methampicillin and brassinolide

24-epibrassinolide overcame the inhibitory effect of brassinazole on the barley growth and the content of brassinosteroids. The present work demonstrates the occurrence of mainly castasterone, brassinolide and cathasterone and lower amounts of 24-epibrassinolide, 24-epicastasterone, 28-homobrassinolide, typhasterol, 6-deoxocastasterone and 6-deoxotyphasterol in 14-day-old de-etiolated barley (Hordeum vulgare L. cv. Golden Promise). We also investigated the endogenous level of brassinosteroids (BRs) in barley seedlings treated with 24-epibrassinolide (EBL) and/or brassinazole (Brz). To our knowledge, this is the first report related to the occurrence of BRs and application of EBL and Brz in terms of the endogenous content of BRs in barley. Brz as a specific inhibitor of BR biosynthetic reactions decreased the level of BRs in the leaves. Application of EBL showed a weak promotive effect on the BR content in Brz-treated seedlings. Brz also inhibited growth of the seedlings; however, addition of EBL overcame the inhibition. The EBL applied alone at 0.01-1 µM increased the BR level in the leaves but at 10 µM lowered the BR content. In opposition to leaves, the Brz in the concentration range from 0.1 to 1 µM did not significantly affect the content of BRs in the roots. However, application of 10 µM Brz caused BRs to decrease, but treatment of EBL concentrations overcame the inhibitory effect of Brz.

Topics: Brassinosteroids; Hordeum; Plant Leaves; Plant Roots; Steroids, Heterocyclic; Triazoles

Exogenous 24-epibrassinolide (BL) and brassinazole (BRZ) have regulatory roles in G-fiber cell wall development and secondary xylem cell wall carbohydrate biosynthesis during tension wood formation in hybrid poplar. Brassinosteroids (BRs) play important roles in regulating gravitropism and vasculature development. Here, we report the effect of brassinosteroids on negative gravitropism and G-fiber cell wall development of the stem in woody angiosperms. We applied exogenous 24-epibrassinolide (BL) or its biosynthesis inhibitor brassinazole (BRZ) to slanted hybrid poplar trees (Populus deltoids × Populus nigra) and measured the morphology of gravitropic stems, anatomy and chemistry of secondary cell wall. We furthermore analyzed the expression levels of auxin transport and cellulose biosynthetic genes after 24-epibrassinolide (BL) or brassinazole (BRZ) application. The BL-treated seedlings showed no negative gravitropism bending, whereas application of BRZ dramatically enhanced negative gravitropic bending. BL treatment stimulated secondary xylem fiber elongation and G-fiber formation on the upper side of stems but delayed G-fiber maturation. BRZ inhibited xylem fiber elongation but induced the production of more mature G-fibers on the upper side of stems. Wood chemistry analyses and immunolocalization demonstrated that BL and BRZ treatments increased the cellulose content and modified the deposition of cell wall carbohydrates including arabinose, galactose and rhamnose in the secondary xylem. The expression of cellulose biosynthetic genes, especially those related to cellulose microfibril deposition (PtFLA12 and PtCOBL4) was significantly upregulated in BL- and BRZ-treated TW stems compared with control stems. The significant differences of G-fibers development and negative gravitropism bending between 24-epibrassinolide (BL) and brassinazole (BRZ) application suggest that brassinosteroids are important for secondary xylem development during tension wood formation. Our findings provide potential insights into the mechanism by which BRs regulate G-fiber cell wall development to accomplish negative gravitropism in TW formation.

Topics: Brassinosteroids; Cellulose; Fluorescent Antibody Technique; Gravitropism; Populus; Seedlings; Steroids, Heterocyclic; Triazoles; Wood

Topics: Arabidopsis; Arabidopsis Proteins; Biomimetic Materials; Brassinosteroids; Cytochrome P-450 Enzyme System; DNA-Binding Proteins; Ecdysterone; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Glycosyltransferases; Hypocotyl; Nuclear Proteins; Oryza; Phosphorylation; Plant Growth Regulators; Real-Time Polymerase Chain Reaction; Seeds; Signal Transduction; Steroid Hydroxylases; Steroids, Heterocyclic; Triazoles

Brassinosteroids (BRs), plant steroid hormones, play essential roles in modulating cell elongation, vascular differentiation, senescence, and stress responses. However, the mechanisms by which BRs regulate plant mitochondria and resistance to abiotic stress remain largely unclear. Mitochondrial alternative oxidase (AOX) is involved in the plant response to a variety of environmental stresses. In this report, the role of AOX in BR-induced tolerance against cold, polyethylene glycol (PEG), and high-light stresses was investigated. Exogenous applied brassinolide (BL, the most active BR) induced, while brassinazole (BRZ, a BR biosynthesis inhibitor) reduced alternative respiration and AOX1 expression in Nicotiana benthamiana. Chemical scavenging of H2O2 and virus-induced gene silencing (VIGS) of NbRBOHB compromised the BR-induced alternative respiratory pathway, and this result was further confirmed by NbAOX1 promoter analysis. Furthermore, inhibition of AOX activity by chemical treatment or a VIGS-based approach decreased plant resistance to environmental stresses and compromised BR-induced stress tolerance. Taken together, our results indicate that BR-induced AOX capability might contribute to the avoidance of superfluous reactive oxygen species accumulation and the protection of photosystems under stress conditions in N. benthamiana.

Topics: Brassinosteroids; Cold Temperature; Light; Mitochondrial Proteins; Nicotiana; Oxidoreductases; Plant Growth Regulators; Plant Proteins; Polyethylene Glycols; Reactive Oxygen Species; Signal Transduction; Steroids, Heterocyclic; Stress, Physiological; Triazoles

Brassinosteroid (BR) signaling is essential for plant growth and development. In Arabidopsis (Arabidopsis thaliana), BRs are perceived by the BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor. Root growth and hypocotyl elongation are convenient downstream physiological outputs of BR signaling. A computational approach was employed to predict root growth solely on the basis of BRI1 receptor activity. The developed mathematical model predicts that during normal root growth, few receptors are occupied with ligand. The model faithfully predicts root growth, as observed in bri1 loss-of-function mutants. For roots, it incorporates one stimulatory and two inhibitory modules, while for hypocotyls, a single inhibitory module is sufficient. Root growth as observed when BRI1 is overexpressed can only be predicted assuming that a decrease occurred in the BRI1 half-maximum response values. Root growth appears highly sensitive to variation in BR concentration and much less to reduction in BRI1 receptor level, suggesting that regulation occurs primarily by ligand availability and biochemical activity.

Topics: Arabidopsis; Arabidopsis Proteins; Brassinosteroids; Computational Biology; Culture Media; Green Fluorescent Proteins; Hypocotyl; Ligands; Models, Theoretical; Plant Roots; Protein Kinases; Receptors, Cell Surface; Signal Transduction; Steroids, Heterocyclic; Triazoles

Brassinosteroids (BRs) can induce plant tolerance to a variety of abiotic stresses by triggering the generation of H(2) O(2) as a signalling molecule in cucumber leaves. Whether nitric oxide (NO) also plays a signalling role and, if so, what is the relationship between NO and H(2) O(2) in BR-induced stress tolerance are unknown. Involvement of NO and H(2) O(2) in BR-induced tolerance was examined. NO accumulation and defence related gene transcripts were monitored by confocal laser-scanning microscopy and qRT-PCR, respectively. NO content was elevated after treatment with 24-epibrassinolide (EBR) and reduced with the inhibition of BR biosynthesis. EBR-induced NO production was blocked by pre-treatment with inhibitor of NADPH oxidase and a reactive oxygen species scavenger. On the other hand, EBR-induced H(2) O(2) generation was not sensitive to NO scavenger or inhibitor of NO production. Scavenging or inhibition of NO production inhibited EBR-induced tolerance to photo-oxidative and cold stress and partly blocked EBR-induced expression and activities of several antioxidant enzymes. Pre-treatment of the exogenous NO precursor, on the other hand, led to both increased stress tolerance and increased expression of antioxidant enzymes. These results strongly suggest that NO plays an important role in H(2) O(2) -dependent induction of plant stress tolerance by BR.

Topics: Adaptation, Physiological; Brassinosteroids; Cucumis sativus; Gene Expression Regulation, Plant; Glutathione Reductase; Hydrogen Peroxide; Microscopy, Confocal; Nitric Oxide; Plant Growth Regulators; Plant Leaves; Plant Proteins; Seedlings; Signal Transduction; Steroids, Heterocyclic; Triazoles

Jasmonate (JA) regulates plant development, mediates defense responses, and induces anthocyanin biosynthesis as well. Previously, we isolated the psc1 mutant that partially suppressed coi1 insensitivity to JA, and found that brassinosteroid (BR) was involved in JA signaling and negatively regulated JA inhibition of root growth in Arabidopsis. In this study it was shown that JA-induced anthocyanin accumulation was reduced in BR mutants or in wild type treated with brassinazole, an inhibitor of BR biosynthesis, whereas it was induced by an application of exogenous BR. It was also shown that the 'late' anthocyanin biosynthesis genes including DFR, LDOX, and UF3GT, were induced slightly by JA in the BR mutants relative to wild type. Furthermore, the expression level of JA-induced Myb/bHLH transcription factors such as PAP1, PAP2, and GL3, which are components of the WD-repeat/Myb/bHLH transcriptional complexes that mediate the 'late' anthocyanin biosynthesis genes, was lower in the BR mutants than that in wild type. These results suggested that BR affects JA-induced anthocyanin accumulation by regulating the 'late' anthocyanin biosynthesis genes and this regulation might be mediated by the WD-repeat/Myb/bHLH transcriptional complexes.

Topics: Anthocyanins; Arabidopsis; Arabidopsis Proteins; Basic Helix-Loop-Helix Transcription Factors; Brassinosteroids; Cyclopentanes; Cytochrome P-450 Enzyme System; Gene Expression Regulation, Plant; Genes, Plant; Mutation; Oxylipins; Pancreatitis-Associated Proteins; Reverse Transcriptase Polymerase Chain Reaction; Seedlings; Signal Transduction; Steroids, Heterocyclic; Triazoles

The phytohormone brassinosteroid (BR) is crucial for plant growth and development. Although genetic and molecular approaches have improved understanding of the cellular BR signaling pathway, we still do not have sufficient knowledge about the function of BR. Therefore, proteomic analysis was used to elucidate BR signaling and gene expression in the nuclei of suspended Arabidopsis cells treated with brassinolide, a bioactive BR, or brassinazole, a BR biosynthesis inhibitor. Interestingly, chromatin remodeling-related proteins, the abundance of which was altered in response to cellular BR levels, were identified. This suggested that BR-induced gene expression is regulated not only by transcription factors directly binding to cis-elements, but also by chromatin remodeling in response to BR signaling. In this addendum, we summarize the functions of our identified nuclear proteins in chromatin remodeling and discuss the need for chromatin remodeling regulated by BR signal transduction for expression of BR-induced genes.

Topics: Arabidopsis; Brassinosteroids; Cell Nucleus; Chromatin Assembly and Disassembly; Gene Expression Regulation, Plant; Plant Growth Regulators; Signal Transduction; Steroids, Heterocyclic; Triazoles

Exogenous applications of brassinolide (BL) increased the number and quality of microspore-derived embryos (MDEs) whereas treatments with brassinazole (BrZ), a BL biosynthetic inhibitor, had the opposite effect. At the optimal concentration (4x10(-6) M) BrZ decreased both embryo yield and conversion to less than half the value of control embryos. Metabolic studies revealed that BL levels had profound effects on glutathione and ascorbate metabolism by altering the amounts of their reduced forms (ASC and GSH) and oxidized forms [dehydroascorbate (DHA), ascorbate free radicals (AFRs), and GSSG]. Applications of BL switched the glutathione and ascorbate pools towards the oxidized forms, thereby lowering the ASC/ASC+DHA+AFR and GSH/GSH+GSSG ratios. These changes were ascribed to the ability of BL to increase the activity of ascorbate peroxidase (APX) and decrease that of glutathione reductase (GR). This trend was reversed in a BL-depleted environment, effected by BrZ applications. These metabolic alterations were associated with changes in embryo structure and performance. BL-treated MDEs developed zygotic-like shoot apical meristems (SAMs) whereas embryos treated with BrZ developed abnormal meristems. In the presence of BrZ, embryos either lacked a visible SAM, or formed SAMs in which the meristematic cells showed signs of differentiation, such as vacuolation and storage product accumulation. These abnormalities were accompanied by the lack or misexpression of three meristem marker genes isolated from Brassica napus (denoted as BnSTM, BnCLV1, and BnZLL-1) homologous to the Arabidopsis SHOOTMERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL). The expression of BnSTM and BnCLV1 increased after a few days in cultures in embryos treated with BL whereas an opposite tendency was observed with applications of BrZ. Compared with control embryos where these two genes exhibited abnormal localization patterns, BnSTM and BnCLV1 always localized throughout the subapical domains of BL-treated embryos in a zygotic-like fashion. Expression of both genes was often lost in the SAM of BrZ-treated embryos. The results suggest that maintenance of cellular BL levels is required to modulate the ascorbate and glutathione redox status during embryogenesis to ensure proper development of the embryos and formation of functional apical meristems.

Topics: Ascorbic Acid; Biomarkers; Brassica napus; Brassinosteroids; Cholestanols; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Genes, Plant; Glutathione; In Situ Hybridization; Meristem; Pollen; Reverse Transcriptase Polymerase Chain Reaction; Seeds; Steroids, Heterocyclic; Triazoles

Brassinosteroids (BRs) are a new group of plant growth substances that promote plant growth and productivity. We showed in this study that improved growth of cucumber (Cucumis sativus) plants after treatment with 24-epibrassinolide (EBR), an active BR, was associated with increased CO(2) assimilation and quantum yield of PSII (Phi(PSII)). Treatment of brassinazole (Brz), a specific inhibitor for BR biosynthesis, reduced plant growth and at the same time decreased CO(2) assimilation and Phi(PSII). Thus, the growth-promoting activity of BRs can be, at least partly, attributed to enhanced plant photosynthesis. To understand how BRs enhance photosynthesis, we have analyzed the effects of EBR and Brz on a number of photosynthetic parameters and their affecting factors, including the contents and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Northern and Western blotting demonstrated that EBR upregulated, while Brz downregulated, the expressions of rbcL, rbcS and other photosynthetic genes. In addition, EBR had a positive effect on the activation of Rubisco based on increased maximum Rubisco carboxylation rates (V (c,max)), total Rubisco activity and, to a greater extent, initial Rubisco activity. The accumulation patterns of Rubisco activase (RCA) based on immunogold-labeling experiments suggested a role of RCA in BR-regulated activation state of Rubisco. Enhanced expression of genes encoding other Calvin cycle genes after EBR treatment may also play a positive role in RuBP regeneration (J (max)), thereby increasing maximum carboxylation rate of Rubisco (V (c,max)). Thus, BRs promote photosynthesis and growth by positively regulating synthesis and activation of a variety of photosynthetic enzymes including Rubisco in cucumber.

Topics: Biomass; Blotting, Western; Brassinosteroids; Carbon Dioxide; Chlorophyll; Cholestanols; Cucumis sativus; Gene Expression Regulation, Plant; Hexoses; Kinetics; Photosynthesis; Plant Growth Regulators; Plant Leaves; Plant Proteins; Plant Roots; Reverse Transcriptase Polymerase Chain Reaction; Ribulose-Bisphosphate Carboxylase; Starch; Steroids, Heterocyclic; Sucrose; Triazoles

Homeostasis of brassinosteroids (BRs) is essential for normal growth and development in higher plants. We examined responsiveness of 11 BR metabolic gene expressions to the decrease or increase of endogenous BR contents in Arabidopsis (Arabidopsis thaliana) to expand our knowledge of molecular mechanisms underlying BR homeostasis. Five BR-specific biosynthesis genes (DET2, DWF4, CPD, BR6ox1, and ROT3) and two sterol biosynthesis genes (FK and DWF5) were up-regulated in BR-depleted wild-type plants grown under brassinazole, a BR biosynthesis inhibitor. On the other hand, in BR-excessive wild-type plants that were fed with brassinolide, four BR-specific synthesis genes (DWF4, CPD, BR6ox1, and ROT3) and a sterol synthesis gene (DWF7) were down-regulated and a BR inactivation gene (BAS1) was up-regulated. However, their response to fluctuation of BR levels was highly reduced (DWF4) or nullified (the other eight genes) in a bri1 mutant. Taken together, our results imply that BR homeostasis is maintained through feedback expressions of multiple genes, each of which is involved not only in BR-specific biosynthesis and inactivation, but also in sterol biosynthesis. Our results also indicate that their feedback expressions are under the control of a BRI1-mediated signaling pathway. Moreover, a weak response in the mutant suggests that DWF4 alone is likely to be regulated in other way(s) in addition to BRI1 mediation.

Topics: Arabidopsis; Arabidopsis Proteins; Brassinosteroids; Cholestanols; Down-Regulation; Feedback, Physiological; Gene Expression Regulation, Plant; Homeostasis; Plant Growth Regulators; Signal Transduction; Steroids; Steroids, Heterocyclic; Triazoles; Up-Regulation

Brassinosteroids (BR) promote the elongation of cotton fibers and may be a factor in determining their final length. To begin to understand the role of BR-mediated responses in the development of cotton fibers we have characterized the BIN 2 genes of cotton. BIN 2 is a member of the shaggy-like protein kinase family that has been identified as a negative regulator of BR signaling in Arabidopsis. Sequence analyses indicate that the tetraploid cotton genome includes four genes with strong sequence similarity to BIN 2. These genes fall into two distinct subclasses based on sequence and expression patterns. Sequence comparisons with corresponding genes from cotton species that have the diploid A and D genomes, respectively, shows that each pair of genes comprises homeologs derived from the A and D sub-genomes. Transgenic Arabidopsis plants that express these cotton BIN 2 cDNAs show reduced growth and similar phenotypes to the semi-dominant bin 2 mutant plants. These results indicate that the cotton BIN 2 genes encode functional BIN 2 isoforms that can inhibit BR signaling. Further analyses of the function of BIN 2 genes and their possible roles in determining fiber yield and quality are underway.

Topics: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Brassinosteroids; Cholestanols; Cloning, Molecular; DNA, Complementary; Gene Expression Regulation, Plant; Genome, Plant; Gossypium; Molecular Sequence Data; Mutation; Plant Proteins; Plants, Genetically Modified; Polyploidy; Protein Kinases; Protein Serine-Threonine Kinases; RNA, Plant; Sequence Homology, Amino Acid; Steroids, Heterocyclic; Triazoles

Treatment of cultured Chlorella vulgaris Beijerinck cells with 0.1-10 microM brassinazole (Brz2001), an inhibitor of brassinosteroid (BR) biosynthesis, inhibits their growth during the first 48 h of cultivation in the light. This inhibition is prevented by the co-application of BR. This result suggests that the presence of endogenous BRs during the initial steps of the C. vulgaris cell cycle is indispensable for their normal growth in the light. In darkness, a treatment with 10 nM brassinolide (BL) promotes growth through the first 24 h of culture, but during the following 24 h the cells undergo complete stagnation. Treatment of dark-grown cells with either Brz2001 alone, or a mixture of 10 nM BL and 0.1/10 microM Brz2001, also stimulates their growth. The effects of treatment with 10 nM BL mixed with 0.1-10 microM of a mevalonate-pathway inhibitor (mevinolin), or a non-mevalonate-pathway inhibitor (clomazone), were also investigated. Mevinolin at these concentrations did not inhibit growth of C. vulgaris; however, clomazone did. Addition of BL overcame the inhibition. These results suggest that the mevalonate pathway does not function in C. vulgaris, and that the non-mevalonate pathway for isopentenyl diphosphate biosynthesis is responsible for the synthesis of one of the primary precursors in BR biosynthesis.

Topics: Brassinosteroids; Chlorella; Cholestanols; Darkness; Hemiterpenes; Isoxazoles; Light; Lovastatin; Organophosphorus Compounds; Oxazolidinones; Steroids, Heterocyclic; Triazoles

Suppression of brassinosteroid (BR) biosynthesis in cotton ovules by treatment with brassinazole inhibits fiber formation, indicating that BR plays an important role in cotton fiber development. Plant responses to brassinosteroids (BR) are mediated through a plasma membrane-bound leucine-rich repeat (LRR) receptor-like protein kinase known as BRI1. Mutations in the BRI1 genes of several species result in dwarfed plants with reduced sensitivity to BR. A single expressed sequence tag (EST) from cotton with strong sequence similarity to Arabidopsis BRI1 ( AtBRI1 ) was identified in a search of publicly available databases. With this EST as a starting point, full-length cDNAs and genomic coding sequences from upland cotton ( Gossypium hirsutum ) BRI1 ( GhBRI1 ) were obtained and characterized. Ectopic expression of this coding sequence in BR-insensitive Arabidopsis plants resulted in recovery of normal growth indicating that GhBRI1 is a functional homologue of AtBRI1. G. hirsutum is an allotetraploid (AADD) derived from diploid ancestors. Analysis of several GhBRI1 cDNAs showed two distinct sequences indicating the presence of two GhBRI1 genes, denoted GhBRI1-1 and GhBRI1-2. Sequence comparisons between these GhBRI1 coding sequences and those from related A and D genome diploid Gossypium species ( G. arboreum and G. thurberi ) indicated that GhBRI1-1 is likely to the A sub-genome orthologue while GhBRI1-2 is from the D sub-genome.

Topics: Amino Acid Sequence; Arabidopsis; Brassinosteroids; Cholestanols; Cloning, Molecular; Cotton Fiber; DNA, Complementary; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Genetic Complementation Test; Genome, Plant; Gossypium; Molecular Sequence Data; Mutation; Phylogeny; Plant Proteins; Polyploidy; Protein Kinases; Protein Serine-Threonine Kinases; Seeds; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Steroids, Heterocyclic; Triazoles

Brassinolide (BL), considered to be the most important brassinosteroid (BR) and playing pivotal roles in the hormonal regulation of plant growth and development, was found to induce disease resistance in plants. To study the potentialities of BL activity on stress responding systems, we analyzed its ability to induce disease resistance in tobacco and rice plants. Wild-type tobacco treated with BL exhibited enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The measurement of salicylic acid (SA) in wild-type plants treated with BL and the pathogen infection assays using NahG transgenic plants indicate that BL-induced resistance does not require SA biosynthesis. BL treatment did not induce either acidic or basic pathogenesis-related (PR) gene expression, suggesting that BL-induced resistance is distinct from systemic acquired resistance (SAR) and wound-inducible disease resistance. Analysis using brassinazole 2001, a specific inhibitor for BR biosynthesis, and the measurement of BRs in TMV-infected tobacco leaves indicate that steroid hormone-mediated disease resistance (BDR) plays part in defense response in tobacco. Simultaneous activation of SAR and BDR by SAR inducers and BL, respectively, exhibited additive protective effects against TMV and Pst, indicating that there is no cross-talk between SAR- and BDR-signaling pathway downstream of BL. In addition to the enhanced resistance to a broad range of diseases in tobacco, BL induced resistance in rice to rice blast and bacterial blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, respectively. Our data suggest that BDR functions in the innate immunity system of higher plants including dicotyledonous and monocotyledonous species.

Topics: Brassinosteroids; Cholestanols; Fungi; Immunity, Innate; Nicotiana; Oryza; Plant Diseases; Pseudomonas; Salicylic Acid; Steroids, Heterocyclic; Time Factors; Tobacco Mosaic Virus; Triazoles

Vascular cell axialization refers to the uniform alignment of vascular strands. In the Arabidopsis cotyledon vascular pattern1 (cvp1) mutant, vascular cells are not arranged in parallel files and are misshapen, suggesting that CVP1 has a role in promoting vascular cell polarity and alignment. Characterization of an allelic series of cvp1 mutations revealed additional functions of CVP1 in organ expansion and elongation. We identified CVP1 and found that it encodes STEROL METHYLTRANSFERASE2 (SMT2), an enzyme in the sterol biosynthetic pathway. SMT2 and the functionally redundant SMT3 act at a branch point in the pathway that mediates sterol and brassinosteroid levels. The SMT2 gene is expressed in a number of developing organs and is regulated by various hormones. As predicted from SMT2 enzymatic activity, the precursors to brassinosteroid are increased at the expense of sterols in cvp1 mutants, identifying a role for sterols in vascular cell polarization and axialization.

Topics: Alleles; Arabidopsis; Arabidopsis Proteins; Biological Transport; Brassinosteroids; Cholestanols; Cloning, Molecular; Cotyledon; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; In Situ Hybridization; Methyltransferases; Mutation; Phytosterols; Plants, Genetically Modified; RNA, Messenger; Signal Transduction; Steroids, Heterocyclic; Triazoles

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. Although insights into the functions of BRs have been provided by recent studies of biosynthesis and sensitivity mutants, the mode of action of BRs is poorly understood. With the use of DNA microarray analysis, we identified BR-regulated genes in the wild type (WT; Columbia) of Arabidopsis and in the BR-deficient mutant, det2. BR-regulated genes generally responded more potently in the det2 mutant than in the WT, and they showed only limited response in a BR-insensitive mutant, bri1. A small group of genes showed stronger responses in the WT than in the det2. Exposure of plants to brassinolide and brassinazole, which is a specific inhibitor of BR biosynthesis, elicited opposite effects on gene expression of the identified genes. The list of BR-regulated genes is constituted of transcription factor genes including the phytochrome-interacting factor 3, auxin-related genes, P450 genes, and genes implicated in cell elongation and cell wall organization. The results presented here provide comprehensive view of the physiological functions of BRs using BR-regulated genes as molecular markers. The list of BR-regulated genes will be useful in the characterization of new mutants and new growth-regulating compounds that are associated with BR function.

Topics: Arabidopsis; Arabidopsis Proteins; Basic Helix-Loop-Helix Transcription Factors; Brassinosteroids; Cell Division; Cell Wall; Chlorophyll; Cholestanols; Cytochrome P-450 Enzyme System; Down-Regulation; Gene Expression Regulation, Plant; Indoleacetic Acids; Mutation; Oligonucleotide Array Sequence Analysis; Phytochrome; Plant Growth Regulators; Protein Kinases; Steroids, Heterocyclic; Triazoles

Screening for brassinosteroid biosynthesis inhibitors was performed to find azole derivatives that induced dwarfism, to resemble brassinosteroid-deficient mutants in Arabidopsis, and which could be rescued by brassinosteroid. Through this screening experiment, propiconazole fungicide was selected as a likely inhibitor of brassinosteroid biosynthesis and, thus, propiconazole derivatives with optimized activity and selectivity were synthesized. The biological activity of these compounds was evaluated by examining cress stem elongation. Among the compounds tested, 2RS,4RS-1-[2-(4-trifluoromethylphenyl)-4-n-propyl-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazole (12) showed the most potent capability to retard cress stem elongation in the light. The compound-induced hypocotyl dwarfism was restored by the coapplication of 10 nM brassinolide but not by 1 microM gibberellin. These results suggest that 12 should affect brassinosteroid biosynthesis. The potency and specificity of 12 were greater than those of brassinazole, a previously reported brassinosteroid biosynthesis inhibitor.

Topics: Brassinosteroids; Cholestanols; Cytochrome P-450 Enzyme Inhibitors; Dioxoles; Gibberellins; Steroids, Heterocyclic; Triazoles

Brassinazole (Brz) is a specific brassinosteroid biosynthesis inhibitor. Cress plants (Lepidium sativum) grown in medium containing Brz exhibited a slight predominance of phloem differentiation at the expense of xylem differentiation and remarkable inhibition of the development of secondary xylem. This result indicates that brassinosteroids function in xylem development in vivo.

Topics: Arabidopsis; Biological Transport; Brassicaceae; Brassinosteroids; Cell Differentiation; Cell Division; Cholestanols; Phytosterols; Plant Growth Regulators; Plant Stems; Steroids, Heterocyclic; Triazoles

Brassinazole is the only known specific brassinosteroid (BR)-biosynthesis inhibitor, and it has been shown to be useful for elucidating the function of BRs. In the course of a structure-activity relationship study of brassinazole, we found a more specific BR-biosynthesis inhibitor, Brz2001. This new inhibitor induced similar morphological changes to those seen in brassinazole-treated plants, including Arabidopsis thaliana (L.) Heynh., Nicotiana tabacum L., and Lepidium sativum L. These changes included dwarfism with altered leaf morphology, including downward curling and dark-green color, and the changes were reversed by brassinolide. Although the structure of Brz2001 is similar to that of uniconazole, a gibberellin-biosynthesis inhibitor, Brz2001-treated plants showed almost no recovery with the addition of gibberellic acid (GA3). Comparison of the responses of both brassinazole- and Brz2001-treated cress to brassinolide and GA3 suggested that Brz2001 is a more specific BR-biosynthesis inhibitor than brassinazole. Unlike the results just described, Brz2001-treated rice did not show any morphological changes. This suggests that the roles of BRs in rice may be different from those in the dicotyledonous plants examined in this study. Brz2001 can be used to clarify the function of BRs in dicots as a complement to BR-deficient mutants, and to elucidate the different roles of BRs in monocots and dicots.

Topics: Arabidopsis; Brassinosteroids; Cholestanols; Cotyledon; Gibberellins; Hypocotyl; Nicotiana; Oryza; Phytosterols; Plant Development; Plant Growth Regulators; Plants; Steroids, Heterocyclic; Structure-Activity Relationship; Triazoles