Biosynthesis of 2,3-epoxybrassinosteroids in seedlings of Secale cereale

Two new brassinosteroids, (22R,23R,24S)-22,23-dihydroxy-24-methyl-5alpha-cholest-2-en-6-one (secasterol) and (22R,23R,24S)-22,23-dihydroxy-2alpha,3alpha-epoxy-24-methyl-5alpha-cholest-6-one (2,3-diepisecasterone) have been identified together with a known 2,3-epoxybrassinosteroid, secasterone, in seedlings of Secale cereale. Deuterated secasterol, teasterone, and typhasterol, upon administration to rye seedlings, were incorporated into secasterone and 2,3-diepisecasterone, indicating a biosynthetic route via teasterone/typhasterol to secasterol to 2,3-epoxybrassinosteroids.     

Stereoisomers of Castasterone, 3-<Emphasis Type="Italic">Epi</Emphasis>castasterone and 2,3-<Emphasis Type="Italic">Diepi</Emphasis>castasterone,in Immature Seeds of <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>

A capillary GC-MS analysis revealed that two stereoisomers of castasterone are contained in immature seeds of Phaseolus vulgaris. 400 MHz proton NMR analysis of the stereoisomers determined they are A ring epimers of castasterone, 3-epicastasterone and 2,3-diepicastasterone. In rice lamina inclination assay, 3-epicastasterone and 2,3-diepicastasterone showed reduced biological activity than that of castasterone. Together with our previous finding that 2-epicastasterone exhibits a less biological activity than CS, this result indicates that epimerization of hydroxyl at C-2 or/and C-3 is/are inactive processes to reduce biological activity of CS after exerting as a bioactive brassinosteroid in P. vulgaris.     

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.     

Blockage of Brassinosteroid Biosynthesis and Sensitivity Causes Dwarfism in Garden Pea

Endogenous brassinosteroids (BRs) in the dwarf mutants lka and lkb of garden pea (Pisum sativum L.) and comparable wild-type plants were quantified by gas chromatography-selected ion monitoring using deuterated internal standards. In young shoots of the lkb mutant, the levels of brassinolide, castasterone, and 6-deoxocastasterone were 23-, 22-, and 9-fold lower, respectively, than those of wild-type plants. Applications of brassinolide, castasterone, typhasterol, 3-dehydroteasterone, and teasterone normalized internode growth of lkb seedlings. These findings indicate that the lkb plants are BR-deficient mutants, probably as a consequence of a block in the BR biosynthetic pathway prior to the production of teasterone. Young shoots of lka plants contained only 50% less brassinolide and 5 times more castasterone than the equivalent wild-type tissues. The lka seedlings were approximately 100 times less responsive to brassinolide than the lkb mutant, and application of castasterone had only a marginal effect on lka internode growth, suggesting that the lka lesion results in impaired sensitivity to BR.     

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.     

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.     

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.     

Biosynthesis of brassinosteroids in cultured cells of Catharanthus roseus

Precursor administration experiments with 2H-labeled 6-oxocampestanol, 6-deoxocastasterone and 6alpha-hydroxycastasterone in cultured cells of Catharanthus roseus were performed and the metabolites were analyzed by GC-MS. [2H6]Cathasterone was identified as a metabolite of [2H6]6-oxocampestanol, whereas [2H6]6alpha-hydroxycastasterone and [2H6]castasterone were identified as metabolites of [2H6]6-deoxocastasterone, and [2H6]castasterone was identified as a metabolite of [2H6]6alpha-hydroxycastasterone, indicating that 6-deoxocastasterone is converted to castasterone via 6alpha-hydroxycastasterone. In addition, 6-deoxocathasterone, a putative biosynthetic intermediate in the late C6-oxidation pathway, was identified as an endogenous brassinosteroid. These studies provide further evidence supporting our proposed biosynthetic pathways for brassinolide.     

Endogenous brassinosteroids in wheat treated with 24-epibrassinolide

The aim of the study was to examine the effect of exogenous 24-epibrassinolide on its uptake and content of endogenous brassinosteroids in wheat seedlings. 24-Epibrassinolide was applied at two concentrations (0.1 and 2.0 μM) and in three different methods: by soaking seeds, by drenching and by spraying plants. Brassinosteroids were determined by high-performance liquid chromatography combined with electrospray mass spectrometry. Three important brassinosteroids, 24-epibrassinolide, brassinolide and castasterone, were detected in the wheat leaves, but their contents varied with leaf insertion and plant age. Increased 24-epibrassinolide content in the leaf tissue was found when this hormone was applied by soaking or drenching. Additionally the seed treatment influenced brassinosteroid balance in seedlings. The growth response of wheat seedlings treated with 24-epibrassinolide has been also investigated.     

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.     

Ameliorative Role of Castasterone on Copper Metal Toxicity by Improving Redox Homeostasis in <Emphasis Type="Italic">Brassica juncea</Emphasis> L.

The transition metal elements like copper act as double-edged sword for living cells. Cu, a redox active metal, is essential for various biological processes, but at higher concentrations it leads to toxicity by inducing production of reactive oxygen species (ROS). Thus, the objective of the present study was to investigate the effects of exogenously applied castasterone on oxidative stress markers and redox homeostasis managers in Brassica juncea plants subject to copper stress for 30 days. Copper-exposed plants showed accumulation of free radicals (H₂O₂ and superoxide anion) and lipid peroxidation. However, the exogenous treatment of seeds via the seed soaking method with different concentrations of castasterone reduced H₂O₂ production, superoxide anion radical content, and lipid peroxidation, thus indicating improved detoxification of ROS. Enzyme activity was increased by 19.19% for guaiacol peroxidase, 16.20% for superoxide dismutase, 35.74% for glutathione peroxidase, 27.58% for dehydroascorbate reductase, and 42.75% for ascorbate peroxidase, with castasterone pre-soaking under copper stress. The levels of non-enzymatic antioxidants were also increased with castasterone pre-treatment under copper stress. It may be concluded that castasterone treatment enhanced redox homeostasis managers in addition to increased levels of osmoprotectants.     

Biosynthetic pathways of brassinolide in Arabidopsis

Our previous studies on the endogenous brassinosteroids (BRs) in Arabidopsis have provided suggestive evidence for the operation of the early C6-oxidation and the late C6-oxidation pathways, leading to brassinolide (BL) in Arabidopsis. However, to date the in vivo operation of these pathways has not been fully confirmed in this species. This paper describes metabolic studies using deuterium-labeled BRs in wild-type and BR-insensitive mutant (bri1) seedlings to establish the intermediates of the biosynthetic pathway of BL in Arabidopsis. The first evidence for the conversion of campestanol to 6-deoxocathasterone and the conversion of 6-deoxocathasterone to 6-deoxoteasterone is provided. The later biosynthetic steps (6-deoxoteasterone --> 3-dehydro-6-deoxoteasterone --> 6-deoxotyphasterol --> 6-deoxocastasterone --> 6alpha-hydroxycastasterone --> castasterone --> BL) were demonstrated by stepwise metabolic experiments. Therefore, these studies complete the documentation of the late C6-oxidation pathway. The biosynthetic sequence involved in the early C6-oxidation pathway (teasterone --> 3-dehydroteasterone --> typhasterol --> castasterone --> BL) was also demonstrated. These results show that both the early and late C6-oxidation pathways are functional in Arabidopsis. In addition we report two new observations: the presence of a new branch in the pathway, C6 oxidation of 6-deoxotyphasterol to typhasterol, and increased metabolic flow in BR-insensitive mutants.     

Ceramide in Rice Grain

A rice lamina inclination test that is simple and specific for brassinosteroids was used as a micro-quantitative bioassay for brassinolide 1 and its 6-keto congener, castasterone 2, in the concentration range of 5 x 10^–5 /ig/ml to 5 x 10^–3μg/ml, when uniform seedlings of the rice cultivars Arborio J-l and Nihonbare were selected. A phytohormone, indole-3-acetic acid (IAA), showed similar activity in this bioassay. Its lowest effective concentration, however, was 50 /ig/μl, about five orders of magnitude greater than that of brassinolide. Other phytohormones, abscisic acid (ABA) and the cytokinins kinetin and A⁶-benzyladenine, inhibited the lamina inclination of rice seedlings. The addition of a cytokinin reduced the promoting effect of brassinolide. Thus, the rice lamina inclination test can be used both as a micro-quantitative bioassay for brassinosteroids and as a method for detecting antibrassinolide compouds.     

Production of (2S,3S)-2,3-butanediol and (3S)-acetoin from glucose using resting cells of Klebsiella pneumonia and Bacillus subtilis

Production of highly pure (2S,3S)-2,3-butanediol ((2S,3S)-2,3-BD) and (3S)-acetoin ((3S)-AC) in high concentrations is desirable but difficult to achieve. In the present study, glucose was first transformed to a mixture of (2S,3S)-2,3-BD and meso-2,3-BD by resting cells of Klebsiella pneumoniae CICC 10011, followed by biocatalytic resolution of the mixture by resting cells of Bacillus subtilis 168. meso-2,3-BD was transformed to (3S)-AC, leaving (2S,3S)-2,3-BD in the reaction medium. Using this approach, 12.5 g l(-1) (2S,3S)-2,3-BD and 56.7 g l(-1) (3S)-AC were produced. Stereoisomeric purity of (2S,3S)-2,3-BD and enantiomeric excess of (3S)-AC was 96.9 and 96.2%, respectively.     

Arabidopsis CYP85A2 catalyzes lactonization reactions in the biosynthesis of 2-deoxy-7-oxalactone brassinosteroids

Brassinolide (BL), a plant 7-oxalactone-type steroid hormone, is one of the active brassinosteroids (BRs) that regulates plant growth and development. BL is biosynthesized from castasterone by the cytochrome P450 monooxygenase, CYP85A2. We showed that a Pichia pastoris transformant that synchronously expresses Arabidopsis P450 reductase gene ATR1 and P450 gene CYP85A2 converts teasterone and typhasterol to 7-oxateasterone and 7-oxatyphasterol, respectively. Thus, CYP85A2 catalyzes the lactonization reactions of not only castasterone but also teasterone and typhasterol. The two 2-deoxy-7-oxalactone-type BRs were identified in Arabidopsis plants. Although the reversible conversion between 7-oxateasterone and 7-oxatyphasterol was observed in vivo, no conversion of 7-oxatyphasterol to BL was observed. The biological activity of 7-oxatyphasterol toward Arabidopsis hypocotyl elongation was nearly the same as that of castasterone. These results suggest that a new BR biosynthetic pathway, a BR lactonization pathway, functions in Arabidopsis and plays an important role in regulating the concentration of active BRs, even though the metabolism of 7-oxatyphasterol to BL is still unknown.     

Castasterone Can be Biosynthesized from 28-homodolichosterone in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

We recently demonstrated the biosynthesis of 24-ethylidene brassinosteroids in Arabidopsis thaliana. To determine the physiological role of biosynthesis of 24-ethylidene brassinosteroids, metabolism of 28-homodolichosterone as the end product of 24-ethylidene brassinosteroids biosynthesis was examined by a crude enzyme solution prepared from A. thaliana. In wild-type plants, dolichosterone and castasterone were identified as enzyme products on GC-MS analysis. In a mutant where DWARF1 was overexpressed (35S-DWF1), the conversion rate of 28-homodolichosterone to castasterone was significantly increased. These results indicate that conversion of 28-homodolichosterone to castasterone is mediated by dolichosterone in Arabidopsis. In the root growth assay, inhibitory activity was enhanced in the order of castasterone > dolichosterone > 28-homodolichosterone, demonstrating that conversion of 28-homodolichosterone to castasterone via dolichosterone is a biosynthetic reaction that increases BR activity in Arabidopsis. Compared to Arabidopsis grown under dark conditions, light-grown Arabidopsis showed up-regulated DWARF1 expression, resulting in an increased conversion rate of 28-homodolichosterone to castasterone, suggesting that light is an important regulatory factor for the biosynthetic connection of 24-ethylidene brassinosteroids and 24-methyl brassinosteroids in A. thaliana. Consequently, 24-ethylidene brassinosteroids biosynthesis to generate 28-homodolichosterone is a lightregulated alternative route for synthesis of the biologically-active BRs, castasterone and brassinolide in Arabidopsis plants.     

Arabidopsis CYP85A2 Catalyzes Lactonization Reactions in the Biosynthesis of 2-Deoxy-7-oxalactone Brassinosteroids

Brassinolide (BL), a plant 7-oxalactone-type steroid hormone, is one of the active brassinosteroids (BRs) that regulates plant growth and development. BL is biosynthesized from castasterone by the cytochrome P450 monooxygenase, CYP85A2. We showed that a Pichia pastoris transformant that synchronously expresses Arabidopsis P450 reductase gene ATR1 and P450 gene CYP85A2 converts teasterone and typhasterol to 7-oxateasterone and 7-oxatyphasterol, respectively. Thus, CYP85A2 catalyzes the lactonization reactions of not only castasterone but also teasterone and typhasterol. The two 2-deoxy-7-oxalactone-type BRs were identified in Arabidopsis plants. Although the reversible conversion between 7-oxateasterone and 7-oxatyphasterol was observed in vivo, no conversion of 7-oxatyphasterol to BL was observed. The biological activity of 7-oxatyphasterol toward Arabidopsis hypocotyl elongation was nearly the same as that of castasterone. These results suggest that a new BR biosynthetic pathway, a BR lactonization pathway, functions in Arabidopsis and plays an important role in regulating the concentration of active BRs, even though the metabolism of 7-oxatyphasterol to BL is still unknown.     

Study of the Toxicity of Quinomethionate (6-Methyl-2,3-dithiolquinoxaline Cyclocarbonate) I. Penetration and Metabolism of Quinomethionate in Cucumber (Cucumis sativus)

Biodegradation of 6-methyl-2,3-dithiolquinoxaline cyclocarbonate or quinomethionate (Q₁) by cucumber seedlings seems to confirm that the thiocarbonate linkage is disrupted during metabolism of the fungicide by the plant.

The sulfur liberated is principally incorporated into sulphates and sulfur amino-acids, while the labeled (2,3-¹⁴C) quinoxaline nucleus is catabolized to ¹⁴CO₂. This work also suggests that microorganisms can largely share in fungicide biodegradation when they are in the immediate environment of seedlings.     

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.     

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.