Brassinosteroid pathway

The future will give us the chance to characterize more enzymes in the brassinosteroid pathway, leading not only to an improved understanding, but also to the possibility of modifying internal brassinosteroid levels, thus getting a new insight in the mode of action of brassinosteroids. 

Figure 8.3 Brassinosteroid biosynthetic pathway. DWF4 and CPD catalyze the 22a- and 23a-hydroxylation reaction, respectively, in both the early and late C-6 oxidation pathways. CYP85 catalyzes the C-6 oxidation of 6-deoxytyphasterol and 6-deoxocastasterone and tentatively the C-6 oxidation of 6-deoxocathasterone and 6-deoxoteasterone (arrows not shown). DDWF1 dark-induced DIP/7-like protein 1 DWF4 DWARF4 CPD CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARF.

CHOE, S, FUJIOKA, S., NOGUCHI, T., TAKATSUTO, S. YOSHIDA, S., FELDMANN, K.A., Overexpression of DWARF4 in the brassinosteroid biosynthetic pathway results in increased vegetative growth and seed yield in Arabidopsis, Plant J., 2001,26, 1-11. 

Clouse, S.D. (2002) Brassinosteroid signal transduction clarifying the pathway from ligand perception to gene expression. Mol. Cell 10, 973-982. 

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. 

Fig. (1). Proposed pathways and genes involved in sterol and brassinosteroid biosynthesis.

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. 

Another dwarf mutant of Arabidopsis, sax], defines a step upstream of DWF1 in the brassinosteroid biosynthesis pathway [27]. Rescue experiments with intermediates showed that saxl is involved in the oxidation and isomerization of 3P-hydroxyl,A5 6 precursors to 3-oxo-A4 5 steroids (Fig. (4)). 

An enormous progress in investigating the brassinosteroid biosynthesis was made by mutant analysis and the use of very sensitive analytical methods, leading within few years to a logical and very attractive putative biosynthetic pathway. 

Nevertheless, there are many questions still open because of problems to detect enzyme activities corresponding to each step of the pathway. The model of biosynthesis pathway was put together by studying the metabolism of exogenously applied intermediates in cell cultures of various origins and combining these results with data of native brassinosteroid patterns. It is more or less accepted that there are three pathways in parallel, the early and the late C6 oxidation pathway, as well as the 24/ -epimers follow ing the same route. Some observations in the analysis of native brassinosteroid patterns suggest a possible connection between the pathways. It was shown that seeds of Arabidopsis contain castasterone and 24-epi-brassinolide [34]. Also members of both 24-epimers, brassinolide and 24-epi-brassinolide were detected in tomato seeds [Winter, unpublished]. 

Figure 33 Biosynthetic pathways of brassinosteroids. The larger the arrows, the more principal the routes. Arabidopsis enzymes involved in the biosynthetic reactions are indicated in bold letters.

Asami, T., Mizutani, M., Fujioka, S., Goda, Fi., Min, Y.K., Shimada, Y., Nakano, T., Takatsuto, S., Matsuyama, T., Nagata, N., Sakata, K. and Yoshida, S. (2001) Selective interaction of triazole derivatives with DWF4, a cytochrome P450 monooxygenase of the brassinosteroid biosynthetic pathway, correlates with brassinosteroid deficiency in planta. /. Biol. Chem., T7B, 25687-91. 

Fujioka, S., Takatsuto, S. and Yoshida, S. (2002) An early C-22 oxidation branch in the brassinosteroid biosynthetic pathway. Plant Physiol., 130, 930-9. 

K, Yoshida S. Selective interaction of triazole derivatives with DWE4, a c)4ochrome P450 monooxygenase of the brassinosteroid biosynthetic pathway, correlates with brassinosteroid deficiency in Planta. J. Biol. Chem. 2001 276 25687-25691. 

Belkhadir Y, Wang X, Chory J. Brassinosteroid signaling pathway. Sci. STKE. 2006 cm4. 

Leubner-Metzger, G. (2000). Brassinosteroids and gibberellins promote tobacco seed germination by distinct pathways. Planta, 213, 758-763. 

Microbial Transformation of Brassinosteroids. Until now, information concerning the microbial transformations of brassinosteroids has not been published. Such biotransformations could open novel pathways to additionally functionalized members for structure-activity investigations as well as provide information about possible metabolic processes of such compounds. Furthermore, the intermediate metabolites may have practical application. 

Like all steroids, brassinosteroids derive from a single common precursor mevalonic acid. Some phytohormones are synthesized totally or in part via the isoprenoid pathway, such as absdsic acid, gibberellins and cytokinins. The importance of this biosynthetic pathway in processes involved with cell cycle regulation and tumorigenesis in mammals is well documented. Having in mind the similarities between certain regulatory systems in plants and animals the question arose whether brassinosteroids as putative plant steroid hormones would show a specific effect on plant tumor cells. 

Two compounds common in plant metabolism are believed to be precursors of isoprenoid cytokinins in plants adenosine-5 -monophosphate (AMP) and A -isopentenylpyrophos-phate (iPP). As a final product of the mevalonate pathway, the latter substance serves also as a precursor for a wide spectrum of metabolites including some other plant hormones, as abscisic acid, gibberellins and brassinosteroids. The hypothetical scheme of reactions resulting in the formation of iPA, Z and DHZ is given in Fig. 2. The enzyme of entry into isoprenoid cytokinin formation is A -isopentenylpyrophosphate 5 -AMP-A -iso-pentenyltransferase (EC 2.5.1.8, trivially named cytokinin synthetase ). This enzyme activity was first detected in a cell-free preparation from the slime mould Dictyostelium discoideum [7,8]. Later the enzyme from higher plants (cytokinin-independent tobacco callus [9,10] and immature Zea mays kernels [11]) was described and the data were recently summarised in [12], The enzyme is very specific as far as the substrate is concerned [13,14] only the nucleotide AMP can be converted and only iPP (with a double bond in A position) may function as a side chain donor. 

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).

Molecular approaches have contributed considerably to our current understanding of phytohormone action and signal transduction. Putative receptors have been identified for ethylene, cytokinin and brassinosteroids, whereas for hormones such as auxin and GAs components acting more downstream in signal transduction pathways have been identified. However, many steps in phytohormone signalling are still unclear and also the cross-talk between hormones is a field that has hardly been touched. 

The biosynthetic pathway for brassinosteroids has not yet been fully elucidated. Models as presented in figure 12.3 for two parallel pathways assigned as the early C-6 oxidation and the late C-6-oxidation pathways have been... 

---