Brassinolide application to Lepidium sativum seeds and the effects on seedling growth

Brassinosteroids have been reported to accelerate plant growth when applied to seeds. We examined the effects of seed treatment with brassinolide on early growth of Lepidium sativum (cress). Submicromolar and micromolar concentrations of brassinolide inhibited root growth within 48 h after seed treatment. Germination of cress was not affected by brassinolide. The inhibition of cress root growth by brassinolide was time specific in terms of eliciting the response. Untreated germinated seeds transferred to filter paper moistened with brassinolide solution did not exhibit the same level of root inhibition as treated seeds. Brassinolide (2 m) had no effect on ethylene levels, suggesting that at this concentration brassinolide is acting independently of ethylene to inhibit cress root elongation. Also, brassinolide had no effect on DNA synthesis within 24 h after seed treatment, but synthesis was reduced after 48 h. The results of this study illustrate a significant specific effect on very early cress root growth by seed treatment with brassinolide.Abbreviations BR brassinosteroid(s) - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - ACC 1-aminocyclopropane-1-carboxylic acid     

Brassinolide affects the rate of cell division in isolated leaf protoplasts of Petunia hybrida

Brassinosteroids are known to promote cell elongation in a wide range of plant species but their effect on cell division has not been as extensively studied. We examined the effect of brassinolide on the kinetics and final division frequencies of regenerating leaf mesophyll protoplasts of Petunia hybrida Vilm v. Comanche. Under optimal auxin and cytokinin conditions, 10–100 nM brassinolide accelerated the time of first cell division by 12 h but had little effect on the final division frequencies after 72–120 h of culture. One micromolar brassinolide showed the same acceleration of first cell division but inhibited the final division frequency by approximately 20%. Under sub-optimal auxin conditions, 10–100 nM brassinolide both accelerated the time of first cell division and dramatically increased the 72- to 120-h final division frequencies. Isolated protoplasts may provide a useful model system to investigate the molecular mechanisms of brassinosteroid action on cell proliferation. Received: 1 December 1997 / Revision received: 13 February 1998 / Accepted: 24 April 1998     

油菜素内酯（BR）促进植物生长机理研究进展

介绍了油菜素内酯促进植物生长,提高作物产量的作用,并简述了促进生长的生理代谢基础,通过比较油菜素内酯与生长素,赤霉素促进生长作用方式的异同,提出油菜素内酯促进生长的信号传导路径不同于其它植物激素,另外从细胞的形态发生,细胞壁扩展的机制和细胞骨架在细胞伸长中的作用等几个方面对油菜素内酯促进植物生长的细胞及分子生物学机制进行了详尽的论述。     

Castasterone is a likely end product of brassinosteroid biosynthetic pathway in rice

Brassinolide is known to be the most biologically active compound among more than 50 brassinosteroids identified to date. However, brassinolide has not been detected in rice. To determine if this is due to the lack of the brassinolide synthase function in the rice CYP85A enzyme, we performed analyses to study metabolic conversion using a yeast strain harboring the rice CYP85A1 gene. In repeated feeding tests where the substrates were used, the biosynthetic pathway progressed only up to the synthesis of castasterone, not of brassinolide. Phylogenetic analysis of the CYP85 amino acid sequences revealed that duplication of the CYP85 gene has occurred in most dicotyledonous plant genomes; further, 1 of the 2 copies of CYP85 is evolving to develop a brassinolide synthase function. However, only a single copy of this gene is found in the currently available genome sequences of graminaceous plants; this is a likely explanation for the absence of an endogenous pool of brassinolide in rice plants.     

The effect of brassinolide on the light-induced growth inhibition in mung bean epicotyl

Growth of the mung bean (Vigna radiata L.) epicotyl was retarded by white (400–700 nm) light, especially by monochromatic red (660 nm) light. Growth promoting effects of brassinolide were observed under those light conditions that retarded growth, but were not evident in the dark or under far-red light. Brassinolide seems to act to overcome the inhibitory affects of lights.     

Structure-activity relationship of brassinosteroids

The plant growth-promoting activities of brassinolide and brassinosteroids with different side chains were investigated by means of the Raphanus an     

Endogenous brassinosteroids in microalgae exposed to salt and low temperature stress

Brassinosteroids are part of the hormonal network that regulates growth processes and stress responses in plants. There is evidence for a similar hormonal network in microalgae. In the present study, six microalgae (Chlorococcum ellipsoideum, Gyoerffyana humicola, Nautococcus mamillatus, Acutodesmus acuminatus, Protococcus viridis and Chlorella vulgaris) were subjected to salt and low temperature stress with the addition of 36 g l^–1 NaCl and transfer from 25°C to 15°C. There was a rapid response to salt stress with the brassinosteroid content (mainly castasterone with lower amounts of brassinolide, homocastasterone and typhasterol) increasing within 30 min of the salt treatment and remaining at these elevated levels after 7 h. The decrease in temperature had little effect on the brassinosteroid content. This was the first study to show that endogenous brassinosteroids increase in response to abiotic stress in a number of microalgae species.     

Synthesis and bioactivity of C-2 and C-3 methyl ether derivatives of brassinolide

The following six novel methyl ether derivatives of brassinolide were prepared and their brassinosteroid activity was measured by means of the rice leaf lamina inclination bioassay: 2-O-methylbrassinolide, 3-O-methylbrassinolide, 2,22,23-tri-O-methylbrassinolide, 3,22,23-tri-O-methylbrassinolide, 2-O-methyl-25-methoxybrassinolide and 3-O-methyl-25-methoxybrassinolide. Brassinolide was used as a standard for comparison. All six compounds were also tested in the presence of 1000 ng of indole-3-acetic acid (IAA), an auxin that synergizes the effects of brassinosteroids. The 2-O-methyl- and 3-O-methylbrassinolide derivatives showed weak activity at high doses, which was enhanced by IAA, especially in the case of the 3-O-methyl derivative. Similarly, the 2,22,23-tri-O-methyl- and 3,22,23-tri-O-methyl derivatives displayed weak bioactivity on their own, but significantly stronger activity when applied with IAA. The 3-O-methyl and 3,22,23-tri-O-methyl analogues plus IAA were comparable in bioacivity to brassinolide alone, but were less active than brassinolide plus IAA. In each case, O-methylation at C-2 resulted in a greater loss of activity than O-methylation at C-3 under the same conditions. The relatively strong activity of 3,22,23-tri-O-methylbrassinolide in the presence of IAA is especially noteworthy as it indicates that free hydroxyl groups at C-3, C-22, and C-23 are not essential for bioactivity. Finally, 2-O-methyl- and 3-O-methyl-25-methoxybrassinolide were essentially inactive alone, and showed only a modest increase in bioactivity when coapplied with IAA.     

A rapid effect of applied brassinolide on abscisic acid concentrations in <Emphasis Type="Italic">Brassica napus</Emphasis> leaf tissue subjected to short-term heat stress

Three-week old canola (Brassica napus L.) seedlings grown at 20/16°C (day/night) were subjected to short-term (4 and 8 h) heat stress (45°C) or maintained at a normal temperature of 20°C. Half of the plants under each treatment received a 10⁻⁶ M solution of brassinolide (BL) 1 h prior to beginning the temperature treatments. The concentration (ng/g dry weight) of endogenous abscisic acid (ABA) was subsequently determined in young leaves via the stable isotope dilution method. Applied BL had no effect on endogenous ABA for plants maintained at normal temperatures. However, ABA concentration was significantly elevated by heat stress alone and doubled by heat stress + BL. These results suggest that the well-known enhancement of tolerance to high temperature stress that can be obtained by BL or 24-epi-BL applications may be caused by a brassinosteroid-induced elevation in endogenous ABA concentration.     

Brassinolide-2,3-acetonide: A brassinolide-induced rice lamina joint inclination antagonist

A novel chemical tool compound that is an antagonist of brassinolide (BL, 1)-induced rice lamina joint inclination was developed. Although 2-O-, 3-O-, 22-O-, or 23-O-methylation of BL causes a critical decrease in biological activity,⁵ a crystal structure of the extracellular leucine-rich repeat (LRR) domain of BRASSINOSTEROID-INSENSITIVE I (BRI1) bound to BL3, 4 indicates that the loss of activity of the O-methylated BL may result from not only the low affinity to BRI1, but also from blocking the interaction with another BR signaling factor, a partner protein of BRI1 (e.g., BRI1-ASSOCIATED KINASE 1, BAK1). On the basis of this hypothesis we synthesized the BL 2,3-acetonide 2, the 22,23-acetonide 3, and the 2,3:22,23-diacetonide 4 to assess the possibility of 2-O- and 3-O- or/and 22-O- and 23-O-alkylated BL as an antagonist in BR signaling evoked by exogenously applied BL. The 2,3-acetonide 2 more strongly inhibited the lamina inclination caused by BL relative to the 22,23-acetonide 3, whereas the diacetonide 4 had no effect most likely due to its increased hydrophobicity. This suggested that the 2,3-hydroxyl groups of BL play a more significant role in the interaction with a BRI1 partner protein rather than BRI1 itself in rice lamina joint inclination. Taken together it was demonstrated that BL, the most potent agonist of BRI1, is transformed into an antagonist by functionalization of the 2,3-dihydroxyl groups as the acetonide. This finding opens the door to the potential development of a chemical tool that modulates protein–protein interactions in the BR signaling pathway to dissect the BR-dependent processes.     

A simple brassinolide analogue 2α, 3α-dihydroxy-17β-(3-methyl-butyryloxy)-7-oxa-B-homo-5α-androstan-6-one which induces bean second internode splitting

A new synthetic brassinolide analogue, 2,3-dihydroxy-17-(3-methylbutyryloxy)-7-oxa-B-homo-5-androstan-6-one (11), has been shown to exhibit typical brassinolide activity characterised by elongation, swelling, twisting and splitting of the bean second internode. It was prepared from the known lactone 2,3,17-trihydroxy-7-oxa-B-homo-5-androstan-6-one (4) which was transformed to an isopropylidenedioxy derivative. After protection of the 2- and 3-hydroxy groups it yielded the 2,3-isopropylidenedioxy-17-(3-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one (7) on treating with 3-methylbutyryl chloride in pyridine. The analogue with a 2-methylbutyric moiety (10, 2,3-dihydroxy-17-(2-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one) in position 17 stimulated only elongation and swelling of the bean second internode. However, in this bioassay 100 times more 10 or 11 compared to 24-epibrassinolide is required to obtain the same effects. Analogues with -oriented hydroxyl groups at C-2 and C-3 (14,15), a 6-ketone (17,18) or 6-oxa-7-oxo-lactone system (12,13) in ring B lack the typical brassinolide activity. In addition, the active brassinosteroids applied to the second internode stimulated a similar, but 30% lower elongation of the first internode. From data presented here we conclude that the presence of two hydroxy groups in the positions 22 and 23 of the brassinolide side chain, which are considered as a key structural requirement, is not absolutely necessary for a compound to exhibit typical brassinosteroid activity. Nevertheless, these compounds have generally 2–10 times lower activity than that having 22,23-vicinal diol in the side chain.     

Brassinolide and [26, 28-2H6]brassinolide are differently demethylated by loss of C-26 and C-28, respectively, in Marchantia polymorpha

Metabolism of brassinolide in Marchantia polymorpha was investigated by use of in vivo suspension cultured cells. GC-MS analysis of metabolites derived from non-labelled brassinolide and [26, 28-2H6] brassinolide revealed that brassinolide was converted to 26-norbrassinolide while [26, 28-2H6]brassinolide to [26-2H3]28-norbrassinolide. It seems that Marchantia cells recognized [26, 28-2H6]brassinolide as a xenobiotic rather than brassinolide and deteriums attached to C-28 significantly affect demethylation reaction due to isotopic effect. Thus, demethylation of brassinolide in planta seems to proceed by loss of C-26 rather than C-28. The present finding is the first evidence for demethylation metabolism of brassinosteroids. The biological activity of 26-norbrassinolide was 10-fold reduced as shown by the rice lamina inclination test. However, because of its high biological activity, it remains difficult to conclude whether or not C-26 demethylation serves as an important deactivation process of brassinolide.     

Structure–Activity Studies of Brassinosteroids and the Search for Novel Analogues and Mimetics with Improved Bioactivity

A number of novel brassinosteroid analogues were synthesized and subjected to the rice leaf lamina inclination bioassay. Modified B-ring analogues included lactam, thiolactone, cyclic ether, ketone, hydroxyl, and exocyclic methylene derivatives of brassinolide. Those derivatives containing polar functional groups retained considerable bioactivity, whereas the exocyclic methylene compounds were devoid of activity. Analogues containing normal alkyl and cycloalkyl substituents at C-24 (in place of the isopropyl group of brassinolide) showed an inverse relationship between activity and chain length or ring size, respectively. The corresponding cyclopropyl and cyclobutyl derivatives were significantly more active than brassinolide and appear to be the most potent brassinosteroids reported to date. When synergized with the auxin indole-3-acetic acid (IAA), their bioactivity can be further enhanced by 1–2 orders of magnitude. The cyclopropyl derivative, when coapplied with the auxin naphthaleneacetic acid, gave a significant increase in yield of wheat in a field trial. Certain 25- and 26-hydroxy derivatives are known metabolites of brassinosteroids. All of the C-25 stereoisomers of 25-hydroxy, 26-hydroxy, and 25,26-dihydroxy derivatives of brassinolide were prepared and shown to be much less active than brassinolide. This indicates that they are likely metabolic deactivation products of the parent phytohormone. A series of methyl ethers of brassinolide was synthesized to block deactivation by glucosylation of the free hydroxyl groups. The most significant finding was that the compound where three of the four hydroxyl groups (at C-3, C-22, and C-23) had been converted to methyl ethers retained substantial bioactivity. This type of modification could, in theory, allow brassinolide or 24-epibrassinolide to resist deactivation and thus offer greater persistence in field applications. A series of nonsteroidal mimetics of brassinolide was designed and synthesized. Two of the mimetics showed significant bioactivity and one had bioactivity comparable to brassinolide, but only when formulated and coapplied with IAA. They thus represent the first nonsteroidal analogues possessing brassinosteroid activity.     

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.     

Optimization of a rice lamina inclination assay for detection of brassinosteroids: I. effect of phytohormones on the inclination activity

Effect of ABA, GA₃, zeatin (Zea) and IAA on the inclination activity was examined by a rice lamina inclination assay using a Korean cultivar, Tongjin. Treatment with ABA, GA₃, Zea or IAA alone failed to increase the inclination response significantly at the concentration tested from 0.1 ppm to 10 ppm. However, treatment with 0.1 and 1 ppm ABA in the presence of brassinolide inhibited the inclination activity induced by treatment with brassinolide alone. And treatment with 0.1 and 1 ppm GA₃ or Zea in the presence of brassinolide strongly inhibited the inclination activity induced by treatment with brassinolide alone. On the other hand, the inclination activity by treatment with brassinolide alone was clearly promoted by treatment with 0.1 and 1 ppm IAA in the presence of brassinolide. Based on the synergistic effect induced by treatment with BR and IAA, we could develope an improved rice lamina inclination assay whose minimum detectable concentration of brassinolide is 0.00001 ppm /petri dish. The minimum detectable concentration in our assay was five times as low as that of the previous rice lamina inclination assay.     

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.     

Effects of brassinolide on drought resistance of <Emphasis Type="Italic">Xanthoceras sorbifolia</Emphasis> seedlings under water stress

We conducted pot experiments to investigate the effects of brassinolide on 1-year-old Xanthoceras sorbifolia B. seedlings. In the experiment, roots were soaked in 0–0.4 mg/l brassinolide. After the seedlings were established, the soil water content in the pots was regulated to simulate drought conditions and various physiological parameters were measured. The results showed that the treatment with 0.2 mg/l brassinolide decreased the malondialdehyde content and electrolyte leakage of seedlings growing under moderate or severe water stress when compared with untreated seedlings. Leaf water content, relative water content, soluble sugar content, soluble protein content, free proline content, ascorbic acid content, glutathione content and superoxide dismutase, peroxidase, catalase and ascorbate peroxidase activities were all greater in water-stressed seedlings in the 0.2 mg/l brassinolide treatment as compared to the control. The results indicate that the application of brassinolide can ameliorate the effects of water stress and enhance drought resistance of Xanthoceras sorbifolia seedlings.     

Bioactivity of 25-hydroxy-, 26-hydroxy, 25,26-dihydroxy- and 25,26-epoxybrassinolide.

The bioactivity of 25-hydroxybrassinolide, (25S)- and (25R)-26-hydroxybrassinolide, (25S)- and (25R)-25,26-dihydroxybrassinolide, and of (25R)-25,26-epoxybrassinolide was tested in the rice leaf lamina inclination assay. The 25- and (25S)-26-hydroxy derivatives are known metabolites of the naturally-occurring phytohormone brassinolide, whereas the other compounds are novel, but closely related, congeners. When tested alone, all showed either no activity or only weak activity at relatively high doses. When coapplied with indole-3-acetic acid (IAA), an auxin that synergizes the effects of brassinosteroids, enhanced bioactivity was observed for each compound. However, even when applied together with IAA, none of the compounds proved more bioactive than brassinolide with or without IAA. We conclude from these results that enzymatic hydroxylation of endogenous brassinolide at C-25 and/or C-26 does not enhance brassinosteroid activity, and so does not comprise an activation pathway in brassinolide biosynthesis. Instead, these hydroxylations result in modest to appreciable metabolic deactivation.     

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.