First published online July 9, 2004; 10.1104/pp.104.043588
Plant Physiology 135:1231-1242 (2004)
© 2004 American Society of Plant Biologists
BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES
Novel Biosynthetic Pathway of Castasterone from Cholesterol in Tomato1
Tae-Wuk Kim,
Soo Chul Chang,
June Seung Lee,
Suguru Takatsuto,
Takao Yokota and
Seong-Ki Kim*
Department of Life Science, Chung-Ang University, Seoul 156–756, Korea (T.-W.K., S.-K.K.); University College, Yonsei University, Seoul 120–749, Korea (S.C.C.); Department of Biological Science, Ewha Womans University, Seoul 120–750, Korea (J.S.L.); Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943–8512, Japan (S.T.); and Department of Biosciences, Teikyo University, Utsunomiya 320–8551, Japan (T.Y.)
Endogenous brassinosteroids (BRs) in tomato (Lycopersicon esculentum) seedlings are known to be composed of C27- and C28-BRs. The biosynthetic pathways of C27-BRs were examined using a cell-free enzyme solution prepared from tomato seedlings that yielded the biosynthetic sequences cholesterol  cholestanol and 6-deoxo-28-norteasterone  6-deoxo-28-nor-3-dehydroteasterone 6-deoxo-28-nortyphasterol 6-deoxo-28-norcastasterone 28-norcastasterone (28-norCS). Arabidopsis CYP85A1 that was heterologously expressed in yeast mediated the conversion of 6-deoxo-28-norCS to 28-norCS. The same reaction was catalyzed by an enzyme solution from wild-type tomato but not by an extract derived from a tomato dwarf mutant with a defect in CYP85. Furthermore, exogenously applied 28-norCS restored the abnormal growth of the dwarf mutant. These findings indicate that the C-6 oxidation of 6-deoxo-28-norCS to 28-norCS in tomato seedlings is catalyzed by CYP85, just as in the conversion of 6-deoxoCS to CS. Additionally, the cell-free solution also catalyzed the C-24 methylation of 28-norCS to CS in the presence of NADPH and S-adenosylmethionine (SAM), a reaction that was clearly retarded in the absence of NADPH and SAM. Thus it seems that C27-BRs, in addition to C28-BRs, are important in the production of more active C28-BRs and CS, where a SAM-dependent sterol methyltransferase appears to biosynthetically connect C27-BRs to C28-BRs. Moreover, the tomato cell-free solution converted CS to 26-norCS and [2H6]CS to [2H3]28-norCS, suggesting that C-28 demethylation is an artifact due to an isotope effect. Although previous feeding experiments employing [2H6]CS suggested that 28-norCS was synthesized from CS in certain plant species, this is not supported in planta. Altogether, this study demonstrated for the first time, to our knowledge, that 28-norCS is not synthesized from CS but from cholesterol. In addition, CS and [2H6]CS were not converted into BL and [2H6]BL, respectively, confirming an earlier finding that the active BR in tomato seedlings is not BL but CS. In conclusion, the biosynthesis of 28-norBRs appears to play a physiologically important role in maintaining homeostatic levels of CS in tomato seedlings.
1 This work was supported by the Korean Science and Engineering Foundation (grant no. R01–2002–000–00367–0 to S.-K.K. and S.C.C.), by the Japan Society for the Promotion of Science (grant no. 1146007 to T.Y.), and by Human Frontier Research Program (grant no. 2000–162 to T.Y.).
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.043588.
* Corresponding author; e-mail skkimbio{at}cau.ac.kr; fax 82–2–820–5206.
Received March 25, 2004;
returned for revision March 29, 2004;
accepted March 29, 2004.
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T.-W. Kim, J.-Y. Hwang, Y.-S. Kim, S.-H. Joo, S. C. Chang, J. S. Lee, S. Takatsuto, and S.-K. Kim
Arabidopsis CYP85A2, a Cytochrome P450, Mediates the Baeyer-Villiger Oxidation of Castasterone to Brassinolide in Brassinosteroid Biosynthesis
PLANT CELL,
August 1, 2005;
17(8):
2397 - 2412.
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