51NISR Research GRANT
2010 Young Investigator Research Grant

Analysis of Plant Secondary Metabolite Transport andApplication to High Production Using Plant Cultured Cells
Nobukazu SHITAN
Laboratory of Natural Medicinal Chemistry, Kobe Pharmaceutical University

Research aims
Plants produce numerous secondary metabolites whichshow various biological activities1). Some of these are used
as medicines, such as anticancer drugs. To effectively harvest these valuable compounds from plant cells, biosynthetic enzymes and cell culturing techniques have beenstudied for many decades2). However, it is difficult toachieve high production of the bioactive secondary metabolites with plant cells. Our recent studies showed that not
only genes involved in biosynthesis but also genes relevantto the transport of these metabolites are important forachieving high production of these compounds3). Hence,
knowledge of membrane transport mechanisms is necessary
for systemic metabolic engineering. Recently, we were able
to isolate the novel vacuolar-type alkaloid transporter, NtJAT1 (Nicotiana tabacum jasmonate-inducible alkaloidtransporter1) by analyzing the vacuolar nicotine transport intobacco cultured cells (BY-2 cells)4). In this study, wesought to identify tobacco proteins that regulate the transport activity of Nt-JAT1 using the yeast two-hybrid screening. We also attempted to overexpress the alkaloid transporter in order to investigate its effect on alkaloid biosynthesis in BY-2 cells.
Methods
To identify regulatory protein factors that control thetransport activity of Nt-JAT1, yeast two-hybrid assays wereperformed. Cultured tobacco cells (BY-2 cells) were treatedwith 50 m M methyl jasmonate to induce the expression ofNt-JAT1. An N. tabacum DNA library was constructedfrom the induced BY-2 cells using pJG4-5 prey vector.
Then, an Nt-JAT1 C-terminal bait was used to screen
120,000 clones of the tobacco cDNA library with theFig. 1

Screening of candidates of Nt-JAT1 regulatory protein

LexA-based GFP two-hybrid system (Grow’n’Glow system; MoBiTec). Positive clones were selected for leucine
prototrophy and green fluorescent protein expression in sequential steps of selection on plates. Colony PCRs wereperformed on potential positive clones, and amplified
DNAs were sequenced.
To investigate the effect of alkaloid transporter overexpression on alkaloid biosynthesis, we transformed BY-2
cells with GFP-chimera constructs of Nt-JAT1 and NtJAT2, the homologue of Nt-JAT1, using an Agrobacteriummediated transformation method. Nt-JAT2-GFP-expression
in BY-2 cells was observed under a Zeiss LSM700 confocal
microscope.
Results
Our yeast two-hybrid screen of approximately 120,000
yeast clones from tobacco cDNA library resulted in isolation of 4 candidate clones for regulators of Nt-JAT1 (No.
326, 759, 778, 870) (Fig. 1). Although we were not able toamplify the cDNA of No. 870, cDNAs of other clones
could be amplified well and were sequenced. The cDNA inserts in the clones No. 759 and No. 778 were ca. 700 bp and
1,400 bp, respectively. BLAST search analysis revealed that
their gene products have homology with ‘hypothetical proteins’. The cDNA insert in the clone No. 326 was ca.
1,500 bp and encoded a protein similar to SEC12P-LIKE 2PROTEIN. This gene was expressed constitutively in BY-2cells during the entire growth period. In the model plant
Arabidopsis thaliana, this protein was reported to be localized to the ER and to regulate the expression of phosphatetransporter5).
Transformation of Nt-JAT1-GFP and Nt-JAT2-GFP constructs into BY-2 cells was performed to examine the effects of the alkaloid transporter overexpression on alkaloid

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to a protein involved in regulation of transporters. Furthermore, transgenic BY-2 cells which express Nt-JAT2-GFP atthe tonoplast were established. Further investigation of alkaloid transport mechanisms using the clones and transgenic cells obtained in this study would provide important
knowledge for transport engineering and enable us toachieve the high production of valuable secondary metabolites in cultured cells.

Fig. 2

Fluorescence of Nt-JAT2-GFP in BY-2 cells

References1)

biosynthesis. We successfully obtained two transgenic lines(OX1 and OX3) overexpressing Nt-JAT2-GFP. The fluorescence of Nt-JAT2-GFP suggests that this protein is localized to tonoplast (Fig. 2).
Conclusion
We isolated three candidate clones (No. 326, 759, 778)
encoding proteins that regulate the function of Nt-JAT1, thenicotine transporter in tobacco. Particularly, clone No. 326
was identified as a strong candidate based on its homology

Croteau, R., Kutchan, T. M., and Lewis, N. G. (2000) In Biochemistry & Molecular Biology of Plants, Buchanan, B., Gruissem, W.,and Jones, R., (eds.), American Society of Plant Physiologists,Maryland, pp. 1250–1318.
2) Yazaki, K. (2004) In Handbook of Plant Biotechnology, Vol. 2,Christou, P., and Klee, H. (eds.), John Wiley & Sons, pp. 811–857.
3) Shitan, N., and Yazaki, K. (2007) Curr Pharm Biotechnol. 8: 244–252.
4) Morita, M., Shitan, N., Sawada, K., Van Montagu, M. C. E., Inzé, D.,Rischer, H., Goossens, A., Oksman-Caldentey, K.-M., Moriyama, Y.,and Yazaki, K. (2009) Proc Natl Acad Sci USA. 106: 2447–2452.
5) González, E., Solano, R., Rubio, V., Leyva, A., and Paz-Ares, J.
(2005) Plant Cell 17: 3500–3512.