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Characterisation of BRH1, a brassinosteroid-responsive RING-H2 gene from Arabidopsis thaliana.

Although many important aspects of plant development are controlled by brassinosteroids (BRs), the early molecular events of their hormonal action are largely unknown. Using a differential-display RT-PCR screen designed to detect early response transcripts, those regulated by BR treatment in the absence of de novo protein synthesis, we identified an Arabidopsis thaliana (L.) Heynh. gene (designated BRH1) that encodes a novel RING finger protein. As deduced from a complete cDNA clone, the 170-amino-acid sequence of BRH1 forms an N-terminal hydrophobic domain and a C-terminal RING-H2 signature. In wild-type Arabidopsis, the level of the BRH1 transcript was rapidly down-regulated by brassinolide, but this effect was abolished in a BR-insensitive mutant deficient in the BRI1 receptor. BRH1 mRNA abundance was not influenced by other phytohormones, but the pathogen elicitor chitin induced a rapid and transient accumulation of the transcript. Antisense expression of BRH1 resulted in transgenic Arabidopsis plants with thicker inflorescence stems and altered leaf morphology, whereas in sense overexpression lines no phenotypic effect could be observed. Considering the potential of the RING proteins to participate in regulatory protein complexes, BR-dependent expression of BRH1 may suggest its involvement in later hormonal effects.

Amino Acid Sequence↗

Effects of brassinazole, an inhibitor of brassinosteroid biosynthesis, on light- and dark-grown Chlorella vulgaris.

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.

Brassinosteroids↗

Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stresses.

In addition to an essential role in plant development, brassinosteroids (BRs) appear to have the ability to protect plants against various environmental stresses. However, studies confirming the ability of BRs to modulate plant responses to different environmental stresses are lacking. Earlier we had demonstrated that treatment with 24-epibrassinolide (EBR), a BR, increases the basic thermotolerance of Brassica napus and tomato seedlings [Plant Mol Biol 40:333-342, 1999]. Here we demonstrate that EBR treatment enhances seedling tolerance to drought and cold stresses in both Arabidopsis thaliana and B. napus, and helps to overcome a salt-stress-induced inhibition of seed germination. The ability of EBR to confer tolerance in plants to a variety of stresses was confirmed through analysis of expression of a subset of drought and cold stress marker genes. Transcriptional changes in these genes were more apparent in EBR-treated A. thaliana, in particular during earlier time points of stress. To see if BR is essential for the heat stress (HS) response, we made use of BR-deficient mutants. Both det2-1 and dwf4 mutants still expressed heat shock proteins (hsps) to high levels during HS, indicating that although BR augments thermotolerance in plants, it is not necessary for hsp expression during HS.

Arabidopsis↗

Brassinosteroids and gibberellins promote tobacco seed germination by distinct pathways.

Seed germination of Nicotiana tabacum L. cv. Havana 425 is determined by the balance of forces between the growth potential of the embryo and the mechanical restraint of the micropylar endosperm. In contrast to the gibberellin GA4, the brassinosteroid (BR) brassinolide (BL) did not release photodormancy of dark-imbibed photodormant seeds. Brassinolide promoted seedling elongation and germination of non-photodormant seeds, but did not appreciably affect the induction of class I beta-1,3-glucanase (betaGLU I) in the micropylar endosperm. Brassinolide, but not GA4, accelerated endosperm rupture of tobacco seeds imbibed in the light. Brassinolide and GA4 promoted endosperm rupture of dark-imbibed non-photodormant seeds, but only GA4 enhanced betaGLU I induction. Promotion of endosperm rupture by BL was dose-dependent and 0.01 microM BL was most effective. Brassinolide and GA4 promoted abscisic acid (ABA)-inhibited dark-germination of non-photodormant seeds, but only GA4 replaced light in inducing betaGLU I. These results indicate that BRs and GAs promote tobacco seed germination by distinct signal transduction pathways and distinct mechanisms. Gibberellins and light seem to act in a common pathway to release photodormancy, whereas BRs do not release photodormancy. Induction of betaGLU I in the micropylar endosperm and promotion of release of 'coat-enhanced' dormancy seem to be associated with the GA-dependent pathway, but not with BR signalling. It is proposed that BRs promote seed germination by directly enhancing the growth potential of the emerging embryo in a GA- and betaGLU I-independent manner.

Abscisic Acid↗

A specific brassinosteroid biosynthesis inhibitor, Brz2001: evaluation of its effects on Arabidopsis, cress, tobacco, and rice.

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.

Arabidopsis↗

Microarray analysis of brassinosteroids- and gibberellin-regulated gene expression in rice seedlings.

In order to understand the molecular mechanisms by which two types of phytohormones, brassinosteroids (BRs) and gibberellins (GAs), regulate plant growth and development, a cDNA microarray was made containing 4000 clones randomly selected from a rice cDNA library constructed from RNA isolated from seedlings treated with GA(3) and brassinolide (BL). The array was then probed to identify differences in expression using labelled cDNAs prepared from treated and control seedlings. Nine and 29 unique cDNA clones were up-regulated, while 32 and 42 unique cDNA clones were down-regulated by BL and GA(3), respectively. The predicted products of these BL- and GA-regulated genes fall into such functional categories as signal transduction, transcription, metabolism, cellular organization, and defense or anti-stress responses. Northern analysis of the selected BL- and GA(3)-regulated genes not only confirmed their BL or GA(3) dose dependent expression, but also revealed significant differences in the degree of expression in different organs and in response to other phytohormones. These results demonstrate that BR and GA influence growth and development by coordinately regulating the expression of specific groups of genes.

Brassinosteroids↗

Unique and overlapping expression patterns of Arabidopsis CYP85 genes involved in brassinosteroid C-6 oxidation.

Brassinosteroids (BRs) are steroid hormones that are essential for plant growth and development. To gain insight into potential sites of BR synthesis, we studied promoter activities of the two Arabidopsis BR C-6 oxidase genes (CYP85A1 and CYP85A2) in transgenic plants carrying promoter fusions with the GUS, GFP or LUC reporter genes. BR-dependent feedback regulation of the GUS reporter constructs indicated that their expression corresponded to those of the native genes. Both the CYP85A1 and CYP85A2 promoters showed maximum activity during the first week following germination, particularly in the vascular tissues. Compared to CYP85A2, CYP85A1 expression was weaker and confined to the early stages of seedling development. Stronger CYP85A2 promoter activity was evident in both juvenile and adult plants. Comparison of the 5'-UTR and TATA box sequences of CYP85A1 and CYP85A2 revealed high homology, indicating a relatively recent gene duplication. We also found that transgenic Arabidopsis plants harbouring the tomato DWARF promoter-GUS fusion had similarities in the expression pattern to the Arabidopsis genes suggesting common transcriptional regulation of CYP85 genes in the two species.

5' Flanking Region↗

Suppression of Wolffia arrhiza growth by brassinazole, an inhibitor of brassinosteroid biosynthesis and its restoration by endogenous 24-epibrassinolide.

The effect of the brassinosteroid (BR) 24-epibrassinolide (epiBL; 10(-13)-10(-6)M) on growth and levels of chlorophylls, carotenoids, sugars and protein in Wolffia arrhiza after 7 days of cultivation is reported. Application of epiBL to W. arrhiza cultures stimulates the growth and increases the content of photosynthetic pigments, sugar and protein. The greatest effect of epiBL is observed at a concentration of 10(-9)M. We tested the action of Brz2001, a specific BR biosynthesis inhibitor, in the range of 10(-6)-10(-4)M. Addition of Brz2001 to W. arrhiza cultures inhibits their growth after 7 days of cultivation. The inhibition of growth could be reversed by the addition of epiBL. Moreover, there was not complete recovery to the level of control, especially at 5 x 10(-5)-10(-4)M Brz2001. The effects of treatment with 10(-9)M epiBL mixed with a mevalonate pathway inhibitor (mevinolin), or a 2-methylerythritol 4-phosphate pathway inhibitor (clomazone), were also investigated. Mevinolin did not inhibit growth of W. arrhiza after 7 days of cultivation. However, clomazone did. Addition of epiBL overcame this inhibition. These results suggest that the mevalonate pathway may not function well in W. arrhiza and that biosynthesis of BRs through the non-mevalonate pathway in W. arrhiza could be possible.

Araceae↗

Synthesis of [26,27-2H6]brassinosteroids from 23,24-bisnorcholenic acid methyl ester.

A number of hexadeuterated brassinosteroids (BS) containing a hydroxy group at C-22 or a 22R,23R-diol function were prepared starting from 23,24-bisnorcholenic acid methyl ester for biosynthetic studies. Synthesis of the cyclic part was accomplished via the initial hydroboration-oxidation of Delta(5)-double bond. The key step in the synthesis of the side chain involved addition of (2S)-[3,4-(2)H(6)]2,3-dimethylbutylphenyl sulfone to the corresponding C-22 aldehydes.

Brassinosteroids↗

Brassinosteroid signal transduction--choices of signals and receptors.

Small signaling molecules that mediate cell-cell communication are essential for developmental regulation in multicellular organisms. Among them are the steroids and peptide hormones that regulate growth in both plants and animals. In plants, brassinosteroids (BRs) are perceived by the cell surface receptor kinase BRI1, which is distinct from the animal steroid receptors. Identification of components of the BR signaling pathway has revealed similarities to other animal and plant signal transduction pathways. Recent studies demonstrated that tomato BRI1 (tBRI1) perceives both BR and the peptide hormone systemin, raising new questions about the molecular mechanism and evolution of receptor-ligand specificity.

Brassinosteroids↗

Antisense inhibition of a BRI1 receptor reveals additional protein kinase signaling components downstream to the perception of brassinosteroids in rice.

Plants express a variety of proteins at the cell surface responsible for the transduction of regulatory information into the cell via receptors. In the present study, an attempt has been made to identify the components of the brassinosteroids (BRs) signaling transduction cascades in transgenic rice (Oryza sativa) expressing the antisense strand of OsBRI1 transcript. A 60 kDa protein, immunologically characterized as mitogen-activated protein kinase (MAPK), showed reduced phosphorylation activity in the membrane fractions of the OsBRI1 antisense rice over control, demonstrating the inhibition in the perception of BRs by the BRI1 receptor, when compared with the exogenously applied brassinolide. The phosphorylation activity of the 50 kDa Ca(2+)-dependent protein kinase was however increased in the cytosolic fractions of OsBRI1 antisense over control. The data obtained suggest that MAPK and Ca(2+)-dependent protein kinase in rice are discrete but parallel signaling cascades and might involve receptors other than BRI1 in response to BR stimulus.

Brassinosteroids↗

Plant hormones: brassinosteroids in the spotlight.

Recent studies on dwarf mutants of the model plant Arabidopsis thallana have provided convincing evidence that brassinosteroids-natural plant products similar to animal steroid hormones-are essential for normal plant growth and development.

Animals↗

Auxin and brassinosteroid differentially regulate the expression of three members of the 1-aminocyclopropane-1-carboxylate synthase gene family in mung bean (Vigna radiata L.).

Indole-3-acetic acid (IAA) markedly increased ethylene production by inducing the expression of three 1aminocyclopropane-1-carboxylate (ACC) synthase cDNAs (pVR-ACS1, pVR-ACS6 and pVR-ACS7) in mung bean hypocotyls. Results from nuclear run-on transcription assay and RNA gel blot studies revealed that all three genes were transcriptionally active displaying unique patterns of induction by IAA and various hormones in etiolated hypocotyls. Particularly, 24-epibrassinolide (BR), an active brassinosteroid, specifically enhanced the expression of VR-ACS7 by a distinct temporal induction mechanism compared to that of IAA. In addition, BR synergistically increased the IAA-induced VR-ACS6 and VR-ACS7 transcript levels, while it effectively abolished both the IAA- and kinetin-induced accumulation of VR-ACS1 mRNA. In light-grown plants, VR-ACS1 was induced by IAA in roots, and VR-ACS6 in epicotyls. IAA- and BR-treatments were not able to increase the VR-ACS7 transcript in the light-grown tissues. These results indicate that the expression of ACC synthase multigene family is regulated by complex hormonal and developmental networks in a gene- and tissue-specific manner in mung bean plants. The VR-ACS7 gene was isolated, and chimeric fusion between the 2.4 kb 5'-upstream region and the beta-glucuronidase (GUS) reporter gene was constructed and introduced into Nicotiana tabacum. Analysis of transgenic tobacco plants revealed the VR-ACS7 promoter-driven GUS activity at a highly localized region of the hypocotyl-root junction of control seedlings, while a marked induction of GUS activity was detected only in the hypocotyl region of the IAA-treated transgenic seedlings where rapid cell elongation occurs. Although there was a modest synergistic effect of BR on the IAA-induced GUS activity, BR alone failed to increase the GUS activity, suggesting that induction of VR-ACS7 occurs via separate signaling pathways in response to IAA and BR. A scheme of the multiple regulatory pathways for the expression of ACC synthase multigene family by auxin and BR is presented.

Brassinosteroids↗

Light-dependent induction of proline biosynthesis by abscisic acid and salt stress is inhibited by brassinosteroid in Arabidopsis.

Osmotic stress-induced accumulation of proline, an important protective osmolyte in higher plants, is dependent on the expression of delta1-pyrroline-5-carboxylate synthase (P5CS) and proline dehydrogenase (PDH) enzymes that catalyze the rate-limiting steps of proline biosynthesis and degradation, respectively. Proline metabolism is modulated by differential regulation of organ specific expression of PDH and duplicated P5CS genes in Arabidopsis. Stimulation of proline synthesis by abscisic acid (ABA) and salt stress correlates with a striking activation of P5CS1 expression. By contrast, P5CS2 is only weakly induced, whereas PDH is inhibited to different extent by ABA and salt stress in shoots and roots of light-grown plants. Proline accumulation and light-dependent induction of PSCS1 by ABA and salt stress is inhibited in dark-adapted plants. During dark adaptation P5CS2 is also down-regulated, whereas PDH expression is significantly enhanced in shoots. The inhibitory effect of dark adaptation on PSCS1 is mimicked by the steroid hormone brassinolide. However, brassinolide fails to stimulate PDH, and inhibits P5CS2 only in shoots. Proline accumulation and induction of P5CS1 transcription are simultaneously enhanced in the ABA-hypersensitive prl1 and brassinosteroid-deficient det2 mutants, whereas P5CS2 shows enhanced induction by ABA and salt only in the det2 mutant. In comparison, the prl1 mutation reduces the basal level of PDH expression, whereas the det2 mutation enhances the inhibition of PDH by ABA. Regulation of P5CS1 expression thus appears to play a principal role in controlling proline accumulation stimulated by ABA and salt stress in Arabidopsis.

1-Pyrroline-5-Carboxylate Dehydrogenase↗

The brassinosteroid signal transduction pathway.

Steroids function as signaling molecules in both animals and plants. While animal steroid hormones are perceived by nuclear receptor family of transcription factors, brassinosteroids (BR) in plants are perceived by a cell surface receptor kinase, BRI1. Recent studies have demonstrated that BR binding to the extracellular domain of BRI1 induces kinase activation and dimerization with another receptor kinase, BAK1. Activated BRI1 or BAK1 then regulate, possibly indirectly, the activities of BIN2 kinase and/or BSU1 phosphatase, which directly regulate the phosphorylation status and nuclear accumulation of two homologous transcription factors, BZR1 and BES1. BZR1 and BES1 directly bind to promoters of BR responsive genes to regulate their expression. The BR signaling pathway has become a paradigm for both receptor kinase signaling in plants and steroid signaling by cell surface receptors in general.

Brassinosteroids↗

Transcription of the Arabidopsis CPD gene, encoding a steroidogenic cytochrome P450, is negatively controlled by brassinosteroids.

The Arabidopsis CPD gene encodes a cytochrome P450 steroid side-chain hydroxylase (CYP90) that plays an essential role in the biosynthesis of the plant hormone brassinolide. Expression of the CPD gene is confined to cotyledons and leaf primordia in etiolated seedlings and detectable in the adaxial parenchyma of expanding leaves in light-grown plants. Transcription of the CPD gene is not affected by the plant growth factors auxin, ethylene, gibberellin, cytokinin, jasmonic acid and salicylic acid, but is specifically down-regulated by brassinolide in both dark and light. Steady-state mRNA levels of a CPD promoter-driven uidA reporter gene correlate with the expression of resident CPD gene in transgenic plants. Intermediates of the early and late C-6 oxidation pathways of brassinolide, carrying C-22 and C-23 side-chain hydroxyls, efficiently inhibit the activity of the CPD promoter. Repression of CPD transcription by brassinosteroids is sensitive to the protein synthesis inhibitor cycloheximide, indicating a requirement for de novo synthesis of a regulatory factor.

Arabidopsis↗

Promotive effect of brassinosteroids on cell division involves a distinct CycD3-induction pathway in Arabidopsis.

Brassinosteroids (BRs) are steroid hormones that play an essential role in plant growth and development. However, the contradictory results of previous studies make their role in cell division unclear. Using a cDNA array, we identified genes that respond to BR in the det2 suspension culture of Arabidopsis, and found that epi-brassinolide upregulated transcription of the CycD3, a D-type plant cyclin gene through which cytokinin activates cell division. RNA gel-blot analysis and cell culturing showed that epi-brassinolide may promote cell division through CycD3, and can substitute cytokinin in culturing of Arabidopsis callus and suspension cells. The CycD3 induction by epi-brassinolide was further shown to involve de novo protein synthesis, but no protein phosphorylation or dephosphorylation. Induction was also found to occur in cells of a BR-insensitive mutant, bri1, suggesting that BR induces CycD3 transcription through a previously unknown signal pathway in plants.

Arabidopsis↗

Expression of a plant gene with sequence similarity to animal TGF-beta receptor interacting protein is regulated by brassinosteroids and required for normal plant development.

Brassinosteroids (BRs) regulate the expression of numerous genes associated with plant development, and require the activity of a Ser/Thr receptor kinase to realize their effects. In animals, the transforming growth factor-beta (TGF-beta) family of peptides acts via Ser/Thr receptor kinases to have a major impact on several pathways involved in animal development and adult homeostasis. TGF-beta receptor-interacting protein (TRIP-1) was previously shown by others to be an intracellular substrate of the TGF-beta type II receptor kinase which plays an important role in TGF-beta signaling. TRIP-1 is a WD-repeat protein that also has a dual role as an essential subunit of the eukaryotic translation initiation factor eIF3 in animals, yeast and plants, thereby revealing a putative link between a developmental signaling pathway and the control of protein translation. In yeast, expression of a TRIP-1 homolog has also been closely associated with cell proliferation and progression through the cell cycle. We report here the novel observation that transcript levels of TRIP-1 homologs in plants are regulated by BR treatment under a variety of conditions, and that transgenic plants expressing antisense TRIP-1 RNA exhibit a broad range of developmental defects, including some that resemble the phenotype of BR-deficient and -insensitive mutants. This correlative evidence suggests that a WD-domain protein with reported dual functions in vertebrates and fungi might mediate some of the molecular mechanisms underlying the regulation of plant growth and development by BRs.

Amino Acid Sequence↗