Novel Biosynthetic Pathway of Castasterone from Cholesterol in Tomato

Abstract

Endogenous brassinosteroids (BRs) in tomato (Lycopersicon esculentum) seedlings are known to be composed of C 27-and C 28-BRs. The biosynthetic pathways of C 27-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 4 6-deoxo-28-nor-3-dehydroteasterone 4 6-deoxo-28-nortyphasterol / 6-deoxo-28-norcastasterone / 28-norcastasterone (28-norCS). Arabi-dopsis 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 C 27-BRs, in addition to C 28-BRs, are important in the production of more active C 28-BRs and CS, where a SAM-dependent sterol methyltransferase appears to biosynthetically connect C 27-BRs to C 28-BRs. Moreover, the tomato cell-free solution converted CS to 26-norCS and [ 2 H 6 ]CS to [ 2 H 3 ]28-norCS, suggesting that C-28 demethylation is an artifact due to an isotope effect. Although previous feeding experiments employing [ 2 H 6 ]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 [ 2 H 6 ]CS were not converted into BL and [ 2 H 6 ]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. Brassinosteroid (BR)-deficient mutants such as det2 (Li et al., 1996), dwarf4 (Choe et al., 1998), and cpd (Szekeres et al., 1996) in Arabidopsis, dwarf in tomato (Lycopersicon esculentum; Bishop et al., 1999), and lkb in pea (Pisum sativum; Nomura et al., 1997, 1999) showed reduced shoot elongation, reduced fertility, delayed senescence, altered vasculature, and photomorpho-genesis. The pleiotropic abnormal developments can be rescued only by application of BRs. Mutants such as

Identification and Biosynthesis of Cholest-4-en-3-one and 6-Oxocholetanol in Young Tomato Plants

“…7 In this study, the presence of a partial biosynthetic sequence for the early C6-oxidation pathway for C27-BRs, CHR → cholest-4-en-3-one → CHN → 6-oxoCHN, was confirmed in the plants. Although the downstream biosynthetic sequence from 6-oxo-CHN to 28-norCS has yet to be clearly elucidated, the results of this study indicate that both the early and late C6-oxidation pathway for C27-BRs biosynthesis can generate 28-norCS in tomato plants.…”

“…7 In the late C6-oxidation pathway for C27-BRs biosynthesis in the tomato, the same C6-oxidation from 6-deoxo-28-norCS to 28-norCS has been shown to be catalyzed by a cytochrome P450, designated as CYP85, 7 thereby suggesting that the conversion of CHN to 6-oxoCHN can also be catalyzed by CYP85 in the tomato. To evaluate this possibility, enzyme solutions were prepared from a wild-type (Alisa) and a dwarf mutant (dx) with a defective CYP85 gene, and the conversion of CHN to 6-oxoCHN was assessed with the enzyme solutions using the same enzyme assay as described above.…”

“…In the aid for S-adenosyl-methionine and NAD-PH, 28-norCS is methylated at C-24 to synthesize active C28-BR, CS in tomato plants. 7 Considering that CS is more biologically active than 28-norCS, the early and late C6-oxidation pathway for C 27 -BRs biosynthesis can be concluded to be alternative routes for the synthesis of biologically active C28-BR, CS in the plants.…”

“…1, CS) and brassinolide, are biosynthesized from campesterol (CR) by two parallel biosynthetic pathways designated the early and late C6-oxidation pathway--in many plants. 7 Recently, we showed that young tomato plants exploit a novel biosynthetic pathway to generate a C27-BR, 28-norCS from cholesterol (CHR). 7 The pathway for C 27 -BRs biosynthesis in the tomato plants was basically identical to that of the late C6-oxidation pathway for C28-BRs biosynthesis, thus indicating that the late C6-oxidation pathway for C27-BRs biosynthesis is functional in the plants.…”

“…7 Recently, we showed that young tomato plants exploit a novel biosynthetic pathway to generate a C27-BR, 28-norCS from cholesterol (CHR). 7 The pathway for C 27 -BRs biosynthesis in the tomato plants was basically identical to that of the late C6-oxidation pathway for C28-BRs biosynthesis, thus indicating that the late C6-oxidation pathway for C27-BRs biosynthesis is functional in the plants. This finding compelled us to assess the possible operation of the early C6-oxidation pathway for C27-BRs biosynthesis in the tomato plants.…”