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.     

Biosynthesis of a cholesterol-derived brassinosteroid, 28-norcastasterone, in Arabidopsis thaliana

A metabolic study revealed that 28-norcastasterone in Arabidopsis is synthesized from cholesterol via the late C-6 oxidation pathway. On the other hand, the early C-6 oxidation pathway was found to be interrupted because cholestanol is converted to 6-oxocholestanol, but further metabolism to 28-norcathasterone was not observed. The 6-oxoBRs were found to have been produced from the respective 6-deoxoBRs administered to the enzyme solution, thus indicating that these 6-oxoBRs are supplied from the late C-6 oxidation pathway. Heterologously expressed CYP85A1 and CYP85A2 in yeast catalysed this C-6 oxidation, with CYP85A2 being much more efficient than CYP85A1. Abnormal growth of det2 and dwf4 was restored via the application of 28-norcastasterone and closer precursors. Furthermore, det2 and dwf4 could not convert cholesterol to cholestanol and cholestanol to 6-deoxo-28-norcathasterone, respectively. It is, therefore, most likely that the same enzyme system is operant in the synthesis of both 28-norcastasterone and castasterone. In the presence of S-adenosyl-L-methionine, the cell-free enzyme extract catalysed the C-24 methylation of 28-norcastasterone to castasterone, although the conversion rates of 28-norteasterone to teasterone and 28-nortyphasterol to typhasterol were much lower; this suggests that 28-norcastasterone is the primary precursor for the generation of C(28)-BRs from C(27)-BRs.     

Isolation and Characterization of Brassinosteroids from Leaves of Camellia sinensis (L.) O. Kuntze

Brassinosteroids are of ubiquitous occurrence in plants and elicit a wide spectrum of physiological responses. In our study, brassinosteroids were isolated and identified in topmost dormant leaves of tea plants. Six brassinosteriods, i.e. 6-deoxocastasterone, 24-epibrassinolide,3-dehydroteasterone, typhasterol, 3-deoxotyphasterol and 28-homodolicholide, were isolated and identified by GC–MS. All the brassinosteroids identified belong to important components of early and late C6 oxidation pathways proposed for brassinosteroids biosynthesis in plants. It suggests that both pathways are operating in tea to produce brassinolide, the most active brassinosteroid biologically.     

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.     

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.     

Isolation and characterization of brassinosteroids from algal cultures of Chlorella vulgaris Beijerinck (Trebouxiophyceae)

The brassinosteroids (BRs) occur ubiquitously in the plant kingdom. The occurrence of BRs has been demonstrated in almost every part of higher plants, such as pollen, flower buds, fruits, seeds, vascular cambium, leaves, shoots and roots. In this study, BRs were isolated and identified in the culture of wild-type Chlorella vulgaris. Seven BRs, including teasterone, typhasterol, 6-deoxoteasterone, 6-deoxotyphasterol, 6-deoxocastasterone, castasterone and brassinolide, were identified by GC–MS. All compounds belong to the BR biosynthetic pathway. The results suggest that early and late C6 oxidation pathways are operating in C. vulgaris. This study represents the first isolation of BRs from C. vulgaris cultures.     

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.     

7Beta-oxygenated limonoids from Trichilia elegans ssp. elegans

Five 7beta- and 7alpha-oxygenated obacunone-type limonoids were obtained on reinvestigation of the seeds of Trichilia elegans ssp. elegans: 7-deoxo-7beta-acetoxykihadanins A and B, 7-deoxo-7beta-hydroxykihadanins A and B and 7-deoxo-7alpha-hydroxykihadanin A, the last three being isolated after acetylation procedures as their mono- and/or diacetate derivatives. This is the first report of the natural occurrence of C-7 beta-substituted limonoids without any oxygenated function at C-6. The structures of these compounds have been established on the basis of 1D- and 2D-NMR spectral techniques, ESI-mass spectrum and X-ray crystallographic data.     

Biosynthesis and metabolism of brassinosteroids

Natural brassinosteroids so far identified from various plant species include biosynthetic congeners of brassinolide, such as cathasterone, teasterone, 3-dehydroteasterone, typhasterol and castasterone as well as another series of 6-deoxoteasterone, 3-dehydro-6-deoxoteasterone, 6-deoxotyphasterol and 6-deoxocastasterone. Using cell culture system of Catharanthus roseus , the outlines of biosynthetic pathways of brassinolide, via plant sterol of campesterol, have now been demonstrated. There are two pathways, named early C6-oxidation pathway and late C6-oxidation pathway, both of which would be operating in wide varieties of plants. Metabolic studies with various plant systems revealed multiple paths of metabolism such as hydroxylation, epimerization, side chain cleavage, reduction and conjugation with glucose and fatty acids. Recent progress of biosynthesis and metabolism of brassinosteroids is described.     

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.     

3Beta-brassinosteroid dehydrogenase activity in Arabidopsis and tomato.

Enzymatic conversion of 24-epiteasterone to 3-dehydro-24-epiteasterone, representing a reversible step in the biosynthesis of 24R-methyl brassinosteroids, was monitored in vitro in Arabidopsis thaliana and Lycopersicon esculentum using fluorescent tagging and HPLC analysis. In both species 24-epiteasterone was metabolized by the cytosolic fraction of induced root-callus suspension cultures in a NAD dependent manner. This 3beta-dehydrogenation was only slightly inhibited by 12-fold excess of other 3beta-hydroxy intermediates of the early (cathasterone) and late C-6 oxidation pathway (6-deoxo-cathasterone, 6-deoxo-teasterone), indicating a rather specific protein. 3-Dehydro-6-deoxoteasterone and 3alpha-hydroxy intermediates of brassinosteroid biosynthesis did not reduce conversion of 24-epiteasterone to 3-dehydro-24-epiteasterone. In contrast to light-grown cultures the reaction was clearly inhibited in the dark.     

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.     

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.     

New Techniques for the Estimation of Naturally Occurring Brassinosteroids

We have developed enzyme-linked immunosorbent assays (ELISAs) for measuring 24-epicastasterone and related brassinolide analogs, with detection ranges of 0.005 to 50 pmoles. Polyclonal antibodies used in these assays were raised against 24-epicastasterone carboxymethyloxime-bovine serum albumin conjugates and were found to have high specificity for 24-epibrassinosteroids. Natural brassinosteroids (BRs), such as brassinolide and 24-epibrassinolide, exhibited relatively high cross-reactivities with the generated antibodies, whereas other BR analogs with β-oriented hydroxyl groups at C-2, C-3, C-22, and C23 lacked immunoreactivity. Through the use of internal standardization, dilution assays, recovery of authentic [³H]24-epicastasterone, and immunohistograms, the ELISAs have been shown to be applicable for estimating 24-epibrassinosteroid levels in crude plant extracts. To analyze brassinosteroids in tissues from young bean (Phaseolus vulgaris L., cv. Pinto), Daucus carota ssp.sativus plants and Arabidopsis thaliana L. Heynh. seedlings, and rape (Brassica napus L.) pollen, the extracts were fractionated by high performance liquid chromatography (HPLC) and the resulting fractions were analyzed by the ELISA method. Immunohistogram ELISA analysis of HPLC fractions indicated that major peaks of immunoreactivity co-chromatographed with the labeled and unlabeled 24-epibrassinolide. A highly sensitive electrospray ionization mass spectrometry (MS) technique (LOD: 50 fmol) was also developed and the results obtained by the HPLC-ELISA and HPLC-MS approaches were compared.     

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.     

油菜素内酯生物合成途径的研究进展

油菜素内酯(BRs)在植物的生长发育过程中具有重要作用。该文首先综述了油菜素甾醇的结构及其生物合成途径的研究方法。之后, 介绍了其化学及生物活性的检测方法。最后, 详细介绍了BR生物合成的早期和晚期C-6氧化途径及早期C-22和C-23羟化与合成途径的调控, 并阐述了近年来植物油菜素内酯生物合成缺失突变体及其合成酶等方面的研究进 展。     

Acceleration of ripening of tomato pericarp discs by brassinosteroids

Brassinosteroids are now considered as the sixth group of hormones in plants. As brassinosteroids influence varied growth and development processes such as growth, germination of seeds, rhizogenesis, flowering, senescence and abscission, they are considered as plant hormones with pleiotropic effects. The effect of 28-homobrassinolide and 24-epibrassinolide on ripening of tomato pericarp discs was studied. Application of brassinosteroids to pericarp discs resulted in elevated levels of lycopene and lowered chlorophyll levels. In addition brassinosteroid-treated pericarp discs exhibited decreased ascorbic acid and increased carbohydrate contents. Fruit ripening as induced by brassinosteroids was associated with increase in ethylene production. The study revealed the ability of brassinosteroids in accelerating fruit-senescence.     

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.     

Brd1 gene in maize encodes a brassinosteroid C-6 oxidase

The role of brassinosteroids in plant growth and development has been well-characterized in a number of plant species. However, very little is known about the role of brassinosteroids in maize. Map-based cloning of a severe dwarf mutant in maize revealed a nonsense mutation in an ortholog of a brassinosteroid C-6 oxidase, termed brd1, the gene encoding the enzyme that catalyzes the final steps of brassinosteroid synthesis. Homozygous brd1-m1 maize plants have essentially no internode elongation and exhibit no etiolation response when germinated in the dark. These phenotypes could be rescued by exogenous application of brassinolide, confirming the molecular defect in the maize brd1-m1 mutant. The brd1-m1 mutant plants also display alterations in leaf and floral morphology. The meristem is not altered in size but there is evidence for differences in the cellular structure of several tissues. The isolation of a maize mutant defective in brassinosteroid synthesis will provide opportunities for the analysis of the role of brassinosteroids in this important crop system.