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.     

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.     

The effect of 24-epibrassinolide and clotrimazole on the adaptation of Cajanus cajan (L.) Millsp. to salinity

The objective of this study was to evaluate the effects of one of brassinosteroids (24-epibrassinolide) and clotrimazole, (an inhibitor of brassinosteroid synthesis) on plant growth parameters, parameters related to leaf gas exchange (photosynthetic and transpiration rates; stomatal conductance; water use efficiency), photosynthetic pigment content and osmolyte (sugars and proline) content in Cajanus cajan exposed to salinity. Salt stress??caused by NaCl treatment??affected values of all parameters analyzed. The effects were ameliorated by 24-epibrassinolide and intensified by clotrimazole. The hormone increased fresh mass of the plant, shoot dry mass, leaf area, water content of leaves and roots, photosynthetic pigments, sugar concentration, photosynthetic rate, and water use efficiency. The effects of hormone were less evident in the absence of salt. However, under this condition the application of clotrimazole affected the values of parameters studied, indicating the importance of brassinosteroid synthesis for the normal development of the plant.     

The effects of brassinosteroids on photosynthetic parameters in leaves of two field-grown maize inbred lines and their F1 hybrid

The effect of foliar spray with 10⁻¹² M aqueous solutions of 24-epibrassinolide or a synthetic androstane analogue of castasterone on the activity of photosystem (PS) 1, the Hill reaction activity, the content of photosynthetic pigments and the specific leaf mass was examined for three different leaves developed after brassinosteroid (BR) treatment in two inbred lines of field-grown maize and their F1 hybrid. The brassinosteroids significantly affected neither the efficiency of photosynthetic electron transport, nor the content of chlorophylls or carotenoids.     

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.     

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     

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.     

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.     

Combined effect of 24-epibrassinolide and salicylic acid mitigates lead (Pb) toxicity by modulating various metabolites in <Emphasis Type="BoldItalic">Brassica juncea</Emphasis> L. seedlings

The present study demonstrated the combined effect of 24-epibrassinolide and salicylic acid against lead (Pb, 0.25, 0.50, and 0.75 mM) toxicity in Brassica juncea seedlings. Various parameters including water status, metal uptake, total water- and lipid-soluble antioxidants, metal chelator content (total thiols, protein-bound thiols, and non-protein-bound thiols), phenolic compounds (flavonoids, anthocyanins, and polyphenols), and organic acids were studied in 10-day-old seedlings. Dry matter content and the heavy metal tolerance index were reduced by 42.24 and 52.3%, respectively, in response to Pb treatment. Metal uptake, metal-chelating compounds, phenolic compounds, and organic acids were increased in Pb-treated seedlings as compared to control plants. The treatment of Pb-stressed seedlings with combination of EBL and SA resulted in enhancement of heavy metal tolerance index by 40.07%, water content by 1.84%, and relative water content by 23.45%. The total water- and lipid-soluble antioxidants were enhanced by 21.01 and 2.21%, respectively. In contrast, a significant decline in dry weight, metal uptake, thiol, and polyphenol contents was observed following the application of 24-epibrassinolide and salicylic acid. These observations indicate that Pb treatment has an adverse effect on B. juncea seedlings. However, co-application of 24-epibrassinolide and salicylic acid mitigates the negative effects of Pb, by lowering Pb metal uptake and enhancing the heavy metal tolerance index, water content, relative water content, antioxidative capacities, phenolic content, and organic acid levels.     

Physiological mechanisms of enhancing salt tolerance of oilseed rape plants with brassinosteroids

The ability of brassinosteroids, such as 24-epibrassinolide (EBL) to increase the resistance of oilseed rape plants (Brassica napus L.) to salt stress (175 mM NaCl) was investigated along with the possible mechanisms of their protective action. Seedlings were grown for three weeks on the Hoagland-Snyder medium under controlled conditions. The experimental plants were treated with either (1) 175 mM NaCl, or (2) 10^?10 M EBL, or (3) 175 mM NaCl plus 10^?10 M EBL by adding the corresponding components to the growth medium. The exposure was 7 and 14 days. As compared to the control, salinization inhibited plant height by 33–35%, reduced leaf area by 2.0–2.5 times, reduced 2.5- and 2-fold plant fresh and dry weight, respectively, reduced water content of plant tissues by 26–31% and, twofold, the content of chlorophylls a and b. Plants responded to NaCl by developing oxidative stress conditions, lowering the osmotic potential of the cell contents down to ?2 MPa, accumulating proline (by 43–52 times) and low-molecular-weight phenolics (by 1.9–2.7 times). Oilseed rape plants were shown to respond to salinization with an increase of endogenous content of steroid hormones: 24-epibrassinosteroids (24-epibrassinolide and 24-epicastasterone), 24S-methyl-brassinosteroids (brassinolide and castasterone), and 28-homobrassinosteroids (28-homobrassinolide and 28-homocastasterone); such evidence indirectly confirms the involvement of brassinosteroids in the development of salt tolerance. Adding EBL to the nutrient medium under optimal growth conditions did not significantly affect the indices under study. Under salt stress, EBL showed a pronounced protective effect: stem growth was fully restored, plant assimilation area increased by as much as 67–76% as compared to the control index, fresh and dry weight largely recovered (up to 85–92% of the control values), and the inhibitory effect of NaCl on photosynthetic pigments was diminished. Exogenous EBL impeded the development of NaCl-dependent lipid peroxidation and increased the osmotic potential of the leaf cell contents. The protective effect of EBL under salt stress was probably associated with EBL antioxidant effect, rather than the hormone-induced accumulation of proline and of low-molecula-weight phenolics, as well as with the ability to regulate water status by maintaining intracellular ion homeostasis.     

Preparation and synthetic application of partially protected brassinosteroids

Preparation of partially protected brassinosteroids is achieved through the reaction of the source material (24-epicastasterone and 24-epibrassinolide) with diol-specific reagents (2,2-dimethoxypropane and methylboronic acid). The obtained products were shown to be useful synthetic intermediates for further preparation of minor representatives of this class of natural phytohormones (such as 3,24-diepicastasterone and 3-dehydro-24-epibrassinolide).     

Sensitized bean first internode bioassay for auxins and brassinosteroids

The bean first internode bioassay ofMeudt andBennett (Physiol. Plant.44: 422–428, 1978) is based on the curvature of bean internode sections after unilateral application of test material near to their base. The test was also used for estimation of biological activity of brassinosteroids which enhance the effect of auxin. Sensitivity of the assay to both IAA and 24-epibrassinolide was increased 1000 and 100 times, respectively, when internode sections were incubated in morphologically inversed position. Under these conditions test material was applied to the young auxin-sensitive morphologically apical internode tissues and auxin was basipetally translocated to the zone of curvature. Using this bioassay as little as 10 fmol of IAA and 1 pmol of 24-epibrassinolide can be estimated.     

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     

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.     

Effect of brassinosteroids on salinity stress induced inhibition of seed germination and seedling growth of rice (Oryza sativa L.)

The effect of 24-epibrassinolide and 28-homobrassinolide on the inhibitionof germination and seedling growth of rice (Oryza sativa) induced bysalinity stress was studied. Brassinosteroids were found to reverse theinhibitory effect on germination and seedling growth. The activation ofseedling growth by brassinosteroids under salinity stress was associatedwith enhanced levels of nucleic acids and soluble proteins.     

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.     

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.     

The role of reactive oxygen species and calcium ions in the implementation of the stress-protective effect of brassinosteroids on plant cells

The effect of the brassinosteroids (BS) 24-epibrassinolide and 24-epicastasterone on the thermoresistance of wheat coleoptiles (Triticum aestivum L.) and their generation of the superoxide anion radical and antioxidant enzymes activity were investigated. The treatment of coleoptiles with 10 nM solutions of BS caused a transient increase in O ₂ ^⊙? generation and a subsequent increase in the activity of superoxide dismutase and catalase and an improvement in heat resistance. Pretreatment of coleoptiles with the NADPH oxidase inhibitor imidazole leveled the increase in production of the superoxide anion radical and prevented an increase in the activity of antioxidant enzymes and the development of cell thermostability. The investigated effects of BS were also depressed by the pretreatment of coleoptile segments with extracellular calcium chelator EGTA and inhibitor of ADP-ribosyl cyclase nicotinamide. A conclusion was made about the participation of calcium ions and reactive oxygen species generated by the action of NADPH oxidase in the implementation of the stress-protective effect of the BS in the cells of wheat coleoptiles.     

镧浸种对盐胁迫下小麦幼苗生长及其生理特征的影响

通过水培方式研究了0、25、50和100 mg/L硝酸镧浸种对盐胁迫条件下小麦品种临抗11和临优2069根系及幼苗生长的影响.结果表明:(1)与对照组相比,盐胁迫处理小麦幼苗植株矮,根系短,叶片叶绿素含量、根系活性吸收面积以及SOD和CAT活性明显降低,叶片MDA与Pro含量水平显著上升;在钠离子浓度相同的情况下,Na2CO3对小麦生长的影响大于NaCl.(2)适当浓度硝酸镧浸种处理增加了盐胁迫下小麦幼苗的株高、总根长、根系活性吸收面积及SOD和CAT活性,且各指标在盐胁迫下增加幅度高于正常水分处理.(3)2个小麦品种对镧处理的敏感程度存在差异,不同小麦品种及不同盐胁迫下最适的镧浸种浓度不同.研究发现,适当浓度镧浸种能有效缓解盐胁迫对小麦幼苗的伤害,具有显著促进小麦根系生长、培育壮苗的作用.