In Vivo and in Vitro Conversion of Teasterone to Typhasterol in Cultured Cells of Marchantia polymorpha

Park, Seung-Hyun; Han, Kwang-Seok; Kim, Tae‐Wuk; Shim, Jae-Kuk; Takatsuto, Suguru; Yokota, Takao; Kim, Seong‐Ki

doi:10.1093/oxfordjournals.pcp.a029628

Cited by 29 publications

(14 citation statements)

References 35 publications

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“…To get information on biosynthesis of BRs in lower plants, we have previously investigated endogenous BRs and their biosynthetic precursors in a liverwort, Marchantia polymorpha, resulting in identification of castasterone (CS) and its two biosynthetic precursors, campesterol (CR) and campestanol (CN) in the lower plant. 8,9 CS, CR and CN are members of the early and late C6-oxidation pathway ( Fig. 1) in higher plants, 10~15 which suggested that the liverwort contained the early and/or late C6-oxidation pathway to produce BRs.…”

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Identification of 6-Deoxocastasterone and Brassinolide from a Liverwort, Marchantia polymorpha

2002

Bulletin of the Korean Chemical Society

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Brassinosteroids (BRs) are steroidal plant hormones which play essential roles in plant growth and morphogenesis. [1][2][3][4] The presence of BRs in lower plants has been already demonstrated, [5][6][7] but biosynthesis of BRs in lower plants has not been well known yet. To get information on biosynthesis of BRs in lower plants, we have previously investigated endogenous BRs and their biosynthetic precursors in a liverwort, Marchantia polymorpha, resulting in identification of castasterone (CS) and its two biosynthetic precursors, campesterol (CR) and campestanol (CN) in the lower plant.8,9 CS, CR and CN are members of the early and late C6-oxidation pathway ( Fig. 1) in higher plants, 10~15 which suggested that the liverwort contained the early and/or late C6-oxidation pathway to produce BRs. To verify that, we attempted to identify other BRs included in the pathway with a large amount of M. polymorpha in the study.Naturally-grown M. polymorpha (5.3 Kg) collected in early July, 2001, at Banpo area, Seoul, was extracted with 80% methanol. After concentrating to aqueous phase in vacuo, 200 ng of deuterium labeled ([26, 28-2 H 6 ]) teasterone (TE), typhasterol (TY), 6-deoxocastasterone (6-deoxoCS), CS and brassinolide (BL) were added to the extract as internal standards for quantitative analysis. By guidance of the rice lamina inclination assay, 16 the extract was reextracted, solvent-partitioned and column-chromatographed using silica gel, Sephadex LH-20 and ODS (RP-18). The resulting active fractions were combined, and further purified by a reversed phase HPLC.After HPLC, BR-like activities were detected in several fractions (Fig. 2). To get hint for the presence of BRs in the active fractions, authentic TE, TY, 6-deoxoCS, CS and BL were analyzed by the same HPLC condition, providing that the authentic BRs were eluted in the fraction 36, 41, 46, 20 and 14-15, respectively. Therefore, it was thought that fraction I, II, III, IV and V may contain [ 2 H 6 ]-BL, -CS, -TE, -TY and -6-deoxoCS added as internal standard possibly accompanied with deuterium unlabeled ([ 2 H 0 ]) endogenous BL, CS, TE, TY and 6-deoxoCS, respectively. To confirm that, the fractions were derivatized to a bismethaneboronates (BMBs) or monomethaneboronate (MB)-trimethylsilyl (TMSi) ethers, and analyzed by GC-MS and/or GC-selected ion monitoring (SIM).In the GC-SIM, BMB of an active compound in fraction I gave mass ions at m/z 534 and 161 which are a molecular and a base peak for [ 2 H 6 ]-BL BMB at the same retention time as that of authentic sample (Table 1). In addition, the other active compound in the same fraction showed ions at m/z 528 and 155 which were 6 mass less than those derived from

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Identification of 6-Deoxocastasterone and Brassinolide from a Liverwort, Marchantia polymorpha

2002

Bulletin of the Korean Chemical Society

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“…The in vivo and in vitro C-3 epimerization of 6-deoxoTE and TE to 6-deoxoTY and TY, respectively, is mediated by two or more reversible enzymes. Park et al, 1999;Kim et al, 2000cKim et al, , 2003bNoguchi et al, 2000). Recently, it was reported that the D2 gene of rice is responsible for the C-3 oxidation involved in C-3 epimerization (Hong et al, 2003).…”

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Novel Biosynthetic Pathway of Castasterone from Cholesterol in Tomato

Kim

Chang²,

Lee³

et al. 2004

Plant Physiology

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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

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“…3 However, the level was too low to identify BRs from the cultured cells of M. polymorpha. Nevertheless, the presence of enzymes responsible for the conversion of teasterone to castasterone, a partial biosynthetic sequence for BRs, was demonstrated in the Marchantia cells.…”

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“…The concentrated residue of the 80% methanol soluble fraction was partitioned again between phosphate buffer (pH 7.8) and ethyl acetate (1 L × 3). The concentrated ethyl acetate soluble fraction (23.1 g) was purified by silica gel column chromatography (150 g, Merck) eluted stepwise with chloroform containing 0, 1, 2, 3,4,5,6,7,8,9,10, 20, 50 and 100% methanol (100 mL each). The fractions eluted with 4-8% methanol in chloroform showed activity based on the rice lamina inclination assay.…”

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“…Nevertheless, the presence of enzymes responsible for the conversion of teasterone to castasterone, a partial biosynthetic sequence for BRs, was demonstrated in the Marchantia cells. 3,4 Furthermore, campesterol (24α-methylcholesteol) and campestanol (24α-cholestanol), which are known to be a biosynthetic precursors of BRs, were recently identified from M. polymorpha. 5 These events strongly suggested the presence of BRs in the lower plant, and led us to investigate the occurrence of BRs in naturally-grown M. polymorpha.…”

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Identification of a Brassinosteroid, Castasterone from Marchantia polymorpha

2002

Bulletin of the Korean Chemical Society

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The presence of steroidal plant hormones, brassinosteroids (BRs), in lower plants has been demonstrated in a green alga and a fern.1,2 Recently, we revealed that liverworts, which are phylogenically higher than algae, contain BRs based on the rice lamina inclination bioassay of the extract from cultured cells of a liverwort, Marchantia polymorpha. 3 However, the level was too low to identify BRs from the cultured cells of M. polymorpha. Nevertheless, the presence of enzymes responsible for the conversion of teasterone to castasterone, a partial biosynthetic sequence for BRs, was demonstrated in the Marchantia cells.3,4 Furthermore, campesterol (24α-methylcholesteol) and campestanol (24α-cholestanol), which are known to be a biosynthetic precursors of BRs, were recently identified from M. polymorpha.5 These events strongly suggested the presence of BRs in the lower plant, and led us to investigate the occurrence of BRs in naturally-grown M. polymorpha.Whole bodies of naturally-grown M. polymorpha (4.3 Kg) were homogenized and extracted with 80% methanol (4 L × 3). The extracts were concentrated to aqueous phase in vacuo and re-extracted with chloroform (1 L × 4). After reducing to dryness in vacuo, the chloroform fraction was solventpartitioned between n-hexane and 80% methanol (1 L × 3). The concentrated residue of the 80% methanol soluble fraction was partitioned again between phosphate buffer (pH 7.8) and ethyl acetate (1 L × 3). The concentrated ethyl acetate soluble fraction (23.1 g) was purified by silica gel column chromatography (150 g, Merck) eluted stepwise with chloroform containing 0, 1, 2, 3,4,5,6,7,8,9,10, 20, 50 and 100% methanol (100 mL each). The fractions eluted with 4-8% methanol in chloroform showed activity based on the rice lamina inclination assay. The fractions were combined (3.9 g), dissolved in 50% methanol (100 mL), and subjected to ODS column (100 mL, Merck LiChroprep RP-18) chromatography. The elution was carried out with aqueous methanol increasing the methanol content every 10% from 50% to 100% (100 mL each). The biologically active fractions eluted with 70-90% methanol were concentrated and loaded on Sephadex LH-20 column (bed volume 340 mL, 22 × 900 mm) eluted with a mixture of methanolchloroform (4 : 1) at a flow rate of 0.5 mL min with the solvent system programmed. 6 As shown in Figure 2, strong activity was detected in HPLC fraction 17-19, whose retention time was similar to that of castasterone (18.2 min), suggesting that castasterone was included in the fraction. In addition, moderate activity was observed in the fraction 12-13 whose retention time was almost equal to that of authentic brassinolide (12.8 min) or 28-norbrassinolide (12.8 min). To verify this, the compounds in the fractions were derivatized into a bismethaneboronate with methaneboronic acid (1 mg) in pyridine (2 mL) at 80 o C for 30 min, and analyzed by GC-MS (EI, 70 eV) using Hewlett-Packard 6890-5973 fitted with a capillary column (HP-5, 0.25 mm × 30 m, 0.25 µm film thickness). 7Bismethaneboronate of the a...

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In Vivo and in Vitro Conversion of Teasterone to Typhasterol in Cultured Cells of Marchantia polymorpha

Cited by 29 publications

References 35 publications

Identification of 6-Deoxocastasterone and Brassinolide from a Liverwort, Marchantia polymorpha

Identification of 6-Deoxocastasterone and Brassinolide from a Liverwort, Marchantia polymorpha

Novel Biosynthetic Pathway of Castasterone from Cholesterol in Tomato

Identification of a Brassinosteroid, Castasterone from Marchantia polymorpha

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