Novel biosynthetic pathway of castasterone from cholesterol in tomato

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.     

Endogenous level of 28-Norcastasterone is strictly regulated in Plant Cells

A cell-free enzyme solution prepared from cultured cells ofPhaseolus vulgaris mediated C-24 methylation of 28-nor-castasterone to castasterone with the aid of S-adenosylmethionine as a co-substrate in the presence of the NADPH cofactor. This enzyme solution also catalyzed conversion of 28-norcastasterone to a demethylated 28-norcastasterone, most likely 26,28-didemethyl-castasterone, when S-adenosylmethionine was not added to the enzyme solution. Furthermore, gene expression ofArabidopsis CYP85A1 andCYP85A2 mediating the conversion of 6-deoxo-28-norcastast-erone to 28-norcastasterone was strongly inhibited by treatment of 28-norcastasterone. These results suggest that 28-norcastasterone, along with castasterone and brassinolide, is an important brassinosteroid whose endogenous level should be strictly controlled to express brassinosteroid activities in plants.     

Accumulation of 6-deoxocathasterone and 6-deoxocastasterone in Arabidopsis, pea and tomato is suggestive of common rate-limiting steps in brassinosteroid biosynthesis

To gain a better understanding of brassinosteroid biosynthesis, the levels of brassinosteroids and sterols related to brassinolide biosynthesis in Arabidopsis, pea, and tomato plants were quantified by gas chromatography-selected ion monitoring. In these plants, the late C-6 oxidation pathway was found to be the predominant pathway in the synthesis of castasterone. Furthermore, all these plant species had similar BR profiles, suggesting the presence of common biosynthetic control mechanisms. The especially high levels of 6-deoxocathasterone and 6-deoxocastasterone may indicate that their respective conversions to 6-deoxoteasterone and castasterone are regulated in planta and hence are important rate-limiting steps in brassinosteroid biosynthesis. Other possible rate-limiting reactions, including the conversion of campestanol to 6-deoxocathasteonre. are also discussed. Tomato differs from Arabidopsis and pea in that tomato contains 28-norcastasterone as a biologically active brassinosteroid, and that its putative precursors, cholesterol and its relatives are the major sterols.     

Roots and shoots of tomato produce 6-deoxo-28-norcathasterone, 6-deoxo-28-nortyphasterol and 6-deoxo-28-norcastasterone, possible precursors of 28-norcastasterone

Roots and shoots of tomato (Lycopersicon esculentum) were investigated for the occurrence of biosynthetic precursors of 28-norcastasterone, a C27 brassinosteroid that we have shown to be present in shoots of tomato. A series of putative precursors, including 6-deoxo-28-norcathasterone, 6-deoxo-28-norteasterone, 3-dehydro-6-deoxo-28-norteasterone, 6-deoxo-28-nortyphasterol and 6-deoxo-28-norcastasterone, were synthesized and used as GC-MS standards, resulting in the identification of 6-deoxo-28-norcathasterone, 6-deoxo-28-nortyphasterol and 6-deoxo-28-norcastasterone in both roots and shoots. These findings indicate that the biosynthesis of 28-norcastasterone may parallel that of castasterone. The endogenous levels of brassinosteroids differed between roots and shoots, indicating that the biosynthesis of brassinosteroids is differently regulated between these tissues. Regulation of root growth by brassinosteroids is also discussed.     

Isolation and characterization of brassinosteroids from immature seeds of Camellia sinensis (O) Kuntze

Brassinosteroids play an important role in growth and development of plants. They have been reported universally in all the plants. The present study deals with the presence of these compounds in immature tea seeds. Five brassinosteroids, i.e. 6-deoxo-28-norcathasterone, 6-deoxo-28-norteasterone, 3-dehydro-6-deoxo-28-norteasterone, 6-deoxo-28-nortyphasterol and 6-deoxo-28-norcastasterone have been isolated and identified by GC–MS. The identified brassinosteroids and their derivatives are active constituents of late C-6 oxidation pathway, thereby suggesting the biosynthesis of brassinosteroids in tea seeds by late C-6 oxidation pathway.     

Arabidopsis CYP85A2, a cytochrome P450, mediates the Baeyer-Villiger oxidation of castasterone to brassinolide in brassinosteroid biosynthesis

The conversion of castasterone (CS) to brassinolide (BL), a Baeyer-Villiger oxidation, represents the final and rate-limiting step in the biosynthesis of BL in plants. Heterologously expressed Arabidopsis thaliana CYP85A2 in yeast mediated the conversion of CS to BL as well as the C-6 oxidation of brassinosteroids (BRs). This indicated that CYP85A2 is a bifunctional enzyme that possesses BR C-6 oxidase and BL synthase activity. CYP85A2 is thus a cytochrome P450 that mediates Baeyer-Villiger oxidation in plants. Biochemical, physiological, and molecular genetic analyses of Arabidopsis CYP85A2 loss-of-function and overexpression lines demonstrated that CS has to be a bioactive BR that controls the overall growth and development of Arabidopsis plants. Mutant studies also revealed that BL may not always be necessary for normal growth and development but that Arabidopsis plants acquire great benefit in terms of growth and development in the presence of BL.     

Tomato cytochrome P450 CYP734A7 functions in brassinosteroid catabolism

Several cytochrome P450 monooxygenases (P450s) catalyze essential oxidative reactions in brassinosteroid (BR) biosynthesis as well as in BR catabolism; however, only limited information exists on the P450s involved in the BR catabolic pathway. Here, we report the characterization of two P450 mRNAs, CYP734A7 and CYP734A8, from Lycopersicon esculentum. These P450s show high homology with Arabidopsis CYP734A1/BAS1 (formerly CYP72B1), which inactivates BRs via C-26 hydroxylation. Transgenic tobacco plants that constitutively overexpressed CYP734A7 showed an extreme dwarf phenotype similar to BR deficiency. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in the transgenic plants showed that the levels of castasterone and 6-deoxocastasterone significantly decreased in comparison with those in wild-type plants. By measuring the Type I substrate-binding spectra using recombinant CYP734A7, the dissociation constants for castasterone, brassinolide, and 6-deoxocastasterone were determined to be 6.7, 12, and 12 microM, respectively. In an in vitro assay, CYP734A7 was confirmed to metabolize castasterone to 26-hydroxycastasterone. In addition, 28-norcastasterone and brassinolide were converted to the hydroxylated products. The expression of CYP734A7 and CYP734A8 genes in tomato seedlings was upregulated by exogenous application of bioactive BRs. These results indicated that CYP734A7 is a C-26 hydroxylase of BRs and is likely involved in BR catabolism in tomato. The presence of the CYP734A subfamily in various plant species suggests that oxidative inactivation of BRs by these proteins is a widespread phenomenon in plants.     

Regulation of castasterone level in primary roots of maize, Zea mays

Gas chromatography–mass spectrometry analysis revealed that primary roots of maize contain 28-norcastasterone (28-norCS) and its biosynthetic precursors, cholesterol, and cholestanol, which suggests that the C₂₇-brassinosteroid (C₂₇-BR) biosynthetic pathway to generate 28-norCS is operative in the roots. A cell-free enzyme solution prepared from maize roots successfully mediated C24-methylation of 28-norCS to produce castasterone (CS) with the aid of S-adenosyl- l -methionine, which indicates that CS can be generated through C₂₇-BR biosynthesis, as well as C₂₈-BR biosynthesis, in maize roots. Enzymatic conversion study using the cell-free enzyme solution demonstrated that CS is converted into 26-norCS in the enzyme solution. Exogenously applied 28-norCS and 26-norCS showed less activity than CS in the activation of gravitropic curvature and inhibition of root elongation. Taken together, a steady-state level of CS, the active BR in maize roots, seems to be strictly controlled by complicated processes such as C₂₈- and C₂₇-BR biosynthesis and biodegradation by C26-demethylation to exert its biological activity.     

A double mutant for theCYP85A1 andCYP85A2 Genes ofArabidopsis exhibits a Brassinosteroid dwarf phenotype

Brassinosteroid (BR)-6-oxidases mediate the bridge reactions that connect the late and early C-6 oxidation pathways by converting 6-deoxoBR to 6-oxoBRs. Two similar genes ofArabidopsis, CYP85A1 (At5g38970) andCYP85A2 (At3g30180), are proposed to encode BR-6-oxidases based on findings that heterologously expressed genes mediate BR-6-oxidation reactions in yeast. However, genetic evidence that both genes are critically involved in the BR-6-oxidation step inArabidopsis has been limited. Here, we show that a double mutant for the two genes displays dwarfism similar to that of typical BR biosynthesis-deficient mutants, suggesting that they are the major BR-6-oxidases inArabidopsis. Examination of endogenous BR levels and metabolism monitoring tests using this double mutant revealed a great reduction in the levels of 6-oxoBRs, e.g., TY and CS, due to a lack in the conversion reactions from 6-deoxoCS to CS, and from 6-deoxoTY to TY. Surprisingly, the double mutant accumulated a significant amount of 6-oxocampestanol, suggesting that the upstream C-6 oxidation of campestanol to 6-oxocampestanol is not catalyzed by the two BR-6-oxidases inArabidopsis, rather, by another enzyme yet to be discovered.     

Brassinosteroids are inherently biosynthesized in the primary roots of maize, Zea mays L

GC-MS analysis revealed that primary roots of maize contain 6-deoxocathasterone, 6-deoxoteasterone and 6-deoxotyphasterol. These brassinosteroids, and the previously identified campesterol, campestanol, 6-deoxocastasterone and castasterone, in the roots are members of a biosynthetic pathway to castasterone, namely the late C-6 oxidation pathway, suggesting that its biosynthetic pathway is operative in the roots. To verify this, a cell-free enzyme extract was prepared from maize roots, and enzymatic conversions from campesterol to castasterone through the aforementioned sterols and brassinosteroids were examined. The presence for the biosynthetic sequences, campesterol-->24-methylcholest-4-en-3beta-ol-->24-methylcholest-4-en-3-one-->24-methylcholest-5 alpha-cholestan-3-one-->campestanol and 6-deoxoteasterone-->6-deoxo-3-dehydroteasterone-->6-deoxotyphasterol-->6-deoxocastasterone-->castasterone were demonstrated. These results indicate that maize roots contain a complete set of enzymes involved in the late C-6 oxidation pathway, thereby demonstrating that endogenous brassinosteroids are biosynthesized in the roots.     

28-Norcastasterone is biosynthesized from castasterone

Metabolic experiments with deuterium-labeled castasterone in seedlings of Arabidopsis thaliana, Oryza saliva and Lycopersicon esculentum, and cultured cells of Catharanthus roseus were performed, and the metabolites were analyzed by GC-MS. In all the plant species examined, [2H3]28-norcastasterone was identified as a metabolite of [26,28-2H6]castasterone, indicating that castasterone is the biosynthetic origin of 28-norcastasterone. Moreover, the natural occurrence of 28-norcastasterone and 28-nortyphasterol in seedlings of A. thaliana has been demonstrated. This is the first report of the natural occurrence of 28-nortyphasterol in plants.     

The last reaction producing brassinolide is catalyzed by cytochrome P-450s, CYP85A3 in tomato and CYP85A2 in Arabidopsis

Brassinosteroids are steroidal hormones essential for the growth and development of plants. Brassinolide, the most biologically active brassinosteroid, has a seven-membered lactone ring that is formed by a Baeyer-Villiger oxidation of its immediate precursor castasterone. Despite its potential key role in controlling plant development, brassinolide synthase has not been identified. Previous work has shown that the formation of castasterone from 6-deoxocastasterone is catalyzed by members of the CYP85A family of cytochrome P-450 monooxygenases. A null mutation in the tomato Dwarf (CYP85A1) gene, extreme dwarf (d(x)), causes severe dwarfism due to brassinosteroid deficiency, but the d(x) mutant still produces fruits. Here, we show that d(x) fruits contain brassinolide at a higher level than wild-type fruits and that a new CYP85A gene, CYP85A3, is preferentially expressed in tomato fruits. Tomato CYP85A3 catalyzed the Baeyer-Villiger oxidation to produce brassinolide from castasterone in yeast, in addition to the conversion of 6-deoxocastasterone to castasterone. We also show that Arabidopsis CYP85A2, which was initially characterized as castasterone synthase, also has brassinolide synthase activity. Exogenous application of castasterone and brassinolide to the Arabidopsis cyp85a1/cyp85a2 double mutant suggests that castasterone can function as an active brassinosteroid but that its conversion into brassinolide is necessary for normal vegetative development in Arabidopsis. We postulate that castasterone is the major active brassinosteroid during vegetative growth in tomato, whereas brassinolide may play an organ-specific role in fruit development in this species.     

Brassinosteroid-6-oxidases from Arabidopsis and tomato catalyze multiple C-6 oxidations in brassinosteroid biosynthesis

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. It has been proposed that BRs are synthesized via two parallel pathways, the early and late C-6 oxidation pathways according to the C-6 oxidation status. The tomato (Lycopersicon esculentum) Dwarf gene encodes a cytochrome P450 that has been shown to catalyze the C-6 oxidation of 6-deoxocastasterone to castasterone. We isolated an Arabidopsis ortholog (AtBR6ox gene) of the tomato Dwarf gene. The encoded polypeptide has characteristics of P450s and is classified into the CYP85 family. The AtBR6ox and tomato Dwarf gene were expressed in yeast and the ability of the transformed yeast cells to metabolize 6-deoxo-BRs was tested. Metabolites were analyzed by gas chromatography-mass spectrometry. Both enzymes catalyze multiple steps in BR biosynthesis: 6-deoxoteasterone to teasterone, 3-dehydro-6-deoxoteasterone to 3-dehydroteasterone, 6-deoxotyphasterol to typhasterol, and 6-deoxocastasterone to castasterone. Our results indicate that the AtBR6ox gene and the tomato Dwarf gene encode steroid-6-oxidases and that these enzymes have a broad substrate specificity. This suggests that the BR biosynthetic pathway consists of a metabolic grid rather than two separate parallel pathways.     

Characterization of two brassinosteroid C-6 oxidase genes in pea

C-6 oxidation genes play a key role in the regulation of biologically active brassinosteroid (BR) levels in the plant. They control BR activation, which involves the C-6 oxidation of 6-deoxocastasterone (6-DeoxoCS) to castasterone (CS) and in some cases the further conversion of CS to brassinolide (BL). C-6 oxidation is controlled by the CYP85A family of cytochrome P450s, and to date, two CYP85As have been isolated in tomato (Solanum lycopersicum), two in Arabidopsis (Arabidopsis thaliana), one in rice (Oryza sativa), and one in grape (Vitis vinifera). We have now isolated two CYP85As (CYP85A1 and CYP85A6) from pea (Pisum sativum). However, unlike Arabidopsis and tomato, which both contain one BR C-6 oxidase that converts 6-DeoxoCS to CS and one BR C-6 Baeyer-Villiger oxidase that converts 6-DeoxoCS right through to BL, the two BR C-6 oxidases in pea both act principally to convert 6-DeoxoCS to CS. The isolation of these two BR C-6 oxidation genes in pea highlights the species-specific differences associated with C-6 oxidation. In addition, we have isolated a novel BR-deficient mutant, lke, which blocks the function of one of these two BR C-6 oxidases (CYP85A6). The lke mutant exhibits a phenotype intermediate between wild-type plants and previously characterized pea BR mutants (lk, lka, and lkb) and contains reduced levels of CS and increased levels of 6-DeoxoCS. To date, lke is the only mutant identified in pea that blocks the latter steps of BR biosynthesis and it will therefore provide an excellent tool to further examine the regulation of BR biosynthesis and the relative biological activities of CS and BL in pea.     

Synthesis and biological activity of 26-norbrassinolide, 26-norcastasterone and 26-nor-6-deoxocastasterone

26-Norbrassinolide, identified as a metabolite of brassinolide in cultured cells of the liverwort, Marchantia polymorpha, as well as 26-norcastasterone and 26-nor-6-deoxocastasterone were synthesized. Synthesis of these new brassinosteroids was conducted by employing the orthoester Claisen rearrangement and asymmetric dihydroxylation as key reactions. The modified rice lamina inclination test indicated that these three 26-norbrassinosteroids were less active than their corresponding C28 brassinosteroids. Growth-promoting activities were also examined by using the brassinosteroid-deficient, dwarf mutant lkb of garden pea (Pisum sativum L.). In this assay, 26-norbrassinolide was as effective as brassinolide and 26-norcastasterone was more effective than castasterone although 26-nor-6-deoxocastasterone was much less effective than 6-deoxocastasterone. Therefore, removal of C-26 of brassinosteroids does not necessarily reduce the biological activity. The role of C-26 removal in Marchantia cells remains unclear.     

Identification and biosynthesis of C-24 ethylidene brassinosteroids in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Isofucosterol is a major 4-demethylsterol which has an ethylidene group at C-24 in Arabidopsis thaliana. To evaluate the presence of brassinosteroids (BRs) with the same carbon skeleton as that of isofucosterol, a large quantity of A. thaliana was extracted and purified. GC-MS/selected ion monitoring analysis verified that 6-deoxohomodolichosterone and homodolichosterone are present in Arabidopsis. An enzyme solution prepared from wild type Arabidopsis successfully mediated conversion of 6-deoxohomodolichosterone to homodolichosterone. However, a double mutant cyp85a1/cyp85a2 could not catalyze the conversion, implying that in A. thaliana the C-6 oxidation of 6-deoxohomodolichosterone to homodolichosterone seems to be catalyzed by CYP85A1 and/or CYP85A2. In yeast, both heterologously expressed CYP85A1 and CYP85A2 catalyzed the C-6 oxidation of 6- deoxohomodolichosterone to homodolichosterone, but the conversion rate in CYP85A2/V60/WAT21 was significantly higher than that in CYP85A1/V60/WAT21, indicating that C-6 oxidation of 6-deoxohomodolichosterone to homodolichosterone is mainly catalyzed by CYP85A2 in A. thaliana. Taken together, this study strongly suggests that a biosynthetic pathway for the production of 6-deoxohomodolichosterone and homodolichosterone is functional, and CYP85As have important roles in 24-ethylidene biosynthesis in A. thaliana.     

Cytochrome P450-catalyzed brassinosteroid pathway activation through synthesis of castasterone and brassinolide in Phaseolus vulgaris

The last reaction in the biosynthesis of brassinolide has been examined enzymatically. A microsomal enzyme preparation from cultured cells of Phaseolus vulgaris catalyzed a conversion from castasterone to brassinolide, indicating that castasterone 6-oxidase (brassinolide synthase) is membrane associated. This enzyme preparation also catalyzed the conversions of 6-deoxocastasterone and typhasterol to castasterone which have been reported to be catalyzed by cytochrome P450s, CYP85A1 of tomato and CYP92A6 of pea, respectively. The activities of these enzymes require molecular oxygen as well as NADPH as a cofactor. The enzyme activities were strongly inhibited by carbon monoxide, an inhibitor of cytochrome P450, and this inhibition was recovered by blue light irradiation in the presence of oxygen. Commercial cytochrome P450 inhibitors including cytochrome c, SKF 525A, 1-aminobenzotriazole and ketoconazole also inhibited the enzyme activities. The present work presents unanimous enzymological evidence that cytochrome P450s are responsible for the synthesis of brassinolide from castasterone as well as of castasterone from typhasterol and 6-deoxocastasterone, which have been deemed activation steps of BRs.     

Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols

Arabidopsis dwf4 is a brassinosteroid (BR)-deficient mutant, and the DWF4 gene encodes a cytochrome P450, CYP90B1. We report the catalytic activity and substrate specificity of CYP90B1. Recombinant CYP90B1 was produced in Escherichia coli, and CYP90B1 activity was measured in an in vitro assay reconstituted with NADPH-cytochrome P450 reductase. CYP90B1 converted campestanol (CN) to 6-deoxocathasterone, confirming that CYP90B1 is a steroid C-22 hydroxylase. The substrate specificity of CYP90B1 indicated that sterols with a double bond at positions C-5 and C-6 are preferred substrates compared with stanols, which have no double bond at the position. In particular, the catalytic efficiency (k(cat)/K(m)) of CYP90B1 for campesterol (CR) was 325 times greater than that for CN. As CR is more abundant than CN in planta, the results suggest that C-22 hydroxylation of CR before C-5alpha reduction is the main route of BR biosynthetic pathway, which contrasts with the generally accepted route via CN. In addition, CYP90B1 showed C-22 hydroxylation activity toward various C(27-29) sterols. Cholesterol (C27 sterol) is the best substrate, followed by CR (C28 sterol), whereas sitosterol (C29 sterol) is a poor substrate, suggesting that the substrate preference of CYP90B1 may explain the discrepancy between the in planta abundance of C27/C28/C29 sterols and C27/C28/C29 BRs.     

Metabolic changes in fruits of the tomato dx mutant

The tomato DWARF cytochrome P450 protein catalyzes the C-6 oxidation of 6-deoxo-castasterone to castasterone. The d(x) mutant does not produce a functional DWARF enzyme, and d(x) shoots display severe symptoms of brassinosteroid-deficiency. However, fruits express the CYP85A3 protein which compensates for the deficiency of the DWARF protein and produce bioactive brassinosteroids. Here, we report on the metabolic characterization of d(x) fruits. Fruit size, fresh weight, and pigment content were not altered. However, d(x) fruits showed reduced dry mass content. Levels of starch and various sugars were reduced, amino acid levels were elevated. BR application to d(x) leaves partially normalized dry mass content, sugar and amino acid levels in d(x) fruits. The data demonstrate that brassinosteroid in shoots is required for fruit development in tomato.