| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online July 9, 2004; 10.1104/pp.104.043588 Plant Physiology 135:1231-1242 (2004) © 2004 American Society of Plant Biologists Novel Biosynthetic Pathway of Castasterone from Cholesterol in Tomato1Department of Life Science, Chung-Ang University, Seoul 156–756, Korea (T.-W.K., S.-K.K.); University College, Yonsei University, Seoul 120–749, Korea (S.C.C.); Department of Biological Science, Ewha Womans University, Seoul 120–750, Korea (J.S.L.); Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943–8512, Japan (S.T.); and Department of Biosciences, Teikyo University, Utsunomiya 320–8551, Japan (T.Y.)
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.
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 photomorphogenesis. The pleiotropic abnormal developments can be rescued only by application of BRs. Mutants such as bri1 (Li and Chory, 1997), bin2 (Li et al., 2001; Li and Nam, 2002), bak1 (Nam and Li, 2002) in Arabidopsis, lka in pea (Nomura et al., 1997, 2003), and curl-3 in tomato (Koka et al., 2000) also exhibited similar abnormal phenotypes as found in BR-deficient mutants; however, the phenotype of the mutants could not be restored to that of the wild type by application of BRs, indicating that they are BR-insensitive mutants generated by a disruption in BR signaling. Consequently, BRs are now thought of as essential chemical signals, plant hormones, the endogenous levels of which must be properly maintained in plant cells to facilitate the normal growth and developments of plants.
To date, over 50 BRs have been identified from the entire plant kingdom (Fujioka, 1999
Several genes or enzymes involved in BR biosynthesis have been characterized by molecular analyses of BR biosynthesis mutants. The Arabidopsis DET2 gene possesses sequence identity to mammalian 5  -reductase, which mediates the reduction of testosterone to dihydrotestosterone, indicating that DET2 encodes a steroid 5-reductase that hydrogenates 24-methylcholest-4-en-3-one in the conversion of campesterol (CR) to CN (Fujioka et al., 1997; Li et al., 1997; Noguchi et al., 1999). Arabidopsis DWF4 and CPD share high amino acid sequence similarity to mammalian steroid cytochrome P450 monooxygenases (Cyt P450s), and DWF4 and CPD encode Cyt P450s designated as CYP90B1 and 90A1 that catalyze the C-22 and C-23 hydroxylation of BRs, respectively (Szekeres et al., 1996; Choe et al., 1998). The C-6 oxidation of 6-deoxoCS to CS has also been shown to be mediated by a Cyt P450, CYP85, through analysis of the tomato DWARF gene which possesses high homology to the mammalian steroid hydroxylase genes (Bishop et al., 1999). DWARF, its Arabidopsis orthologs AtBR6ox1 (CYP85A1) and AtBR6ox2 (CYP85A2), and its rice ortholog OsDWARF (CYP85A1) have been shown to catalyze the C-6 oxidation of not only 6-deoxoCS but also 6-deoxoTE, 6-deoxo-3-DHT, and 6-deoxoTY (Shimada et al., 2001, 2003; Hong et al., 2002). The pea gene DDWF1 (Dark-induced DWF-like protein 1), which encodes a Cyt P450 (CYP92A6), was claimed to catalyze the C-2 hydroxylation of TY to CS and of 6-deoxoTY to 6-deoxoCS through the action of a small GTP-binding protein, Pra2 (Kang et al., 2001). 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. (Choi et al., 1997; Park et al., 1999; Kim et al., 2000c, 2003b; Noguchi 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). We recently found that in Phaseolus vulgaris, CS 6-oxidase (BL synthase), responsible for the conversion of CS to BL, is located in the endoplasmic reticulum membrane, requires molecular oxygen and NADPH, and is inhibited by Cyt P450 inhibitors including carbon monoxide, the effect of which was reversed by irradiation with blue light (Kim et al., 2004).
C27-BRs such as 28-norcastasterone (28-norCS) occur in various plant species ranging from lower to higher plants (Fujioka, 1999
Some investigations have indicated that 28-norCS frequently cooccurs with CS (Fujioka, 1999 In this work, we investigated the biosynthetic pathways of 28-norCS from CHR and related C27 intermediates, in addition to the biosynthetic relationship between 28-norCS and CS, using cell-free preparations derived from tomato seedlings.
28-norCS Is Converted from CHR via C27-BRs in Tomato Enzyme Preparations Tomato seedlings were homogenized, centrifuged, and precipitated with acetone. The precipitates were dissolved in assay buffer and used as a crude enzyme extract for the in vitro conversion experiments. Since isotope-labeled substrates were not available, unlabeled ([2H0]) CHR, CHN, 6-deoxo-28-norCT, 6-deoxo-28-norteasterone (6-deoxo-28-norTE), 6-deoxo-28-nor-3-dehydroteasterone (6-deoxo-28-nor-3-DHT), 6-deoxo-28-norTY, or 6-deoxo-28-norCS were used as substrates. Enzyme products were purified by HPLC and analyzed by GC-MS or GC-SIM following derivatization. Prior to use in the enzymatic incubations, the absence of expected products in the original enzyme preparations was confirmed by gas chromatography- mass spectrometry (GC-MS) and GC-selected ion monitoring (SIM).
As summarized in Table I, the CHR metabolite, derivatized to the trimethylsilyl ether, yielded an identical mass spectrum and GC retention time to those of authentic CHN trimethylsilyl ether, demonstrating that CHR was converted to CHN in the tomato cell-free system. When 6-deoxo-28-norTE was used, two metabolites consisting of 6-deoxo-28-nor-3-DHT and 6-deoxo-28-norTY were detected, suggesting the conversion of 6-deoxo-28-norTE to 6-deoxo-28-norTY via 6-deoxo-28-nor-3-DHT. 6-Deoxo-28-nor-3-DHT was converted to two metabolites, one of which was identified as 6-deoxo-28-norTE while the other metabolite was identified as 6-deoxo-28-norTY. When 6-deoxo-28-norTY was added to the enzyme mixture, three metabolites were detected. Of these, two metabolites were determined to be 6-deoxo-28-nor-3-DHT and 6-deoxo-28-norTE, indicating that the C-3 epimerization from 6-deoxo-28-norTE to 6-deoxo-28-norTY via 6-deoxo-28-nor-3-DHT is a reversible reaction. The third metabolite was identified as 6-deoxo-28-norCS, demonstrating the C2
CS Is Converted to 26-norCS But Not 28-norCS in Tomato Enzyme Preparations The metabolism of CS and [26,28-2H6]CS was investigated separately with the tomato cell-free enzyme solution. HPLC analysis and rice bioassay of the metabolic products derived from CS and [2H6]CS revealed that only fractions 18 and 19, in which both CS and [2H6]CS were eluted, were biologically active. However, in thin-layer chromatography analysis, BR-like purple-bluish spots appeared in fractions 13 and 14. Consequently, these were combined, derivatized, and subsequently analyzed by GC-MS. Although BL was assumed to be the most likely product of CS, no trace amounts of BL or [2H6]BL were detected in fractions 13 and 14 pertaining to CS and [2H6]CS feedings, even by GC-SIM analysis (data not shown). Instead, fractions 13 and 14 derived from CS feedings contained a metabolite with mass-to-charge ratio (m/z) 358, 328/327 and 287 ions representative of a bismethaneboronate (BMB), characteristic of the 6-ketone ring structure such as CS BMB. Further peaks were observed corresponding to a molecular ion at m/z 498 and the most abundant ion at m/z 141 due to fission of C-20/C-22. These 2 ions are 14 mass units less than those of CS BMB, suggesting the presence of either 26-norCS or 28-norCS. Under our GC-MS conditions, the 26-norCS and 28-norCS BMBs showed basically the same mass spectrum, although the former displayed a longer retention time than the latter (Table I). Since the CS metabolite BMB had the same retention time as 26-norCS BMB, the metabolite was determined to be 26-norCS. The activity of the enzyme(s) mediating the conversion of CS to 26-norCS was approximately 55.3 ng mg protein–1 min–1. When [2H6]CS was used, the BMB derivative of the [2H6]CS metabolite obtained yielded characteristic ions for a 6-ketone ring structure at m/z 358, 327/328, and 287, indicating that the metabolite has the same ring structure as [2H6]CS. A molecular ion at m/z 501 and an ion at m/z 144 due to fission of C-20/C-22, both being 17 mass units less than those of [2H6]CS, were also observed, suggesting that the metabolite is either [28-2H3]26-norCS or [26-2H3]28-norCS. The fact that the retention time of the BMB metabolite was 5 s shorter than 28-norCS BMB suggests that the metabolite is [2H3]28-norCS, since under our GC conditions deuterated BRs migrate approximately 5 s faster than the same unlabeled species (Table I). The activity of the enzyme(s) catalyzing the conversion of [2H6]CS to [2H3]28-norCS was approximately 276.6 ng mg protein–1 min–1. Taken together, CS was not converted to BL in the tomato cell enzyme extract, and CS and [26,28-2H6]CS were differentially metabolized to 26-norCS and [26-2H3]28-norCS, respectively (Fig. 2).
CS Is Also Converted from 28-norCS in Tomato Enzyme Preparations
The conversion of 28-norCS to CS represents a reaction in which a methyl group is incorporated at the C-24 position. In plant sterol biosynthesis, alkylation is mediated by a sterol methyltransferase (SMT) with the aid of S-adenosyl-L-Met (SAM; Benveniste, 2002
Biological Activity of C27-BRs
The biological activity of C27-BRs was examined using the rice lamina inclination assay which is based on the specific elongation of adaxial cells of rice lamina joint. As shown in Fig. 4A, 6-deoxo-28-norCT, 6-deoxo-28-norTE, 6-deoxo-28-nor-3-DHT, 6-deoxo-28-norTY, and 6-deoxo-28-norCS displayed almost no biological activity up to 2 x 10–8 M. At 2 x 10–8 M, 6-deoxo-28-norTY and 6-deoxo-28-norCS exhibited slight activity, but much less activity than CS, suggesting that hydroxylation at C-22 and C-23 of the side chain, C-3 epimerization, and C-2
CYP85 Is Responsible for the C-6 Oxidation of 6-deoxo-28-norCS to 28-norCS in Tomato
Studies based on a heterologous functional assay of enzymes involved in the biosynthesis and catabolism of C28-BRs have been limited only to 5
In an effort to determine whether tomato CYP85 possesses the activity, the in vitro conversion of 6-deoxo-28-norCS to 28-norCS was investigated using crude enzyme extracts obtained from wild-type and dwarf mutant tomato plants. Prior to feeding, the absence of 28-norCS in the enzyme preparation of wild-type tomato was confirmed. As expected, enzyme activity for the conversion was detected in the wild-type tomato (Fig. 6A) but not in the dwarf mutant (Fig. 6B). Therefore, it is concluded that tomato CYP85 is responsible for the C-6 oxidation of both 6-deoxoCS and 6-deoxo-28-norCS.
Rescue of the Tomato dwarf Mutant by 28-norCS The physiological importance of C27-BRs biosynthesis was examined in the tomato dwarf mutant that possesses a defect in the C-6 oxidation of both C27- and C28-BRs. As shown in Figure 7, application of 28-norCS clearly reversed the dwarfism in both dark- and light-grown seedlings of the mutants (Fig. 7, A and C). In particular, it was found that the length of hypocotyls in the dark-grown seedlings recovered by up to 80% compared to that of the wild type (Fig. 7B). Therefore, it may be concluded that C27-BRs biosynthesis plays an important role in the BR physiology of tomato plants.
The conversion of CS to BL has been demonstrated in cultured cells of C. roseus and M. polymorpha and the seedlings of C. roseus and Arabidopsis (Suzuki et al., 1993 , 1995; Noguchi et al., 2000; Kim et al., 2003a). Consequently, CS is now considered to be a direct biosynthetic precursor of BL. In certain plants, however, where a large amount of CS is present, no trace amount of BL was detected (Yokota, 1997; Fujioka, 1999). Additionally, the conversion rate of CS to BL is extremely low even in plants where the conversion was confirmed (Suzuki et al., 1995; Sakurai, 1999; Noguchi et al., 2000). These findings suggest the possibility that CS is not only a biosynthetic precursor of BL, but also exerts its own biological activity. Although a higher concentration of CS is needed compared to BL, CS exogenously applied to plants displays the same biological activity as that induced by BL and can rescue abnormal pleiotropic phenotypes in BR-deficient mutants. Moreover, it has recently been demonstrated, albeit to a lesser extent compared to BL, that CS and BL can bind to BRI1, a plasma membrane located receptor of BL, supporting the notion that CS in addition to BL possess biological activity (Wang et al., 2001).
Tomato dwarf and dumpy mutants showing abnormalities in growth and differentiation were rescued only by application of BRs (Bishop et al., 1999
The present cell-free study revealed that CS is metabolized into a less active C27-BR, namely 26-norCS, indicating that C-26 demethylation represents one of the deactivation steps of CS in tomato. The same C-26 demethylation deactivates BL in P. vulgaris and M. polymorpha (Kim et al., 2000a
In tomato, it has been suggested that 28-norCS is synthesized from CHR, the most abundant sterol, through late C-6 oxidation as is the case with the biosynthesis of CS from CR (Nomura et al., 2001
In tomato shoots, C28 6-oxo-intermediates involved in the early C-6 oxidation pathway are not synthesized (Bishop et al., 1999
As stated in the introductory remarks, an earlier indication that 28-norCS is synthesized from CS (Fujioka et al., 2000
Conversely, this study using a tomato cell-free system resulted in the unexpected finding that CS is synthesized from 28-norCS in the presence of NADPH and SAM which act as proton and methyl donors, respectively. This is the first demonstration, to our knowledge, that CS is biosynthesized from cholesterol via C27 intermediates. To our knowledge, C27 BRs have been identified from as many as 12 species (Fujioka et al., 2000
Plant Materials and Chemicals
Tomato (Lycopercicon esculentum) plants (Poong-kwang) were grown in an environmental growth chamber at 28°C under a 16-h light/8-h dark cycle. Young plants grown for 3 to 4 weeks were harvested and stored at –80°C until required. The tomato dwarf mutant and its corresponding wild type (Alisa) were grown under the same conditions and used in enzyme assay to examine CYP85 function. All chemicals used in the biochemical analyses were obtained from Sigma Chemicals (St. Louis). The C27-BRs, 6-deoxo-28-norCT, 6-deoxo-28-norTE, 6-deoxo-28-nor-3-DHT, 6-deoxo-28-norTY, 6-deoxo-28-norCS, 28-norCS, and 26-norCS were synthesized according to literature procedures (Takatsuto et al., 1984
Harvested plants (30 g) were ground in a prechilled mortar and pestle in 80 mL of 0.1 M sodium phosphate (pH 7.4) containing 15 mM 2-mercaptoethanol, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 40 mM ascorbate, 250 mM Suc, and 10% (v/v) glycerol. The homogenate was filtered and centrifuged at 8,000g for 15 min. The supernatant was recentrifuged at 20,000g for 30 min. The resulting supernatant was precipitated by the addition of cold acetone to 40% (v/v) final concentration. The supernatant-acetone mixture was kept at –25°C for 10 min and then centrifuged at 13,000g for 10 min. The resulting precipitate was dissolved in 10 mL of 0.1 M sodium phosphate (pH 7.4) containing 1.5 mM 2-mercaptoethanol and 20% (v/v) glycerol and was used as the cell-free enzyme solution. The protein concentration of the enzyme solution was estimated with a microassay from Bio-Rad (Cambridge, MA) using bovine serum albumin as a standard. Enzyme assays for the conversion of CHR to 28-norCS were initiated by the addition of substrates (5 µg each) CHR, CHN, 6-deoxo-28-norCT, 6-deoxo-28-norTE, 6-deoxo-28-nor-3-DHT, 6-deoxo-28-norTY, and 6-deoxo-28-norCS to the cell-free enzyme solution (3–4 mg protein mL–1) in the presence of NADP/NADPH (for the conversion of CHR to CHN) or NADPH (for the conversion of CHN to 6-deoxo-28-norCT and 6-deoxo-28-nor TY to 28-norCS via 6-deoxo-28-norCS). Following incubation at 37°C for 30 min, enzyme reactions were terminated by the addition of ethyl acetate (1.2 mL x 3). The ethyl acetate-soluble fractions were concentrated in vacuo, dissolved in 50% methanol (MeOH), loaded onto a C18 SepPak cartridge column (SepPak plus C18, Waters, Milford, MA), and eluted with 50% and 100% MeOH (7 mL each). The fraction eluted with 100% MeOH was dried, dissolved in a small amount of MeOH, and then subjected to RP-HPLC (Senshu Pak C18, 10 x 150 mm) eluted at a flow rate of 2.5 mL min–1 with 100% MeOH for the CHR metabolite or acetonitrile (MeCN)-water gradients: 0 to 20 min, 45% MeCN; 20 to 40 min, 45% to 100% MeCN; 40 to 70 min, 100% MeCN for other metabolites. Fractions were collected every minute. Under the same RP-HPLC conditions eluted with 100% MeOH, authentic CHN was detected in fractions 35 to 37. Under the same RP-HPLC conditions eluted with the MeCN-water gradient, authentic 6-deoxo-28-norCT, 6-deoxo-28-norTE, 6-deoxo-28-nor-3-DHT, 6-deoxo-28-norTY, 6-deoxo-28-norCS and 28-norCS were detected in fractions 36 to 38, 49 to 51, 46 to 48, 44 to 46, 59 to 61, and 13 to 15, respectively. Fractions were then analyzed by capillary GC-MS or GC-SIM following suitable derivatization. The conversion of 28-norCS to CS was carried out with the cell-free enzyme solution containing 0.8 mM NADPH, 0.8 mM SAM, and 5 µg 28-norCS in a total volume of 1.2 mL. Following incubation at 37°C for 30 min, [2H6]-CS was added for quantitative analysis, the enzyme product was extracted with ethyl acetate and then loaded onto a C18 SepPak cartridge column as described above. Further purification was achieved by RP-HPLC (NovaPak C18, 8 x 100 mm, Waters) at a flow rate of 1 mL min–1 with 40% MeCN. Fractions were collected every minute and fractions 18 and 19, which showed biological activity in the rice lamina inclination assay, were analyzed by GC-MS following methaneboronation. The specific enzyme activity was calculated by the ratio of the product/the [2H6]CS added as an internal standard. Metabolism of CS and [2H6]CS were also examined with the same enzyme solution in the presence of NADPH cofactor. Following the enzyme assays, products were extracted with ethyl acetate (1.2 mL), chromatographed on a SepPak cartridge column, and finally purified by RP-HPLC as described for the conversion of 28-norCS to CS. In the rice lamina inclination assay, only fractions 18 and 19, where CS and [2H6]CS were added as substrates, showed biological activity. However, in the thin-layer chromatography (Merck HPTLC F254) analysis, a blue-purplish spot at Rf 0.30 was detected in fractions 13 to 15 obtained from both assays after treatment with 70% sulfuric acid followed by heating. Fractions 13 to 15 were combined and subsequently analyzed by GC-MS following methaneboronation.
Rice lamina inclination assays using cv Koshihikari were carried out in an effort to examine the biological activity of BRs (Arima et al., 1984
The GC-MS and GC-SIM analyses were carried out on a Hewlett-Packard (Palo Alto, CA) 5973 mass spectrometer (electron impact ionization, 70 eVage) coupled to 6890 gas chromatography fitted with a fused silica capillary column (HP-5, 0.25 mm x 30 m, 0.25 µm film thickness). The oven temperature was maintained at 175°C for 2 min, elevated to 280°C at a rate of 40°C min–1 and then maintained at 280°C. Helium was used as the carrier gas at a flow rate of 1 mL min–1, and samples were introduced using an on-column injection mode. Methaneboronation was carried out by heating samples dissolved in pyridine containing methaneboronic acid (2 mg mL–1) at 70°C for 30 min. N-methyl-N-TMS-trifluoroacetamide (MSTFA) was used to effect trimethylsilylation.
Total RNAs were extracted from Arabidopsis seedlings using the TRI Reagent (Sigma). cDNA was synthesized from 1 ug of total RNA using the MMLV-reverse transcription system (Promega, Madison, WI) according to the manufacturer's instructions. CYP85A1 (At5g38970) cDNA was amplified by PCR using specific primers to delete the 5'- and 3'-noncoding regions of CYP85A1 cDNA. Specific primers were designed to introduce a BamHI restriction site immediately upstream of the initiation codon and a KpnI site following the stop codon: forward primer 5'-ggatccTGGGAGCAATGATGGTGATGAT-3'; reverse primer 5'-ggtaccTTAGT AGGGTGAAATCCTAAGATG-3'. CYP85A1 cDNA was amplified using 36 thermal cycles (94°C 20 s, 61°C 30 s, 72°C 2 min) with Ex Taq polymerase (Takara Shuzo, Shiga, Japan) and cloned into the pGEM-T easy vector (Promega). PCR error in the nucleotide sequence was checked by DNA sequencing using T7 and Sp6 universal primers. CYP85A1 cDNA fragments digested by BamHI and KpnI were subcloned into the BamHI and KpnI sites of a yeast expression vector (pYeDP60) that contained two selection markers (ADE2 and URA3). The CYP85A1/pYeDP60 construct (85A1/V60) was transformed into WAT21 strain according to the method of Gietz et al. (1992)
For the dark-grown seedlings, tomato seeds (Alisa and dwarf) were surface sterilized in 10% bleach for 30 min. The seeds were rinsed with sterilized water, incubated at 4°C for 2 d, and then planted on an agar-solidified plate containing 1% Suc-MS medium with or without 1 µM 28-norCS. Following 9 d at 22°C under continuous darkness, the seedlings were photographed using a digital camera. For the light-grown seedlings, sterile tomato seeds were planted on 1% Suc-MS medium for 4 d at 22°C under a 16-h/d light cycle. Following this, 1 µM 28-norCS in a 95% ethanol solution was applied to shoot apexes and the roots of the dwarf mutant using a µ-drop method. Seedlings were photographed after 3 d.
The authors are grateful to Dr. D. Pompon and Dr. P. Urban for providing pYeDP60 and the yeast strain WAT21 and to Dr. Gerard B. Bishop for supplying tomato seeds (dwarf and Alisa). Received March 25, 2004; returned for revision March 29, 2004; accepted March 29, 2004.
1 This work was supported by the Korean Science and Engineering Foundation (grant no. R01–2002–000–00367–0 to S.-K.K. and S.C.C.), by the Japan Society for the Promotion of Science (grant no. 1146007 to T.Y.), and by Human Frontier Research Program (grant no. 2000–162 to T.Y.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.043588. * Corresponding author; e-mail skkimbio{at}cau.ac.kr; fax 82–2–820–5206.
Arima M, Yokota T, Takahashi N (1984) Identification and quantification of brassinolide-related steroids in the insect gall and healthy tissues of the chestnut plant. Phytochemistry 23: 1587–1591[CrossRef] Bajguz A, Tretyn A (2003) The chemical characteristic and distribution of brassinosteroids in plants. Phytochemistry 62: 1027–1046[CrossRef][Web of Science][Medline] Benveniste P (2002) Sterol metabolism. In The Arabidopsis Book. American Society of Plant Biologists, Rockville, MD
Bishop GJ, Nomura T, Yokota T, Harrison K, Noguchi T, Fujioka S, Takatsuto S, Jones JD, Kamiya Y (1999) The tomato DWARF enzyme catalyzes C-6 oxidation in brassinosteroid biosynthesis. Proc Natl Acad Sci USA 96: 1761–1766
Bishop GJ, Yokota T (2001) Plants steroid hormones, brassinosteroids: current highlights of molecular aspects on their synthesis/metabolism, transport, perception and response. Plant Cell Physiol 42: 114–120
Choe S, Dilkes BP, Fujioka S, Takatsuto S, Sakurai A, Feldmann KA (1998) The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22 Choi Y-H, Fujioka S, Nomura T, Harada A, Yokota T, Takatsuto S, Sakurai A (1997) An alternative brassinolide biosynthetic pathway via late C-6 oxidation. Phytochemistry 44: 609–613[CrossRef]
Diener AC, Li H, Zhou WX, Whoriskey WJ, Nes WD, Fink GR (2000) Sterol methyltransferase 1 controls the level of cholesterol in plants. Plant Cell 12: 853–870 Fujioka S (1999) Natural occurrence of brassinosteroids in the plant kingdom. In A Sakurai, T Yokota, SD Clouse, eds, Brassinosteroids: Steroidal Plant Hormones. Springer-Verlag, Tokyo, pp 21–45 Fujioka S, Li J, Choi Y-H, Seto H, Takatsuto S, Noguchi T, Watanabe T, Kuriyama H, Yokota T, Chory J, et al (1997) The Arabidopsis deetiolated2 mutant is blocked early in Brassinosteroid biosynthesis. Plant Cell 9: 1951–1962[Abstract] Fujioka S, Noguchi T, Sekimoto M, Takatsuto S, Yoshida S (2000) 28-Norcastasterone is biosynthesized from castasterone. Phytochemistry 55: 97–101[Medline] Fujioka S, Yokota T (2003) Biosynthesis and metabolism of brassinosteroids. Annu Rev Plant Biol 54: 137–164[CrossRef][Medline]
Gietz D, St Jean A, Woods RA, Schiestl RH (1992) Improved methods for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20: 1425 Hong Z, Ueguchi-Tanaka M, Shimizu-Sato S, Inukai Y, Fujioka S, Shimada Y, Takatsuto S, Agematsu M, Yoshida S, Watanabe Y, et al (2002) Loss-of-function of a rice brassinosteroid biosynthetic enzyme, C-6 oxidase, prevents the organized arrangement and polar elongation of cells in the leaves and stem. Plant J 32: 495–508[CrossRef][Web of Science][Medline]
Hong Z, Ueguchi-Tanaka M, Umemura K, Uozu S, Fujioka SY, Takatsuto S, Yoshida S, Ashikari M, Kitano H, Matsuoka M (2003) A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450. Plant Cell 15: 2900–2910 Kang JG, Yun J, Kim DH, Chung KS, Fujioka S, Kim JI, Dae HW, Yoshida S, Takatsuto S, Song PS, et al (2001) Light and brassinosteroid signals are integrated via a dark-induced small G protein in etiolated seedling growth. Cell 105: 625–636[CrossRef][Web of Science][Medline]
Kim T-W, Chang SC, Choo J, Watanabe T, Takatsuto S, Yokota T, Lee JS, Kim SY, Kim S-K (2000a) Brassinolide and [26,28-2H6] brassinolide are differently demethylated by loss of C-26 and C-28, respectively, in Marchantia polymorpha. Plant Cell Physiol 41: 1171–1174 Kim T-W, Chang SC, Lee JS, Hwang B, Takatsuto S, Yokota T, Kim S-K (2004) Cytochrome P450-catalyzed brassinosteroid pathway activation through synthesis of castasterone in Phaseolus vulgaris. Phytochemistry 65: 679–689[CrossRef][Medline] Kim T-W, Han K-S, Joo S-H, Kang M-W, Kim S-K (2000b) Metabolism of brassinolide in suspension cultured cells of Phaseolus vulgaris. B Kor Chem Soc 21: 1044–1046 Kim Y-S, Kim T-W, Kim S-K (2003a) Conversion of 6-deoxocastasterone to brassinolide in a Liverwort, Marchantia polymorpha. B Kor Chem Soc 24: 1385–1388 Kim Y-S, Kim T-W, Kim S-K (2003b) Biotransformation of 6-Deoxotyphasterol in a liverwort, Marchantia polymorpha. B Kor Chem Soc 24: 1541–1543 Kim T-W, Park S-H, Han K-S, Choo J, Lee JS, Hwang S, Kim S-K (2000c) Occurrence of teasterone and typhasterol, and their enzymatic conversion in Phaseolus vulgaris. B Kor Chem Soc 21: 373–374
Koka CV, Cerny RE, Gardner RG, Noguchi T, Fujioka S, Takatsuto S, Yoshida S, Clouse SD (2000) A putative role for the tomato genes DUMPY and CURL-3 in brassinosteroid biosynthesis and response. Plant Physiol 122: 85–98
Li J, Biswas M, Chao A, Russel D, Chory J (1997) Conservation of function between mammalian and plant steroid 5a-reductase. Proc Natl Acad Sci USA 94: 3554–3559 Li J, Chory J (1997) A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90: 929–938[CrossRef][Web of Science][Medline] Li J, Nagpal P, Vitart V, McMorris TC, Chory J (1996) A role for brassinosteroids in light-dependent development of Arabidopsis. Science 272: 398–401[Abstract]
Li J, Nam KH (2002) Regulation of brassinosteroid signaling by a GSK3/SHAGGY-like kinase. Science 295: 1299–1301
Li J, Nam KH, Vafeados D, Chory J (2001) BIN2, a new brassinosteroid-insensitive locus in Arabidopsis. Plant Physiol 127: 14–22 Mathur J, Molnár G, Fujioka S, Takatsuto S, Sakurai A, Yokota T, Adam G, Voigt B, Nagy F, Maas C, et al (1998) Transcription of the Arabidopsis CPD gene, encoding a steroidogenic cytochrome P450, is negatively controlled by brassinosteroids. Plant J 14: 593–602[CrossRef][Web of Science][Medline] Nakajima N, Fujioka S, Tanaka T, Takatsuto S, Yoshida S (2002) Biosynthesis of cholestanol in higher plants. Phytochemistry 60: 275–279[Medline] Nam KH, Li J (2002) BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell 110: 203–212[CrossRef][Web of Science][Medline]
Neff MM, Nguyen SM, Malancharuvil EJ, Fujioka S, Noguchi T, Seto H, Tsubuki M, Honda T, Takatsuto S, Yoshida S, et al (1999) BAS1: a gene regulating brassinosteroid levels and light responsiveness in Arabidopsis. Proc Natl Acad Sci USA 96: 15316–15323
Noguchi T, Fujioka S, Choe S, Takatsuto S, Tax FE, Yoshida S, Feldmann KA (2000) Biosynthetic pathways of brassinolide in Arabidopsis. Plant Physiol 124: 201–209
Noguchi T, Fujioka S, Takatsuto S, Sakurai A, Yoshida S, Li J, Chory J (1999) Arabidopsis det2 is defective in the conversion of (24R)-24-methylcholest-4-en-3-one to (24R)-24-methyl-5 Nomura T, Bishop GJ, Kaneta T, Reid JB, Chory J, Yokota T (2003) The LKA gene is a brassinosteroids insensitive1 homolog of pea. Plant J 36: 291–300[CrossRef][Web of Science][Medline]
Nomura T, Kitasaka Y, Takatsuto S, Reid JB, Fukami M, Yokota T (1999) Brassinosteroid/sterol synthesis and plant growth as affected by lka and lkb mutations of pea. Plant Physiol 119: 1517–1526 Nomura T, Nakayama M, Reid JB, Takeuchi Y, Yokota T (1997) Blockage of brassinosteroid biosynthesis and sensitivity causes dwarfism in garden pea. Plant Physiol 113: 31–37[Abstract] Nomura T, Sato T, Bishop GJ, Kamiya Y, Takatsuto S, Yokota T (2001) Accumulation of 6-deoxocathasterone and 6-deoxocastasterone in Arabidopsis, pea and tomato is suggestive of common rate-limiting steps in brassinosteroid biosynthesis. Phytochemistry 57: 171–178[CrossRef][Web of Science][Medline]
Park S-H, Han K-S, Kim T-W, Shim J-K, Takatsuto S, Yokota T, Kim S-K (1999) In vivo and in vitro conversion of teasterone to typhasterol in cultured cells of Marchantia polymorpha. Plant Cell Physiol 40: 955–960 Pompon D, Louerat B, Bronine A, Urban P (1996) Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol 272: 51–64[CrossRef][Web of Science][Medline] Sakurai A (1999) Biosynthesis. In A Sakurai, T Yokota, SD Clouse, eds, Brassinosteroids: Steroidal Plant Hormones. Springer-Verlag, Tokyo, pp 137–161 Schaeffer A, Bronner R, Benveniste P, Schaller H (2001) The ratio of campesterol to sitosterol that modulates growth in Arabidopsis is controlled by STEROL METHYLTRANSFERASE 2:1. Plant J 25: 605–615[CrossRef][Web of Science][Medline] Shimada Y, Fujioka S, Miyauchi N, Kushiro M, Takatsuto S, Nomura T, Yokota T, Kamiya Y, Bishop GJ, Yoshida S (2001) Brassinosteroid 6-oxidases from Arabidopsis and tomato catalyze multiple C-6 oxidations in brassinosteroid biosynthesis. Plant Physiol 26: 770–779
Shimada Y, Goda H, Nakamura A, Takatsuto S, Fujioka S, Yoshida S (2003) Organ-specific expression of brassinosteroid-biosynthetic genes and distribution of endogenous brassinosteroids in Arabidopsis. Plant Physiol 131: 287–297 Suzuki H, Fujioka S, Takatsuto S, Yokota T, Murofushi N, Sakurai A (1993) Biosynthesis of brassinolide from castasterone in cultured cells of Catharanthus roseus. J Plant Growth Regul 12: 101–106 Suzuki H, Fujioka S, Takatsuto S, Yokota T, Murofushi N, Sakurai A (1995) Biosynthesis of brassinosteroids in seedlings of Catharanthus roseus, Nicotiana tabacum, and Oryza sativa. Biosci Biotech Bioch 59: 168–172 Suzuki Y, Saso K, Fujioka S, Yoshida S, Nitasaka E, Nagata S, Nagasawa H, Takatsuto S, Yamaguchi I (2003) A dwarf mutant strain of Pharbitis nil, Uzukobito (kobito), has defective brassinosteroid biosynthesis. Plant J 36: 401–410[Medline] Szekeres M, Nemeth K, Koncz-Kalman Z, Mathur J, Kausch-mann A, Altmann T, Redei GP, Nagy F, Schell J, Koncz C (1996) Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85: 171–182[CrossRef][Web of Science][Medline] Takatsuto S, Yazawa N, Ishiguro M, Morisaki M, Ikekawa N (1984) Stereoselective synthesis of plant growth-promoting steroids, brassinolide, castasterone, typhasterol, and their 28-nor analogues. J Chem Soc Perkin Trans I: 139–146[CrossRef]
Turk E, Fujioka S, Seto H, Shimada Y, Takatsuto S, Yoshida S, Denzel M, Torres Q, Neff MM (2003) CYP72B1 inactivates brassinosteroid hormones. An intersection between photomorphogenesis and plant steroid signal transduction. Plant Physiol 133: 1643–1653
Urban P, Mignotte C, Kazmaier M, Delorme F, Pompon D (1997) Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5. J Biol Chem 272: 19176–19186 Wang ZY, Seto H, Fujioka S, Yoshida S, Chory J (2001) BRI1 is a critical component of a plasma-membrane receptor for plant steroids. Nature 410: 380–383[CrossRef][Medline] Watanabe T, Noguchi T, Yokota T, Shibata K, Koshino H, Seto H, Kim S-K, Takatsuto S (2001) Synthesis and biological activity of 26-norbrassinolide, 26-norcastasterone and 26-nor-6-deoxocastasterone. Phytochemistry 58: 343–349[Medline] Yokota T (1997) The structure, biosynthesis and function of brassinosteroids. Trends Plant Sci 2: 137–143[CrossRef][Web of Science]
Yokota T, Nomura T, Nakayama M (1997) Identification of brassinosteroids that appear to be derived from campesterol and cholesterol in tomato shoots. Plant Cell Physiol 38: 1291–1294 Yokota T, Sato T, Takeuchi Y, Nomura T, Uno K, Watanabe T, Takatsuto S (2001) Roots and shoots of tomato produce 6-deoxo-28-norcathasterone, 6-deoxo-28-nortyphasterol and 6-deoxo-28-norcastasterone, possible precursors of 28-norcastasterone. Phytochemistry 58: 233–238[Medline] This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
24 double bond in the side chain, and this moiety is required by SMT1 to facilitate the introduction of a methyl group to C-24 using SAM. Supposing that SMT1 is involved in the conversion of 28-norCS to CS, the introduction of a 
