Physiological and molecular effects of brassinosteroids on Arabidopsis thaliana

We examined the effects of brassinosteroids on Arabidopsis thaliana (L.) Henyh. ecotype Columbia in order to develop a model system for studying gene regulation by plant steroids. Submicromolar concentrations of two brassinosteroids, brassinolide and 24-epibrassinolide, stimulated elongation of Arabidopsis peduncles and inhibited root elongation, respectively. Furthermore, brassinolide altered the abundance of specific in vitro translatable mRNAs from peduncles and whole plants of Arabidopsis. Root elongation in the auxin-insensitive Arabidopsis mutant axr1 was inhibited by 24-epibrassinolide but not by 2,4-D, indicating an independent mode of action for these growth regulators in this physiological response.Abbreviations BR brassinolide - EBR 24-epibrassinolide; 2.4-D,2,4-dichlorophenoxyacetic acid - KPSC 10 mM potassium phosphate, pH 6.0, 2% sucrose, 50 g/ml chloramphenicol - PAGE polyacrylamide gel electrophoresis     

Physiological effects and transport of 24-epibrassinolide in heat-stressed barley

This paper presents a study of the metabolic response (dark respiration intensity, photosystem II efficiency, metabolic activity) and the yield of barley treated with 24-epibrassinolide and subjected to high-temperature stress. Transport of exogenously applied 24-epibrassinolide in barley and changes in the profile of brassinosteroids that may occur in tissues after 24-epibrassinolide application were also studied. The water solution of 24-epibrassinolide (0.005 and 0.25 mg dm⁻³) was applied via infiltration of the first and second leaves of 12-day-old seedlings. Control plants were treated with water solution of hormone solvent (ethanol). Fifteen-day-old plants were subjected to high-temperature stress (42°C for 3 h). The influence of hormone treatment and stress conditions was investigated in the first and second leaves based on measurements of PSII efficiency. The aftereffect of plant treatment was investigated in the seventh leaf (measurements of PS II efficiency, dark respiration intensity, metabolic activity). The transport efficiency of 24-epibrassinolide exogenously applied to the first and second leaves, as well as the profile of other brassinosteroids, was also measured on the seventh leaf. Finally, yield formation was estimated. 24-epibrassinolide showed protective action, which manifested itself in the improved functioning of PSII, but this was observed in case of higher hormone concentration and only for the first, older leaf. The PSII efficiency of the seventh leaf was similar in plants treated with brassinosteroid and in the control plants, whereas the respiration intensity and metabolic activity decreased in plants previously treated with higher concentration of 24-epibrassinolide. The use of a higher hormone concentration at the seedling phase ultimately resulted also in lower crop yield. Brassinosteroids—brassinolide and castasterone—were detected in barley leaves. 24-epibrassinolide was found only in trace amounts in control plants. Its exogenous application directly to the apoplast of the first and second leaves resulted in an increase in the 24-epibrassinolide content in the seventh leaf, but did not depend on whether a high or low concentration had been applied to the plants.     

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.     

Treatment with 24-epibrassinolide,a brassinosteroid,increases the basic thermotolerance of Brassica napus and tomato seedlings

Brassinosteroids are plant growth-promoting compounds that exhibit structural similarities to animal steroid hormones. Recent studies have indicated that brassinosteroids are essential for proper plant development. In addition to a role in development, several lines of evidence suggest that brassinosteroids exert anti-stress effects on plants. However, the mechanism by which they modulate plant stress responses is not understood. We show here that Brassica napus and tomato seedlings grown in the presence of 24-epibrassinolide (EBR) are significantly more tolerant to a lethal heat treatment than are control seedlings grown in the absence of the compound. Since a preconditioning treatment of seedlings was not required to observe this effect, we conclude that EBR treatment increases the basic thermotolerance of seedlings. An analysis of heat shock proteins (HSPs) in B. napus seedlings by western blot analysis indicated that the HSPs did not preferentially accumulate in EBR-treated seedlings at the control temperature. However, after heat stress, HSP accumulation was higher in EBR-treated than in untreated seedlings. The results of the present study provide the first direct evidence for EBR-induced expression of HSPs. The higher accumulation of HSPs in EBR-treated seedlings raises the possibility that HSPs contribute, at least in part, to thermotolerance in EBR-treated seedlings. A search for factors other than HSPs, which may directly or indirectly contribute to brassinosteroid-mediated increase in thermotolerance, is underway.     

Modulation of growth,photosynthetic capacity and water relations in salt stressed wheat plants by exogenously applied 24-epibrassinolide

Brassinosteroids promote the growth of plants and are effective in alleviating adverse effects of abiotic stresses such as salinity and drought. Under saline conditions, improvement in grain yield is more important than simple growth. Previously it was found that although foliar application of brassinosteroids improved growth of wheat plants, it did not increase grain yield. In present study, influence of root applied 24-epibrassinolide was assessed in improving growth and yield of two wheat cultivars. Plants of a salt tolerant (S-24) and a moderately salt sensitive (MH-97) were grown at 0 or 120 mM NaCl in continuously aerated Hoagland’s nutrient solution. Different concentrations of 24-epibrassinolide (0, 0.052, 0.104, 0.156 μM) were also maintained in the solution culture. Exogenous application of 24-epibrassinolide counteracted the salt stress-induced growth and grain yield inhibition of both wheat cultivars. Of the varying 24-epibrassinolide concentrations used, the most effective concentrations for promoting growth were 0.104 and 0.052 μM under normal and saline conditions, respectively. However, root applied 0.052 μM 24-epibrassinolide enhanced the total grain yield and 100 grain weight of salt stressed plants of both cultivars and suggested that total grain yield was mainly increased by increase in grain size which might have been due to 24-epibrassinolide induced increase in translocation of more photoassimilates towards grain. Growth improvement in both cultivars due to root applied 24-epibrassinolide was found to be associated with improved photosynthetic capacity. Changes in photosynthetic rate due to 24-epibrassinolide application were found to be associated with non-stomatal limitations, other than photochemical efficiency of PSII and photosynthetic pigments. Leaf turgor potential found not to be involved in growth promotion.     

Brassinosteroids promote root growth in Arabidopsis

Although brassinosteroids (BRs) are known to regulate shoot growth, their role in the regulation of root growth is less clear. We show that low concentrations of BRs such as 24-epicastasterone and 24-epibrassinolide promote root elongation in Arabidopsis wild-type plants up to 50% and in BR-deficient mutants such as dwf1-6 (cbb1) and cbb3 (which is allelic to cpd) up to 150%. The growth-stimulating effect of exogenous BRs is not reduced by the auxin transport inhibitor 2,3,5-triidobenzoic acid. BR-deficient mutants show normal gravitropism, and 2,3,5-triidobenzoic acid or higher concentrations of 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid inhibit root growth in the mutants to the same extent as in wild-type plants. Simultaneous administration of 24-epibrassinolide and 2,4-dichlorophenoxyacetic acid results in largely additive effects. Exogenous gibberellins do not promote root elongation in the BR-deficient mutants, and the sensitivity to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid is not altered. Thus, the root growth-stimulating effect of BRs appears to be largely independent of auxin and gibberellin action. Furthermore, we analyzed BR interactions with other phytohormones on the gene expression level. Only a limited set of auxin- and ethylene-related genes showed altered expression levels. Genes related to other phytohormones barely showed changes, providing further evidence for an autonomous stimulatory effect of BR on root growth.     

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.     

The multi-protein family of Arabidopsis sulphotransferases and their relatives in other plant species

All members of the sulphotransferase (SOT, EC 2.8.2.-) protein family use 3'-phosphoadenosine 5'-phosphosulphate (PAPS) as the sulphuryl donor and transfer the sulphonate group to an appropriate hydroxyl group of several classes of substrates. These enzymes have highly conserved domains and can be found in eubacteria and eukaryotes. In mammals, sulphate conjugation catalysed by SOTs constitutes an important reaction in the transformation of xenobiotics, and in the modulation of the biological activity of steroid hormones and neurotransmitters. In plants, sulphate-conjugation reactions seem to play an important role in plant growth, development, and adaptation to stress. To date only a few plant SOTs have been characterized in detail. The flavonol 3- and 4'-SOTs from Flaveria species (Asteraceae), which catalyse the sulphonation of flavonol aglycones and flavonol 3-sulphates, respectively, were the first plant SOTs for which cDNA clones were isolated. The plasma membrane associated gallic acid SOT of Mimosa pudica L. pulvini cells may be intrinsic to signalling events that modify the seismonastic response. In Brassica napus L. a SOT catalyses the O-sulphonation of brassinosteroids and thereby abolishes specifically the biological activity of 24-epibrassinolide. The fully sequenced genome of Arabidopsis thaliana Heynh. contains in total 18 genes that are likely to encode SOT proteins based on sequence similarities of the translated products with an average identity of 51.1%. So far only one SOT from A. thaliana (At5g07000) was functionally characterized: the protein was shown to catalyse the sulphonation of 12-hydroxyjasmonate and thereby inactivate excess jasmonic acid in plants. The substrates and, therefore, the physiological roles of SOTs are very diverse. By using the numerous informative databases and methods available for the model plant A. thaliana, the elucidation of the functional role of the SOT protein family will be accelerated.     

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.     

Biological activities of new monohydroxylated brassinosteroid analogues with a carboxylic group in the side chain

Thirteen monohydroxylated brassinosteroids analogues were synthesized and tested for their biological activity in plant and animal systems. The cytotoxic activity of the products was studied using human normal and cancer cell lines with 28-homocastasterone as positive control, their brassinolide type activity was established using the bean second-internode test with 24-epibrassinolide as standard.     

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.     

Evaluation of biological activity of new synthetic brassinolide analogs

The responses of plants to exogenous treatment with new synthetic brassinosteroids (BRs) were assessed and compared with the activity of natural 24-epibrassinolide (24-EPI). Morphological experiments on plants of pea and flax showed that the boundary between stimulatory and inhibitory concentrations of individual BRs and 24-EPI used is very narrow and differs also with the plant species. Moreover brassinosteroids can exhibit effects similar to various other plant hormones. This was proven also in our experiments, where auxin, anti-auxin and cytokinin like effects were achieved by BRs application. One of the explanations of the different morphological effects could be the influence of brassinosteroid application on the level of endogenous hormones. There are changes in the levels of indole-3-acetic acid, 6-benzylaminopurine, trans-zeatin and dihydrozeatin in rape and wheat plants caused by BR 4 and 24-EPI application, but there is no general trend explaining unequivocally their influence. The fact that all tested BRs significantly increased the dry weight accumulation in comparison with non-treated reference rape plants can be accounted for the known BRs characteristics to avoid biotic stresses.     

Physiological and Molecular Effects of 24-Epibrassinolide, a Brassinosteroid on Thermotolerance of Tomato

Brassinosteroids are naturally occurring plant growth regulators, which exhibit structural similarities to animal steroid hormones. Recent studies have indicated that besides an essential role in plant growth and development, brassinosteroids also exert anti-stress effects on plants. We show here that tomato plants treated with 24-epibrassinolide (EBR) are more tolerant to high temperature than untreated plants. An analysis of mitochondrial small heat shock proteins (MT-sHSP) in tomato leaves by western blotting revealed that the MT-sHSP did not preferentially accumulate in EBR treated plants at 25 °C. However, treatment of plants at 38 °C induced much more accumulation of MT-sHSP in EBR treated than in untreated plants. Results of this study provide the first direct evidence for EBR induced expression of MT-sHSP, which possibly induced thermotolerance in tomato plants. EBR treated tomato plants had better photosynthetic efficiency. We also observed significantly higher in vitro pollen germination, enhanced pollen tube growth and low pollen bursting in the presence of EBR at 35 °C, a temperature high enough to induce heat-stress symptoms in tomato, indicating a possible role of EBR during plant reproduction.     

Effect of brassinosteroids on growth and proton extrusion in the alga Chlorella vulgaris Beijerinck (Chlorophyceae)

Brassinolide, as a plant hormone, promotes growth of a number of plant species. Similar effects are induced by its epimer 24-epibrassinolide. In this paper we discuss the effects of brassinosteroids on the growth and proton extrusion in the green alga Chlorella vulgaris (Chlorophyceae). At concentrations between 10^–15 and 10^–8 m, brassinolide and 24-epibrassinolide induce a significant stimulation of growth and H⁺ extrusion. The growth was associated with an increase in the capability of algal cells to acidify the medium, where brassinolide is biologically more active than 24-epibrassinolide.Abbreviations BL brassinolide - BR(s) brassinosteroid(s) - epiBL 24-epibrassinolide - DW dry weight - IAA indole-3-acetic acid     

Effect of brassionosteriods on nodulation and nitrogenase activity in groundnut (Arachis hypogaea L.)

The effect of brassinolide, 24-epibrassinolide and 28-homobrassinolide on nodulation and nitrogenase activity of groundnut was studied. The tested brassinosteroids substantially increased both nodulation and nitrogenase activity.     

Influence of Brassinosteroids on Antioxidant Enzymes Activity in Tomato Under Different Temperatures

The effect of brassinosteroids (BRs) on catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7) and superoxide dismutase (SOD, EC 1.15.1.1) activity in tomato leaf discs was analyzed at 25 and 40 °C. Tomato leaf discs were preincubated for 24 h in Petri dishes with 24-epibrassinolide (EBR) or a polyhydroxylated spirostanic analogue of brassinosteroids (MH5). Both concentrations (10.60 and 2.12 nM) of EBR and MH5 stimulated the activity of SOD at 25 and 40 °C, the MH5-stimulated increase of this enzyme activity was greater. Peroxidase activity was unaffected at 25 °C, while at 40 °C this activity was enhanced by both compounds. The changes in catalase activity markedly depended on the structure BRs, doses and temperature. The results suggest a possible role of EBR and MH5 in the reduction of cell damage produced by heat stress due to induction of enzymatic antioxidants.     

An Hsp90 Inhibitor, Geldanamycin, as a Brassinosteroid Antagonist: Evidence from Salt-Exposed Roots of Vigna radiata

Abstract: Treatment of non-stressed mung bean cuttings with 24-epibrassinolide (BR) inhibited elongation of adventitious roots, but it stimulated root elongation in plants exposed to 70 mM NaCl. These findings confirm that brassinosteroids are involved in control of division and elongation of root cells. We found that both positive and negative effects are counteracted by the presence of geldanamycin in the root medium. Because of the high specificity of geldanamycin for the chaperone Hsp90, this suggests that, as in animal cells, Hsp90 is involved in expression or transduction of the brassinosteroid signals in plants. Moreover, a detailed analysis of root ranking showed an additional geldanamycin-insensitive mode of action of BR, especially observed under salt stress conditions. These two modes differently affected elongation of the adventitious roots, depended on the phases of differentiation and on the presence of an environmental constraint.