Biosynthesis of brassinosteroids

Among a variety of BRs, the biosynthetic pathways of brassinolide from campesterol have been elucidated using both transformed and normal cells of Catharanthus roseus (Fig. 3). First reaction toward brassinolide is the conversion of campesterol to campestanol. Then 

Campesterol is converted to campestanol by hydrogenation. Recently it was postulated that this reaction proceeds via (24/ )-24-methylcholest-4-en-3-one and (24/ )-24-methyl- 

5a-cholestan-3-one based on the analogy of androgen metabolism [37] (Fig. 4). And it was demonstrated that the dwarf mutant det2 of Arabidopsis is BR-deficient and has a defect in the 5a-reductase which converts (24R)-24-methylcholest-4-en-3-one to (24R)24-methyl-5a-cholestan-3-one [13]. Hence, the DET2 gene has the same function of mammalian steroid 5a-reductase which reduces the A -double bond of testosterone to form dihydrotestosterone [38,39]. Recently, the dwarf pea mutant Ik was found to have a defect in the conversion of campesterol to campestanol [40]. 

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The biosynthesis of brassinosteroids has been investigated by in vivo feeding of plant cell cultures, in addition to the analysis of native brassinosteroid-pattems in diverse plant species. The proposed pathway (Fig. (1)) leads from unpolar sterol precursors to the polyhydroxylated phytohormone brassinolide, that is known to exhibit the highest biological activity. 

Most known brassinosteroid mutants are defective in genes that code for enzymes required for the biosynthesis of brassinosteroid precursors. This chapter will summarize the upstream part of brassinosteroid biosynthesis pathway. 

The key step in the biosynthesis of brassinosteroids is the conversion of castasterone to brassinolide. This reaction is a lactonization of the steroidal B-ring or a Bayer-Villiger-oxidation. Tomato cell suspension cultures have been extensively studied in respect to the metabolism of 24-< / i-castasterone and 24-ep/-brassinolide [21, 22, 32, 33]. A microsomal fraction of tomato cell cultures, induced by 24-epi-castasterone, was able to convert this substrate into 24-epi-brassinolide (Fig. (11)). The specific enzyme activity was determined to be 230 fkat/mg protein with NADPH serving as the only accepted electrondonor [24],... 

Suzuki, H., Inoue, T., Fujika, S., Saito, T., Takatsuko, S., Yokota, T., Murofushi, N., Yanagisawa, T. and Sakurai, A. (1995) Conversion of 24-methylcholesterol to 6-oxo-24-methylcholestanol, a putative intermediate of the biosynthesis of brassinosteroids, in cultured cells of Catharanthus roseus. Phytochemistry, 40,1391-7. 

Sakurai A, Fujioka S (1997) Studies on biosynthesis of brassinosteroids. Biosci Biotechnol Biochem 61 757-762... 

Fujioka S, Noguchi T, Watanabe B, Takatsuto S, Yoshida S (2000) Biosynthesis of brassinosteroids in cultured cells of Catharanthus roseus. Phytochemistry 53 549-553... 

The first investigations carried out on a genomic basis led to the revision of canonical pathways and to the accelerated analysis of new routes (e.g. biosynthesis of brassinosteroids [3-5]). They have also revealed new and so far overlooked pathways and an extensive crosstalk in plant metabohsm (e.g. [6-8]). 

Some biochemical functions defined by the Arabidopsis dwarf mutants were later confirmed by heterologous expression of genes and by in vivo conversion of postulated substrates [17-20]. As part of these physiological and biochemical studies, tomato cell suspension cultures have also been established to investigate intermediates and enzymes of brassinosteroid biosynthesis and metabolism [21-23]. Enzyme activities from partially purified protein extracts were first detected in this model system [24]. 

The Arabidopsis mutant dwf7/ste 1 is defective in C5-desaturation of episterol (Fig. (2)) [18], thus impaired in an enzyme function involved in a very early step of brassinosteroid precursor biosynthesis. The enzymatic block of dwf7/stel was determined by feeding experiments using l3C-labelled mevalonic acid and a subsequent analysis of endogenous sterol and brassinosteroid precursors. The mutant accumulates episterol with a simultaneous decrease of downstream intermediates (24-methylenecholesterol, campesterol, castasterone, brassinolide). 

This chapter deals with the conversion from cathasterone to brassinolide, the biologically most active brassinosteroid. Only two mutants have been found to be involved in the downstream subpathway. Nevertheless, enzyme activities from enriched protein fractions have first been detected in this part of brassinosteroid biosynthesis. 

Figure 32 Structures of triazole inhibitors of brassinosteroid biosynthesis.

Takao Yokota is Professor of the Department of Biosciences, Teikyo University. He received his Ph.D. degree from the University of Tokyo in 1971. He became an Assistant Professor in the University of Tokyo in 1970, then a Lecturer in 1988. In 1990, he was assigned to the present position. He has been involved in brassinosteroid research for nearly 30 years. His major research interests concern the biosynthesis and physiological roles of brassinosteroids. 

Fujioka, S. and Yokota, T. (2003) Biosynthesis and metabolism of brassinosteroids. Annu. Rev. Plant Biol, 54,137-64. 

Wang, Z., Nakano, T., Gendron, J., He, J., Chen, M., Vafeados, D., Yang, Y., Fu-jioka, S., Yoshida, S., Asami, T. and Chory, J. (2002) Nuclear-localized BZRl mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev. Cell, 2, 505-13. 

The effect of homoBR on flowering tissues was to produce bisexual and pistillate flowers on a staminate inflorescence. Also, sepals were deformed (24), and one would suspect some of these effects were due to induced ethylene biosynthesis, as the dosage of the brassinosteroid used was very high. Excess hormone levels are known to induce the biosynthesis of ethylene BR can also do this, and it interacts with auxin and cytokinin in the induction (43). BR can also affect endogenous auxin and abscisic acid levels in treated tissue (21,44,45). Thus BR does have multiple and modulatory effects. 

In contrast to the biosynthesis, aspects of interconversion and metabolism of brassinosteroids have been poorly investigated until now. As assumed for classical phytohormones, also in the case of brassinosteroids, different types of conjugates may be involved in the biosynthesis,... 

Fig. 2. Biosynthesis of typical plant sterols. Boxed sterols are probable precursors of various brassinosteroids. Among these sterols, sitosterol, stigmasterol, campesterol, 24-epicampesterol and cholesterol are the most common end-of-pathway sterols in plants. Italic letters refer to the lesions in the biosynthesis mutants of Arabidopsis (dim) and pea (Ikb).

Figure 12.2. A model of the regulatory machinery for brassinosteroid sensing and biosynthesis. Upon perception of brassinosteroids, the receptor BRII signals via a phosphorylation cascade to regulate gene expression and cell expansion. Reprinted with permission from Thummel and Chory (2002) .

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