. As Fig. 1D shows, berberine uptake of these cultured plant cells was also different and uptake activity was lower than that of C. japonica cells. Cell growth was not affected at concentrations of up to 1 mM berberine. Berberine is not soluble at higher concentrations.
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Characterization of berberine uptake by C. japonica cells
As shown in Fig. 2A, C. japonica cells constantly absorbed berberine added to the medium and it had almost completely disappeared after 12 h. The berberine content in the treated cells was 4.82 µmol/0.25 g fresh weight, whereas that in the control cells was 4.10 µmol/0.25 g fresh weight. The increase is accounted for by the amount of berberine added to the medium, 0.75 µmol, which indicates that berberine was taken up by the cells from the medium and stably accumulated, as reported previously (Sato et al., 1992). The berberine uptake activity of C. japonica cells, however, showed some variability during the culture period, i.e. high activity was found in cells in the logarithmic phase of growth (Fig. 2B), while the cells in the stationary phase revealed a lower uptake activity, although endogenous berberine still accumulated in cells at the stationary phase. Since medium pH is an important factor for the uptake of compounds from the medium and also affects the ionic form of berberine, the change in medium pH was monitored (Fig. 2C). Immediately after cell inoculation, the pH of the medium was 5.6. After a transient decrease, it shifted to almost 7.0 in the late logarithmic phase, and then it dropped to 5.5–6.0 in the stationary phase. This change in medium pH almost coincided with that of berberine uptake activity.
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The dependence of berberine uptake activity (Fig. 2B) on the condition of the cultured medium was examined by interchanging the cultured cells and the conditioned medium at two growth phases: (1) 14 d after inoculation (logarithmic phase) and (2) 30 d after inoculation (stationary phase). The berberine uptake by these cell cultures is shown in Fig. 3. The cells on the 14th day of culture actively absorbed berberine from the conditioned medium in both the logarithmic and stationary phases, whereas cells on the 30th day of culture did not absorb berberine from either conditioned medium. These results clearly indicate that the berberine uptake activity is independent of the conditioned medium, but exclusively dependent on the cell condition.
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Inhibition of berberine uptake
Several compounds which inhibit the activity of various transporters were examined by addition to the medium in the concentration range conventionally used (Fig. 4A-D). Figure 4E shows a summary of the effect of the inhibitors tested. Vanadate, azide, cyclosporine A, and nifedipine strongly inhibited berberine uptake in a dose-dependent manner. Vanadate is a membrane ATPase inhibitor (Ambudkar et al., 1992). Azide is known to reduce internal ATP when cells are treated (Wigler and Patterson, 1994) (Fig. 4B). On the other hand, both cyclosporine A and nifedipine are inhibitors of an ABC transporter, P-glycoprotein, which functions as a drug efflux pump in human cancer cells (Ueda et al., 1987). These data suggest that an ABC transporter might be involved in the uptake of berberine from the medium. Because some secondary metabolites are translocated as their glutathione conjugates into plant vacuoles by members of another subclass of ABC transporters, multi-drug resistance-associated protein (MRP) (Zaman et al., 1995), the dependence of berberine uptake on glutathione was also examined using buthionine sulphoximine, a specific gamma-glutamylcysteine synthetase inhibitor which causes depletion of intracellular glutathione (Campbell et al., 1991). As shown in Fig. 4E, neither the addition of glutathione nor buthionine sulphoximine affected berberine uptake by C. japonica cells.
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Inhibition of berberine uptake by P-glycoprotein inhibitors was further examined at various concentrations of nifedipine and quinidine, two common inhibitors of P-glycoprotein. Both compounds clearly and constantly inhibited berberine uptake during the incubation in a dose-dependent manner (Fig. 5A, B). The negative value observed at 100 µM nifedipine suggests the leakage of endogenous berberine from the cells. It may be because of the strong inhibition of the transport system on the tonoplast membrane, which is responsible for the sequestration of berberine in the vacuoles. Similar dose-dependent inhibition was also observed in the time-course experiments using vanadate and azide (data not shown). These results support the idea that an ABC transporter belonging to the multi-drug resistance protein (MDR) subfamily is involved in berberine uptake by C. japonica cells.
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Detection of ABC proteins in C. japonica cells
A photoaffinity labelling experiment was carried out to detect ABC proteins that interact with berberine in C. japonica cells. ABC proteins show ATPase activity and the catalytic sites are known to be of low affinity and specificity for nucleotides. The binding affinity for nucleotides of the ABC protein greatly increased in the presence of vanadate (Urbatsch et al., 1995). A catalytic site produces a stable form of the inhibited ABC protein by trapping of nucleotide with vanadate which is labelled specifically with 8-azido-[
-32P] ATP by UV irradiation (Taguchi et al., 1997). When this vanadate-induced nucleotide trapping method is applied to the crude cell membranes of C. japonica, three bands of 120–150 kDa were photoaffinity-labelled (Fig. 6).
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In the presence of Mg2+ and vanadate, prominent nucleotide trapping was observed in the upper band (lane 1), whereas it largely decreased in the absence of either Mg2+ or vanadate (lanes 2–4). The middle band also shows strong nucleotide trapping in the presence of both Mg2+ and vanadate (lane 1), but it almost disappeared when Mg2+ or vanadate was removed from the reaction mixture (lanes 2–4). This is a characteristic of such ABC transporters as P-glycoprotein (Ueda et al., 1997), suggesting that both bands represented ‘full-sized’ ABC transporters. The lower band, however, required Mg2+ ion for ATP-binding, but still strongly bound ATP even in the absence of vanadate (lanes 1, 2). This suggests that this protein did not hydrolyse ATP to ADP, in a similar way as a member of the ABC proteins, sulphonylurea receptor 1 (Ueda et al., 1997).
The addition of berberine to the reaction mixture apparently reduced the nucleotide trapping of the upper and middle bands in a dose-dependent manner (lanes 5–8), whereas the lower band was unaffected. These data suggest that both the upper and middle bands interact with berberine. Moreover, the size of the middle band in the vanadate trap experiment coincided with that of an ABC protein, Cjmdr1, of C. japonica cells detected by Western blotting (data not shown), which was recently cloned as a potential alkaloid transporter of this plant (Yazaki et al., 2001).
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In this study, it has been demonstrated that berberine shows strong cytotoxicity to plant cells that do not produce berberine, while the berberine-producing plant cells of C. japonica have a clear tolerance to it (Fig. 1). This suggests that there is a species-specific detoxification mechanism for plant cells, which produce cytotoxic alkaloids. When berberine is exogenously added to C. japonica cultures it is actively absorbed by the cells and disappears from the medium. The added berberine was transported into the vacuoles and stably accumulated (Sato et al., 1992, 1994). These observations suggest that vacuolar transport may be one of the mechanisms for the detoxification of the endogenous alkaloid in such alkaloid-producing plant cells.
Berberine uptake depended on the growth phase of the cultured C. japonica cells (Fig. 2). Because endogenous berberine production is active in the logarithmic phase (Takeshita et al., 1995), berberine transport into the vacuoles is also most active in this phase, as shown by the uptake activity of endogenously added berberine. The low uptake activity in the stationary phase may also be attributable to the low level of intracellular ATP that is a general energy source for active transport, since the ATP level in the cells decreases in the stationary phase when the carbon source in the medium is depleted.
The pH value of the medium during the culture period varied from 5.1 to 6.9, but berberine uptake was independent of the change of the medium pH (Figs 2, 3). It is often reported that the medium pH affects the uptake of organic compounds (Komor et al., 1981; Verstappen et al., 1991), and the change in pH of the medium affects the ionic condition of the berberine molecule. The effect of pH value in the medium was, therefore, tested further by raising the pH to 7.5, but the berberine uptake activity was not affected at all (data not shown). Thus, it was concluded that the growth phase-dependent change in berberine uptake activity during the culture was a phenomenon exclusively dependent on the physiological condition of C. japonica cells, but not on the medium.
The transport systems for secondary metabolites in plant cells are often reported to be energy-dependent (Yamamoto et al., 1987; Deus-Neumann and Zenk, 1986; Mende and Wink, 1987). Indeed, the uptake activity of berberine by C. japonica cells was very sensitive to the membrane ATPase inhibitor, vanadate. Furthermore, various inhibitors of P-glycoprotein decreased berberine transport into the cells (Figs 4, 5). P-glycoprotein, a representative of the ABC transporters, was discovered in human cancer cells as a large membrane protein responsible for multiple drug resistance against anti-cancer drugs. Its expression confers on the cancer cells multidrug resistance by effluxing the chemicals of divergent structures, which results in a decrease of their intracellular concentration (Ueda et al., 1987). In C. japonica, however, an ABC transporter is thought to be potentially involved in the inward transport of berberine (Figs 4, 5), which seems to play a role in the sequestration or translocation of this endogenous alkaloid.
In the movement of berberine from the medium to vacuole, two transport events are involved, one through the plasma membrane and the other through the tonoplast membrane. The uptake of berberine by isolated vacuoles of C. japonica has been measured. However, berberine uptake by intact vacuoles does not show a clear dependence on ATP, and uptake was not influenced by the addition of the non-hydrolysable ATP analogue, ATP-
-S (data not shown). This observation suggests that the putative ABC transporter responsible for the uptake of berberine may be localized at the plasma membrane and be implicated in the influx of this alkaloid. If this assumption is correct, this is a novel case for eukaryotic ABC transporters, because influx of substrates by ABC transporters is only known in prokaryotes (Higgins, 2001).
The results of vanadate-induced nucleotide trapping experiments suggested the existence of ABC transporters in C. japonica cells, and two of the three bands (the upper and the middle) showed the characteristic properties of the drug efflux pump, P-glycoprotein (Fig. 6). The interaction of these two putative ABC transporters with berberine suggests that they are involved in the transport of berberine in this plant cell (Fig. 6). As a candidate of such berberine transporters, a cDNA of ABC protein (Cjmdr1) encoding 1289 amino acids, which belongs to the MDR subfamily, has recently been isolated from cultured C. japonica cells (Yazaki et al., 2001). Heterologously expressed protein in yeast showed the size of c. 135 kDa on SDS-PAGE (data not shown), and this coincided with the middle band detected with a vanadate trapping experiment. The characterization of berberine transport activity of Cjmdr1 in yeast is ongoing.
The involvement of ABC transporters in the uptake of berberine from the medium is speculative, but endogenously biosynthesized berberine might be transported in a different way. Zenk et al. (1985) reported that the biosynthetic berberine precursors in Fumaria capreolata cells were localized in membrane vesicles and then the final product berberine was transported to the vacuolar matrix. The uptake of various alkaloids by isolated plant vacuoles has been demonstrated to be highly specific for plant species and is energy-dependent (Deus-Neumann and Zenk, 1984, 1986; Mende and Wink, 1987; Wink and Mende, 1987). The transport processes of solutes across the vacuolar membrane of higher plants are complicated, and many membrane proteins of divergent classes are involved (Martinoia et al., 2000). ABC proteins could be responsible for alkaloid accumulation in plant vacuoles, as is the case for other metabolites (Lu et al., 1998; Marrs et al., 1995; Klein et al., 1996).
Plant ABC protein is a newly developing research area (Theodoulou, 2000; Davies and Coleman, 2000; Sánchez-Fernández et al., 2001). Both MDR and MRP-type transporters have been intensively studied and their biochemical functions and physiological roles have recently been reported (Sidler et al., 1998; Rea, 1999; Martinoia et al., 2000). Molecular characterization of berberine transport in C. japonica cells should provide further information about the ABC proteins involved in alkaloid transport in plant cells.
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Acknowledgements |
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The authors are grateful to Dr H Sato for critical reading of the manuscript. This work was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 10217206) to KY.
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N. Kato, E. Dubouzet, Y. Kokabu, S. Yoshida, Y. Taniguchi, J. G. Dubouzet, K. Yazaki, and F. Sato Identification of a WRKY Protein as a Transcriptional Regulator of Benzylisoquinoline Alkaloid Biosynthesis in Coptis japonica Plant Cell Physiol., January 1, 2007; 48(1): 8 - 18. [Abstract] [Full Text] [PDF]
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N. Shitan, K.-i. Horiuchi, F. Sato, and K. Yazaki Bowman-Birk Proteinase Inhibitor Confers Heavy Metal and Multiple Drug Tolerance in Yeast Plant Cell Physiol., January 1, 2007; 48(1): 193 - 197. [Abstract] [Full Text] [PDF]
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 K. Terasaka, J. J. Blakeslee, B. Titapiwatanakun, W. A. Peer, A. Bandyopadhyay, S. N. Makam, O. R. Lee, E. L. Richards, A. S. Murphy, F. Sato, et al. PGP4, an ATP Binding Cassette P-Glycoprotein, Catalyzes Auxin Transport in Arabidopsis thaliana Roots PLANT CELL, November 1, 2005; 17(11): 2922 - 2939. [Abstract] [Full Text] [PDF]
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X. Wu, J. Lin, Q. Lin, J. Wang, and L. Schreiber Casparian Strips in Needles are More Solute Permeable than Endodermal Transport Barriers in Roots of Pinus bungeana Plant Cell Physiol., November 1, 2005; 46(11): 1799 - 1808. [Abstract] [Full Text] [PDF]
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M. Otani, N. Shitan, K. Sakai, E. Martinoia, F. Sato, and K. Yazaki Characterization of Vacuolar Transport of the Endogenous Alkaloid Berberine in Coptis japonica Plant Physiology, August 1, 2005; 138(4): 1939 - 1946. [Abstract] [Full Text] [PDF]
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N. Samanani, S.-U. Park, and P. J. Facchini Cell Type-Specific Localization of Transcripts Encoding Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis PLANT CELL, March 1, 2005; 17(3): 915 - 926. [Abstract] [Full Text] [PDF]
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 N. Shitan, I. Bazin, K. Dan, K. Obata, K. Kigawa, K. Ueda, F. Sato, C. Forestier, and K. Yazaki Involvement of CjMDR1, a plant multidrug-resistance-type ATP-binding cassette protein, in alkaloid transport in Coptisjaponica PNAS, January 21, 2003; 100(2): 751 - 756. [Abstract] [Full Text] [PDF]
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