Castasterone Research Articles

Cell-free Conversion of Castasterone in Cultured Cells of Phaseolus vulgaris and Marchantia polymorpha

Brassinosteroids (BRs) are steroidal plant hormones, which are required for the normal growth and development of plants. Castasterone (CS) and brassinolide (BL) are the most frequently identified BRs in the plant kingdom. Feeding experiments using isotope-labeled substrates revealed that BL is biosynthesized from CS by 7-oxalactonation. However, many plants which possess a fair amount of CS contain very low levels of BL. In addition, in plants which can convert CS to BL, the conversion rate is extremely low, yielding high levels of CS in the plants. Because CS, as well as BL, is known to induce feedback regulation of earlier steps in BR biosynthesis pathways, accumulation of CS may limit or alter BR biosynthesis in plants. Therefore, endogenous levels of CS should be reduced, after BL production, to a level below that at which feedback regulation can occur. This is a difficult proposition, as little is yet known about the catabolism of CS. This dearth of available data prompted us to investigate the catabolism of CS in cultured Phaseolus vulgaris and Marchantia polymorpha cells, in which the presence of CS and BL and conversion of CS to BL have been demonstrated. Cultured cells (5 g) of P. vulgaris and M. polymorpha were homogenized and centrifuged at 8, 000 × g for 10 minutes, and the resulting supernatants were re-centrifuged at 20,000 × g for 30 min. Cold acetone was then added to the obtained supernatants (final volume 40%), and the acetone precipitates were re-suspended in 0.1 M Na phosphate buffer (pH 7.4) containing 1.5 mM 2-mercaptoethanol and 30% glycerol for crude enzyme solutions. Non-labeled CS and NADPH (4.8 mM) were added to the enzyme solutions as a substrate and a cofactor, respectively, to examine catabolism of CS in the plants. After incubating at 37 C for 30 minutes, the assay mixtures were extracted with ethyl acetate (1.2 mL × 3). The obtained ethyl acetate soluble fractions were loaded on a Sep-Pak C18 cartridge eluted with 50%, and 100% methanol (5 mL each). The 100% methanol fractions were further purified by reversed phase HPLC (Nova Pak, C18, 8 × 100 mm) and eluted with 40% acetonitrile at a flow rate of 1 mL min−1. Fractions were collected every min, and analyzed by a preparative TLC (Merck, HPTLC F254) developed with a 6 : 1 mixture of chloroform and methanol. Besides fraction 19-21, which contained CS (added as the substrate), fraction 13-15 exhibited a BR-like blue-purplish spot at Rf 0.30. The metabolite in the fractions was analyzed by GC-MS/-SIM after methaneboronation. In GC-MS, bismethaneboronate (BMB) of the metabolite showed a molecular ion at m/z 498 and the most abundant ion at m/z 141, due to the fission of C20/C22, which was reduced in mass compared with CS BMB. The mass reduction suggests that a methyl in CS was eliminated in the metabolite. Therefore, the metabolite was proposed to be either 26-norCS or 28-norCS. In GC-MS, BMB of 26-norCS and 28-norCS showed basically the same mass spectrum, but their retention times on GC were clearly different. As shown in Table 1, GC retention time of BMB of the metabolite was equal to that of 26-norCS BMB. Consequently, the metabolite was characterized as 26-norCS. 26-NorCS showed approximately one-tenth the level of activity of CS, indicating that 26-norCS is a catabolite of CS in plant cells. Because isotope-labeled 26-norCS is not available, activity for the enzyme catalyzing the conversion of CS to 26-norCS, namely CS C-26 demethylase, was measured by GC-SIM based quantification method, yielding 0.90 and 0.31 ng mg−1

Conversion of 6‐Deoxocastasterone to Brassinolide in a Liverwort, Marchantia polymorpha.

Department of Life Science, Chung-Ang University, Seoul 156-756, KoreaReceived June 4, 2003Key Words : Biosynthesis, Brassinosteroids, in vivo and in vitro conversion, Liverwort, MarchantiapolymorphaWe have recently demonstrated that a liverwort, Marchantiapolymorpha , contained steroidal plant hormones, collectivelynamed brassinosteroids (BRs), such as 6-deoxocastasterone(6-deoxoCS), castasterone (CS) and brassinolide (BL).

옥수수 유식물 신초에서 Brassinosteroid류의 동정 및 생합성 경로 추정

GC-MS/SIM를 통하여 옥수수 유식물 신초로부터 해 castasterone (CS)과 6-deoxocastasterone (6-deoxoCS)을 동정하였다. 또한 BRs의 생합성 전구물질인 24<TEX>$\alpha$</TEX>-methylcholesterol과 24<TEX>$\alpha$</TEX>-methylcholestanol이 동정하였다. 이들 BRs와 생합성 전구물질은 BRs의 생합성과정 중 후기 C6산화과정에 속하는 화합물로서, 옥수수 유식물 줄기에서는 BRs의 생합성 과정으로 후기 C6산화과정이 주된 생합성 과정임을 알 수 있었다. 다음으로 옥수수 유식물 줄기내의 BRs가 발아와 더불어 종자에서부터 이동해 온 것인지, 아니면 줄기 생장 시 줄기 자체가 생합성된 것인지를 조사하기 위하여 몇몇 중요한 후기 C6산화과정의 반응을 촉매하는 효소의 활성이 유식물 줄기에 존재하는지를 조사하였다. 그 결과 후기 C6산화과정에 포함되는 24<TEX>$\alpha$</TEX>-methylcholestanol에 서 6-deoxocathasterone (6-deoxoCT), 6-deoxoteasterone (6-deoxoTE) 에서 6-deoxo-3-dehydroteasterone (6-deoxo-3-DHT)을 거쳐 6-deoxotyphasterol (6-deoxoTY),그리고 6-deoxoCS에서 CS로의 과정을 촉매하는 24<TEX>$\alpha$</TEX>-methylcholestanol 22(R)-hydroxlyase, 6-deoxoTE dehydrogenase/6-deoxo-3-DHT reductase, 6-deoxoCS oxidase의 활성이 옥수수 유식물 줄기에 존재하고 있음이 확인되었다. 이는 유식물 줄기 생장 시 필요한 BRs가 발아종자로부터 이동되는 것이 아니라, 유식물 줄기 자체에서 생합성 될 가능성이 높음을 나타내는 결과라 할 수 있다. 또한 옥수수 유식물 줄기에서 CS를 brassinolide(BL)로 전환하는 BL synthase의 활성이 검출되지 않아, 옥수수 유식물 줄기에서의 활성형 BR은 BL이 아닌 CS임을 밝혔다. The potent biosynthetic precursors, 24<TEX>$\alpha$</TEX>-methylcholesterol and 24<TEX>$\alpha$</TEX>-methylcholestanol, and the endogenous brassinosteroids (BRs), castasterone (CS) and 6-deoxocastasterone (6-deoxoCS), were identified from shoots of maize seedlings. In addition, the presence for activities of several enzymes involved in the late C6-oxida-lion pathway from 24<TEX>$\alpha$</TEX>-methylcholestanol to CS was demonstrated in the plants. However, activity for brassinolide (BL) synthase which catalyze the conversion of CS to BL, the last step of the late C6-oxidation pathway, was not detected in the enzyme solution obtained from the maize shoots. Together with the fact that BL was not identified from the maize shoots, these results strongly suggested that BRs in the maize shoots are biosynthesized during seedling growth and the active BR in the shoots is not BL but CS.

Conversion of 6-Deoxocastasterone to Brassinolide in a Liverwort, Marchantia polymorpha

Department of Life Science, Chung-Ang University, Seoul 156-756, KoreaReceived June 4, 2003Key Words : Biosynthesis, Brassinosteroids, in vivo and in vitro conversion, Liverwort, MarchantiapolymorphaWe have recently demonstrated that a liverwort, Marchantiapolymorpha , contained steroidal plant hormones, collectivelynamed brassinosteroids (BRs), such as 6-deoxocastasterone(6-deoxoCS), castasterone (CS) and brassinolide (BL).

Identification and Transformation of Campestanol in Cultured Cells of Phaseolus vulgaris

Among them,brassinolide (BL, see structure in Figure 1) and its direct bio-synthetic precursor, castasterone (CS), have been consideredas the most important BRs because of their strong biologicalactivity and wide-distribution in plant kingdom. Recentfeeding experiments using isotope-labeled substrates andmolecular genetic analyses of BR-related mutants haverevealed that both BRs are biosynthesized from campesteol(CR), a phytosterol that has the same carbon skeleton asthose of the BRs, via two biosynthetic pathways, namely theearly and late C6-oxidation pathway.

Uncoupling brassinosteroid levels and de-etiolation in pea.

The suggestion that brassinosteroids (BRs) have a negative regulatory role in de-etiolation is based largely on correlative evidence, which includes the de-etiolated phenotypes of, and increased expression of light-regulated genes in, dark-grown mutants defective in BR biosynthesis or response. However, we have obtained the first direct evidence which shows that endogenous BR levels in light-grown pea seedlings are increased, not decreased, in comparison with those grown in the dark. Similarly, we found no evidence of a decrease in castasterone (CS) levels in seedlings that were transferred from the dark to the light for 24 h. Furthermore, CS levels in the constitutively de-etiolated lip1 mutant are similar to those in wild-type plants, and are not reduced as is the case in the BR-deficient lkb plants. Unlike lip1, the pea BR-deficient mutants lk and lkb are not de-etiolated at the morphological or molecular level, as they exhibit neither a de-etiolated phenotype or altered expression of light-regulated genes when grown in the dark. Similarly, dark-grown WT plants treated with the BR biosynthesis inhibitor, Brz, do not exhibit a de-etiolated phenotype. In addition, analysis of the lip1lkb double mutant revealed an additive phenotype indicative of the two genes acting in independent pathways. Together these results strongly suggest that BR levels do not play a negative-regulatory role in de-etiolation in pea.

The Promotive Effect of Brassinolide on Lamina Joint-Cell Elongation, Germination and Seedling Growth under Low-Temperature Stress in Rice (Oryza sativa L.)

SummaryThe cell elongation-promoting activities of brassinolide (BR) and indoleacetic acid (IAA) in the lamina-inclination test using etiolated rice seedlings were compared at a low temperature. IAA promoted the lamina joint-cell elongation at 30°G, but the effect was markedly lowered with decreasing temperature and reached null at 15°C. On the other hand, BR and castasterone (CAS), a brassinosteroid, promoted this cell elongation even at 15°G. Homobrassinolide (HBR), a brassinosteroid, also promoted this cell elongation, although the activity was weak. The combination of BR and IAA synergistically promoted this cell elongation at a low temperature (15°G).Immersion of the dry seeds in 2 X 10-8 M BR for the first 24 h promoted the early growth of rice seedlings. This treatment also enhanced the germination rate and the growth after direct sowing in submerged paddy pots in a greenhouse or phytotron at a low temperature (15°C). Leaf spraying of BR on the rice seedlings at the 4th leaf stage increased plant height and the fresh weights of tops and roots even at a low temperature. BR sprayed on the completely expanded 4th leaves did not increase their blade length, but that sprayed on the expanding 5th and 6th leaves strikingly increased their blade length, and the effect tended to be stronger at a low temperature.These results suggest that BR promotes cell elongation in young rice seedlings under low-temperature stress, although IAA had no such effect, and that BR may promote germination and the early growth of rice seedlings at a low temperature in direct sowing in the submerged paddy field and in the rice nursery.

Effect of brassinosteroids on nucleic acids and protein content in cultured cells of Chlorella vulgaris

This study was conducted to investigate changes of nucleic acids and protein levels in response to brassinosteroid (BR) effect in the green alga Chlorella vulgaris Beijerinck. BRs had the greatest effect on growth and metabolism of algae between 24 and 36 h after treatment in the range from 10 –12 to 10 –8 M. The highest growth of the content of nucleic acids and proteins was observed in the case of brassinolide (BL) at the concentration 10 –8 M in the 36th hour of cultivation. BL and its derivatives are biologically more active than castasterone (CS). The lowest stimulatory activity was shown by homoCS. The stimulatory effect of BRs in the algae was arranged in the following order: BL > 24-epiBL > homoBL > CS > 24-epiCS > homoCS. The relationship between the structure and the biological activity of BRs in stimulation of growth and changes in the content of DNA, RNA and proteins are discussed.

The tomato DWARF enzyme catalyses C-6 oxidation in brassinosteroid biosynthesis.

Brassinosteroids (BRs) are steroidal plant hormones essential for normal plant growth and development. Mutants in the biosynthesis or perception of BRs are usually dwarf. The tomato Dwarf gene (D), which was predicted to encode a cytochrome P450 enzyme (P450) with homology to other P450s involved in BR biosynthesis, was cloned previously. Here, we show that DWARF catalyses the C-6 oxidation of 6-deoxocastasterone (6-deoxoCS) to castasterone (CS), the immediate precursor of brassinolide. To do this, we first confirmed that the D cDNA complemented the mutant light- and dark-grown phenotypes of the extreme dwarf (dx) allele of tomato. To identify a substrate for the DWARF enzyme, exogenous application of BR intermediates to dx plants was carried out. C-6 oxoBR intermediates enhanced hypocotyl elongation whereas the C-6 deoxoBR, 6-deoxoCS, had little effect. Quantitative analysis of endogenous BR levels in tomato showed mainly the presence of 6-deoxoBRs. Furthermore, dx plants were found to lack CS and had a high level of 6-deoxoCS in comparison to D plants that had CS and a lower level of 6-deoxoCS. Confirmation that DWARF catalyzed the C-6 oxidation of 6-deoxoCS to CS was obtained by functional expression of DWARF in yeast. In these experiments, the intermediate 6alpha-hydroxycastasterone was identified, indicating that DWARF catalyzes two steps in BR biosynthesis. These data show that DWARF is involved in the C-6 oxidation in BR biosynthesis.

Physiological and Biochemical Role of Brassinosteroids and Their Structure-Activity Relationship in the Green Alga Chlorella vulgaris Beijerinck (Chlorophyceae)

This paper studies the influence of the 7-oxalactone type of brassinosteroids (BRs) and 6-ketone upon the biological activity of the alga Chlorella vulgaris (Chlorophyceae). The results of the study indicate significant differences in the growth and metabolism of C. vulgaris cells caused by the different chemical structures of the BRs used. The most significant differences in the stimulation of the growth of the biomass and metabolites contained in it were caused by structural differences in the B ring of BRs. It was found that in C. vulgaris 7-oxalactone type of BRs [brassinolide (BL) and its derivatives] are more active than 6-ketone type of BRs [castasterone (CS) and its derivatives]. It was found that BRs used within the range of concentration of 10−12 to 10−8 m stimulate two- to threefold the growth and division of C. vulgaris cells. The most stimulating influence upon the number of the algal cells and the phosphorus, chlorophyll, and monosaccharides contained in the alga, as well as the intensity of the photosynthesis, and sugar and glycolate excretion was demonstrated by BL at a concentration 10−8 m in the 36th h of cultivation. HomoCS was characterized by the lowest biological activity. In turn, after the 48th h an inhibition of the rate of growth and development of the alga takes place. In the range from 10−7 to 10−6 m the inhibition of growth and development of the alga was manifested by BRs. During the further toxic activity of BRs the cells of C. vulgaris undergo complete degradation. In turn, in concentrations lower than 10−12 m, BRs do not exert any biologically significant influence upon C. vulgaris cells. On the basis of the study, the biological activity of BRs was arranged in the following order: BL > 24-epiBL > homoBL > CS > 24-epiCS > homoCS.

Biosynthesis of Brassinosteroids in Seedlings ofCatharanthus roseus,Nicotiana tabacum, andOryza sativa

The biosynthesis of brassinosteroids was investigated by feeding 2H-labeled teasterone (TE), 2H-labeled typhasterol (TY), and a mixture of 2H-labeled and 3H-labeled castasterone (CS) to seedlings of Catharanthus roseus, Nicotiana tabacum, and Oryza sativa. In all the seedlings examined, the conversion of TE to TY, and the conversion of TY to CS and to TE was demonstrated. In the seedlings of C. roseus, the conversion of CS to brassinolide (BL) and to 3-epicastasterone (3-epiCS) was observed, while the conversion of CS to only 3-epiCS was found in the seedlings of N. tabacum and O. sativa.These results demonstrate the presence of the biosynthetic pathway of TE→TY →CS→BL in seedlings of C. roseus, and the biosynthetic pathway of TE→TY→CS in seedlings of N. tabacum and O. sativa. A reversible conversion between TE and TY was also found. This is the first evidence of CS being the biosynthetic origin of 3-epiCS.

Biosynthesis of brassinolide from teasterone via typhasterol and castasterone in cultured cells of Catharanthus roseus

The biosynthesis of brassinolide (BL) in crown gall and nontransformed cells of Catharanthus roseus in which BL, castasterone (CS), typhasterol (TY), and teasterone (TE) are endogenous was investigated using deuterated TY and TE as substrates. The metabolites were analyzed by gas chromatography-mass spectrometry (GC-MS) and/or GC-selected ion monitoring (SIM). It was found that these cells converted TY to CS and BL, as well as TE to TY and CS. Because the pathway from CS to BL in the cells has already been confirmed, a biosynthetic sequence of TE → TY → CS → BL was established. Reversible conversion between TE and TY was observed.

Biosynthesis of brassinolide from castasterone in cultured cells of Catharanthus roseus

Feeding experiments with tritium- and deuterium-labeled castasterone (CS) were conducted with three cell lines of Catharanthus roseus, including crown gall cells and nontransformed cells. In all three cell lines, the conversion of CS to brassinolide (BL) was observed and unequivocally confirmed by gas chromatography/mass spectrometry (GC/MS). This is the first conclusive evidence that CS is the biosynthetic precursor of BL.