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. 2004 Jul;135(3):1231-42.
doi: 10.1104/pp.104.043588. Epub 2004 Jul 9.

Novel biosynthetic pathway of castasterone from cholesterol in tomato

Tae-Wuk Kim  1 , Soo Chul Chang, June Seung Lee, Suguru Takatsuto, Takao Yokota, Seong-Ki Kim
Affiliations

Novel biosynthetic pathway of castasterone from cholesterol in tomato

Tae-Wuk Kim et al. Plant Physiol. 2004 Jul.

Abstract

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.

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Figures

Figure 1.
Figure 1.
BR biosynthetic pathways in tomato seedlings. The solid arrows indicate the biosynthetic steps that have been demonstrated. The dotted arrows indicate the biosynthetic steps that have yet to be verified. The presence of BRs with an asterisk has not been demonstrated in plants.
Figure 2.
Figure 2.
Metabolism of CS and [2H6]CS in young tomato plants. CS and [2H6]CS were not converted to BL and [2H6]BL, respectively, but were catabolized to 26-norCS and [2H3]28-norCS, respectively.
Figure 3.
Figure 3.
Distribution of biological activity following RP-HPLC analysis of the 28-norCS metabolite extant in the young tomato enzyme preparation. Biological activity was based on results of the rice lamina inclination assay. HPLC was carried out using a NovaPak C18 column (8 × 100 mm) at a flow rate of 1 mL min−1 with 40% acetonitrile. Fractions were collected every minute. The arrow indicates the elution points of authentic BRs.
Figure 4.
Figure 4.
Biological activity of C27-BRs in the rice lamina inclination assay. The activity of 6-deoxo-28-norCT, 6-deoxo-28-norTE, 6-deoxo-28-nor-3-DHT, 6-deoxo-28-norTY, and 6-deoxo-28-norCS was measured in the concentration range from 2 × 10−9 m to 2 × 10−7 m (A), while 26-norCS, 28-norCS, and CS was measured in the concentration range 2 × 10−10 m to 2 × 10−8 m (B).
Figure 5.
Figure 5.
Total ion chromatogram of the product of CYP85A1, which was heterologously expressed in the transformed yeast strain, CYP85A1/pYeDP60/WAT21. The arrowed peaks in the upper (A) and lower (B) chromatograms show the products of [2H6]6-deoxoCS and 6-deoxo-28-norCS, respectively.
Figure 6.
Figure 6.
GC-SIM analysis for the conversion of 6-deoxo-28-norCS to 28-norCS in wild type (A) and dwarf mutant (B) of young tomato plants. Enzyme activity was detected in the wild type but not in the dwarf mutant.
Figure 7.
Figure 7.
Growth recovery of the tomato dwarf mutant following treatment with 28-norCS. A, Dark-grown seedlings. B, Average length (cm) of hypocotyls of dark-grown seedlings. C, Light-grown seedlings. Alisa is the wild-type tomato. Bar represents 1 cm.
Figure 8.
Figure 8.
A hypothetical scheme representing the C-24 methylation of 28-norCS to CS in young tomato plants. S indicates the same ring structure as that of 28-norCS and CS.
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