Brassinosteroids metabolism

The results of the rice lamina inclination test (RLIT) indicated an extraordinary high activity of 25-hydroxy-24-epibrassinolide (90). This compound is about ten times more active than 24-epibrassinolide (12), indicating that the hydroxylation at C-25 is an activating step in the brassinosteroid metabolism. Therefore, 25-hydroxy-24-epibrassinolide (90) is, next to brassinolide (1), one of the most active brassinosteroids known until now. In comparison with 25-hydroxy-24-epibrassinolide (90), the 26-hydroxylated metabolite (91) was clearly less active. As in other groups of steroidal hormones, for instance vitamine D metabolites, hydroxylation at C-25 seems to be essential for high activity. ... 

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 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],... 

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]. 

Adam, G. and Schneider, B. (1999) Uptake, transport and metabolism, in Brassinosteroids - Steroidal Plant Hormones (eds A. Sakurai, T. Yokota and S. Clouse). Springer, Tokyo, Japan, pp. 113-36. 

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

The other steroids, such as bile acids (cholane), vitamin D, saponin steroids, steroid alkaloids, cardiac glycosides, and brassinosteroids, also have biologically important activities. Due to the metabolic versatility of steroid molecules, extremely complex mixtures are often encountered, necessitating the use of chromatographic methods (HPLC, TLC, GC) for their analyses. 

Structural characteristics of unknown brassinosteroids in immature seed of P. vulgaris have been elucidated by GC/MS analyses. They are either stereoisomers of known brassinosteroids, or brassinosteroids that have been oxidized during metabolism [ 23-keto-brassinosteroids ( 34 ), brassinosteroids bearing either carbonyl (32 ) or hydroxyl ( 33 )], or brassinosteroids bearing an extra carbon atom [ CO ( 35 ) or COO ( 36 ) ] on the A ring ( Fig. 3 ). However, their complete structures remain to be characterized (30). 

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. 

The rice lamina inclination assay is very sensitive to brassinosteroids (11). In the second set of studies, we examined the metabolism of radioactive castasterone in the rice lamina assay (Yokota, T. et. al., unpublished data). The fate of tritiated castasterone was monitored for 72 hr. During incubation, again brassinolide was not detected. However, polar metabolites accumulated and the amount increased during 72 hr. (Figure 7). The polar metabolites seemed not to be changed after hydrolysis using either enzyme, hydrochloric acid or sodium hydroxide. 

Further studies on transport, distribution and metabolism of brassinosteroids using labelled molecules are necessary. 

It is concluded that brassinosteroids applied to insects with food may be resorbed and may have effects. Apparently the effects depend on the species as well as the dose and type of brassinosteroid applied. Selective metabolism of brassinosteroids in the gut cannot be excluded. The overall effect of an active brassinosteroid is a lengthening of the larval stage by a delay of moulting. 

In the following recent advances in new brassinosteroid structures, synthesis and metabolism with special consideration of results of our laboratory are discussed. 

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,... 

The lack of knowledge on the interconversion and the fate of brassinosteroids in plant systems prompted us to study the metabolism of selected compounds of this type in detail. At the beginning of the metabolic studies on brassinosteroids we had to choose the compounds to be investigated as well as suitable plant systems. 

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. 

---