Journal of Experimental Botany, Vol. 52, No. 357, pp. 877-879,
April 15, 2001
© 2001 Oxford University Press
Gene Notes |
A novel Coptis japonica multidrug-resistant protein preferentially expressed in the alkaloid-accumulating rhizome1
2 Laboratory of Molecular and Cellular Biology of Totipotency, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Kyoto 606-8502, Japan
3 Laboratory of Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Received 9 November 2000; Accepted 17 December 2000
Abstract
A full-length cDNA, Cjmdr1, which belongs to the multidrug-resistant (mdr) gene family, was isolated by nested RT-PCR from alkaloid-producing cultured cells of Coptis japonica. The cDNA is 4192 nucleotides long and has an ORF of 1289 amino acids. Northern analysis of the intact plant showed a clear preference in its expression in the rhizome, where alkaloids are highly accumulated compared to other organs.
Key words: ABC protein, mdr, rhizome-specific expression, Coptis japonica.
Plant cells produce a large variety of secondary metabolites which play important roles in protecting against pathogen attack or in attracting insects for pollination. Some of them show strong biological activities, and are often used as medicines, for example, taxol. While they are potentially toxic to the plant cells themselves, the plants are able to grow without serious disruption of their basic metabolism (Sato et al., 1990
, 1994).
The regulatory mechanism of alkaloid accumulation in plants has been studied using a high berberine-producing cultured cell line of Coptis japonica (Ranunculaceae), a medicinal plant in Asian countries, as a model system (Sato and Yamada, 1984). Its medicinal part is the rhizome where the active principal berberine and its derivatives are highly accumulated. Although berberine is highly toxic to various bacteria and to cultured plant cells which do not produce berberine, cultured C. japonica cells accumulate berberine in their vacuoles and very clearly tolerate it (Sato et al., 1993). The preliminary studies using various inhibitors of human P-glycoprotein (Ueda et al., 1987), a representative ATP-binding cassette (ABC) protein responsible for multiple drug-resistance of cancer cells, suggested that an mdr-like gene product was involved in the uptake and accumulation of berberine by the cells (unpublished data). In the present study, cDNA cloning of an mdr-like gene from C. japonica is reported as a potential alkaloid transporter in plant cells.
Total RNA prepared from C. japonica cells cultured in liquid medium (Murashige and Skoog, 1962) was reverse-transcribed with M-MLV reverse transcriptase (NEB). Nested PCR was carried out with Taq DNA polymerase (Takara), with the DNA-RNA hybrid as a template, and two sets of degenerate primers designed from the highly conserved amino acid sequences in the nucleotide-binding fold (NBF) which is the domain responsible for the ATP-binding property of ABC proteins; Fwd-primer1, 5'-MARACIYTIGCHYTIGTIGG-3' (n- (Q/K)TTLALVG -c) and Rv-primer1, 5'-GCRCTXHTXGCYTCRTCXARXAG-3' (n- LLDEATSA -c): Fwd-primer2, 5'-GGITGYGGIAARWSIWSIGT-3' (n- GCGKS(T/S)V -c) and Rv-primer2, 5'-GCDATXCKYTGYTTYTGXCCXCC-3' (n- GGQKQRIA -c). As the PCR products, two 320 bp fragments for the NBF domain of mdr-like protein were isolated. The general tandem repeat structure of ABC protein implied that these two DNA sequences were derived from two NBFs of one gene. Thus, using two specific primers corresponding to the internal sequences of these DNA fragments in an inverted direction, RT-PCR was performed again using a heat-stable DNA polymerase of high-fidelity KOD (TOYOBO), resulting in the amplification of a DNA fragment (2.2 kb) which was significantly similar to known mdr-like proteins. To obtain a full-length cDNA clone, 5' and 3' RACE using a cDNA amplification kit (Clontech) was used.
The full-length cDNA, designated Cjmdr1, was 4129 bp long and encoded a putative polypeptide composed of 1289 amino acids, which showed typical features of ‘full-sized’ ABC protein, i.e. two ATP-binding domains and two transmembrane domains. Three motifs characteristic of ABC protein, i.e. ‘Walker A’, ‘Walker B’, and ‘ABC signature’, were highly conserved in Cjmdr1, and are shown in Fig. 1. A protein database search revealed that Cjmdr1 polypeptide belonged to the mdr-like gene family, but not to another subfamily of ABC protein, MRP (multidrug resistance-associated protein) (Rea, 1999). Its hydrophobicity profiles calculated by a Kyte and Doolittle plot and the program on CBS servers (http://www.cbs.dtu.dk/services/TMHMM-1.0/) showed features nearly identical to those of human P-glycoprotein (Loo and Clarke, 1993), although the proteins showed relatively low identity (38%).
|
The expression of Cjmdr1 in various organs of intact C. japonica plant was monitored by Northern hybridization. The highest accumulation was observed in the rhizome, which is the main organ for berberine accumulation in C. japonica, whereas only a low level of transcripts was detected in other organs (Fig. 2). Since other members of the plant mdr-like gene family have not shown such a clear organ-specificity (Dudler and Hertig, 1992; Wang et al., 1996; Davies et al., 1997), this is the first plant mdr-like gene product that is expressed preferentially in rhizome, and demonstrates a correlation between gene expression and alkaloid accumulation in a plant organ.
|
In the phylogenetic relationship of plant mdr-like proteins, the sequences of PMDR1 from potato (StPGP) are very similar to those of AtPGP1 despite the taxonomic difference between the two plant species, while Arabidopsis AtPGP1 to 3 share moderate sequence similarity (Fig. 3). The large divergence in HvPGP of barley may reflect the evolutional distance between monocotyledon and dicotyledon.
|
The physiological function of a plant mdr-like gene has been elucidated only for AtPGP1 (Sidler et al., 1998), but the substrate to be transported by this ABC protein is not yet known. The Cjmdr1 cDNA isolated in this study demonstrates for the first time a close correlation between its expression and the accumulation of berberine. Further studies to clarify the role of the plant mdr family members in alkaloid accumulation in plant cells will be possible using the cDNA as a tool.
Acknowledgments
We thank Kyoto Botanical Garden of Takeda Chemical Industries for the intact plant of C. japonica. 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 (KY).
Notes
1 The sequence of the cDNA reported here will appear in the DDBJ/GenBank DNA databases under Accession No. AB043999. 
4 To whom correspondence should be addressed. Fax: +81 75 753 6398. E-mail: fumihiko{at}kais.kyoto\|[hyphen]\|u.ac.jp
References
Davies TGE, Theodoulou FL, Hallahan DL, Forde BG.1997. Cloning and characterization of a novel P-glycoprotein homologue from barley. Gene 199, 195–202.[Web of Science][Medline]
Dudler R, Hertig C.1992. Structure of an mdr-like gene from Arabidopsis thaliana: Evolutionary implications. Journal of Biological Chemistry 267, 5882–5888.
Loo TW, Clarke DM.1993. Functional consequences of phenylalanine mutations in the predicted transmembrane domain of P-glycoprotein. Journal of Biological Chemistry 268, 19965–19972.
Murashige T, Skoog F.1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497.
Rea P.1999. MRP subfamily ABC transporters from plant and yeast. Journal of Experimental Botany 50, 895–943.[Abstract]
Sato F, Takeshita N, Fujiwara H, Katagiri Y, Huan L, Yamada Y.1994. Characterization of Coptis japonica cells with different alkaloid productivities. Plant Cell, Tissue and Organ Culture 38, 249–256.
Sato F, Yamada Y.1984. High berberine-producing cultures of Coptis japonica cells. Phytochemistry 23, 281–285.
Sato H, Kobayashi Y, Fukui H, Tabata M.1990. Specific differences in tolerance to exogenous berberine among plant cell cultures. Plant Cell Reports 9, 133–136.
Sato H, Tanaka S, Tabata M.1993. Kinetics of alkaloid uptake by cultured cells of Coptis japonica. Phytochemistry 34, 697–701.
Sidler M, Hassa P, Hasan S, Ringli C, Dudler R.1998. Involvement of an ABC transporter in a developmental pathway regulating hypocotyl cell elongation in the light. The Plant Cell 10, 1623–1636.
Ueda K, Cardarelli C, Gottesman MM, Pastan I.1987. Expression of a full-length cDNA for the human ‘MDR1’ gene confers resistance to colchicine, doxorubicin and vinblastine. Proceedings of the National Academy of Sciences, USA 84, 3004–3008.
Wang W, Takezawa D, Poovaiah BW.1996. A potato cDNA encoding a homologue of mammalian multidrug resistant P-glycoprotein. Plant Molecular Biology 31, 683–687.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. J. Blakeslee, A. Bandyopadhyay, O. R. Lee, J. Mravec, B. Titapiwatanakun, M. Sauer, S. N. Makam, Y. Cheng, R. Bouchard, J. Adamec, et al. Interactions among PIN-FORMED and P-Glycoprotein Auxin Transporters in Arabidopsis PLANT CELL, January 1, 2007; 19(1): 131 - 147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 M. Jasinski, E. Ducos, E. Martinoia, and M. Boutry The ATP-Binding Cassette Transporters: Structure, Function, and Gene Family Comparison between Rice and Arabidopsis Plant Physiology, March 1, 2003; 131(3): 1169 - 1177. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Terasaka, N. Shitan, F. Sato, F. Maniwa, K. Ueda, and K. Yazaki Application of Vanadate-Induced Nucleotide Trapping to Plant Cells for Detection of ABC Proteins Plant Cell Physiol., February 15, 2003; 44(2): 198 - 200. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 K. Sakai, N. Shitan, F. Sato, K. Ueda, and K. Yazaki Characterization of berberine transport into Coptis japonica cells and the involvement of ABC protein J. Exp. Bot., September 1, 2002; 53(376): 1879 - 1886. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||