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The first brassinosteroid biosynthesis inhibitor is reported. Among newly synthesized triazole derivatives, 4-(4-chlorophenyl)-2-phenyl-3-(1,2,4-triazoyl)butan-2-ol (6) was found to inhibit the growth of cress seedlings, and this inhibition was recovered by the treatment of brassinolide, suggesting that compound 6 primarily inhibits brassinosteroid biosynthesis.
Continued genetic screening and analysis of Arabidopsis mutants has extended our view of brassinosteroid signaling beyond hormone perception to downstream events involving a negative cytoplasmic regulator and nuclear localized positive activators of the brassinosteroid response.
The dwarf ucu (ultracurvata) mutants of Arabidopsis display vegetative leaves that are spirally rolled downwards and show reduced expansion along the longitudinal axis. We have previously determined that the UCU1 gene encodes a SHAGGY/GSK3-like kinase that participates in the signaling pathways of auxins and brassinosteroids. Here, we describe four recessive alleles of the UCU2 gene, whose homozygotes display helical rotation of several organs in addition to other phenotypic traits shared with ucu1 mutants. Following a map-based strategy, we identified the UCU2 gene, which was found to encode a peptidyl-prolyl cis/trans-isomerase of the FK506-binding protein family, whose homologs in metazoans are involved in cell signaling and protein trafficking. Physiological and double mutant analyses suggest that UCU2 is required for growth and development and participates in auxin and brassinosteroid signaling.
The rice (Oryza sativa) dwarf mutant d61 phenotype is caused by loss of function of a rice BRASSINOSTEROID INSENSITIVE1 ortholog, OsBRI1. We have identified nine d61 alleles, the weakest of which, d61-7, confers agronomically important traits such as semidwarf stature and erect leaves. Because erect-leaf habit is considered to increase light capture for photosynthesis, we compared the biomass and grain production of wild-type and d61-7 rice. The biomass of wild type was 38% higher than that of d61-7 at harvest under conventional planting density because of the dwarfism of d61-7. However, the biomass of d61-7 was 35% higher than that of wild type at high planting density. The grain yield of wild type reached a maximum at middensity, but the yield of d61-7 continued to increase with planting density. These results indicate that d61-7 produces biomass more effectively than wild type, and consequently more effectively assimilates the biomass in reproductive organ development at high planting density. However, the small grain size of d61-7 counters any increase in grain yield, leading to the same grain yield as that of wild type even at high density. We therefore produced transgenic rice with partial suppression of endogenous OsBRI1 expression to obtain the erect-leaf phenotype without grain changes. The estimated grain yield of these transformants was about 30% higher than that of wild type at high density. These results demonstrate the feasibility of generating erect-leaf plants by modifying the expression of the brassinosteroid receptor gene in transgenic rice plants.
Plant steroid hormones, brassinosteroids (BRs), are essential for normal photomorphogenesis. However, the mechanism by which light controls physiological functions via BRs is not well understood. Using transgenic plants carrying promoter-luciferase reporter gene fusions, we show that in Arabidopsis (Arabidopsis thaliana) the BR-biosynthetic CPD and CYP85A2 genes are under diurnal regulation. The complex diurnal expression profile of CPD is determined by dual, light-dependent, and circadian control. The severely decreased expression level of CPD in phytochrome-deficient background and the red light-specific induction in wild-type plants suggest that light regulation of CPD is primarily mediated by phytochrome signaling. The diurnal rhythmicity of CPD expression is maintained in brassinosteroid insensitive 1 transgenic seedlings, indicating that its transcriptional control is independent of hormonal feedback regulation. Diurnal changes in the expression of CPD and CYP85A2 are accompanied by changes of the endogenous BR content during the day, leading to brassinolide accumulation at the middle of the light phase. We also show that CPD expression is repressed in extended darkness in a BR feedback-dependent manner. In the dark the level of the bioactive hormone did not increase; therefore, our data strongly suggest that light also influences the sensitivity of plants to BRs.
BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this study we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro, which might be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated on serine (Ser) and threonine (Thr) residues with p-Ser predominating. Matrix-assisted laser desorption/ionization mass spectrometry identified a minimum of 12 sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in the juxtamembrane region (N-terminal to the catalytic KD), five in the KD (one each in sub-domains I and VIa and three in sub-domain VIII), and two in the carboxy terminal region. Five of the sites were uniquely identified (Ser-838, Thr-842, Thr-846, Ser-858, and Thr-872), whereas seven were localized on short peptides but remain ambiguous due to multiple Ser and/or Thr residues within these peptides. The inability of an active BRI1-KD to transphosphorylate an inactive mutant KD suggests that the mechanism of autophosphorylation is intramolecular. It is interesting that recombinant BRI1-KD was also found to phosphorylate certain synthetic peptides in vitro. To identify possible structural elements required for substrate recognition by BRI1-KD, a series of synthetic peptides were evaluated, indicating that optimum phosphorylation of the peptide required R or K residues at P - 3, P - 4, and P + 5 (relative to the phosphorylated Ser at P = 0).
Cell elongation is a developmental process that is regulated by light and phytohormones and is of critical importance for plant growth. Mutants defective in their response to light and various hormones are often dwarfs. The dwarfed phenotype results because of a failure in normal cell elongation. Little is known, however, about the basis of dwarfism as a common element in these diverse signaling pathways and the nature of the cellular functions responsible for cell elongation. Here, we describe an Arabidopsis mutant, dwarf4 (dwf4), whose phenotype can be rescued with exogenously supplied brassinolide. dwf4 mutants display features of light-regulatory mutants, but the dwarfed phenotype is entirely and specifically brassinosteroid dependent; no other hormone can rescue dwf4 to a wild-type phenotype. Therefore, an intact brassinosteroid system is an absolute requirement for cell elongation.
BRASSINOSTEROID-INSENSITIVE 1 (BRI1) is a Leu-rich-repeat (LRR) receptor kinase that functions as a critical component of a transmembrane brassinosteroid (BR) receptor. It is believed that BRI1 becomes activated through heterodimerization with BAK1, a similar LRR receptor kinase, in response to BR signal. A yeast two-hybrid screen using the kinase domain of BRI1 identified an Arabidopsis thaliana Transthyretin-Like protein (TTL) as a potential BRI1 substrate. TTL interacts with BRI1 in a kinase-dependent manner in yeast and is phosphorylated by BRI1 in vitro. TTL displays a similar expression pattern with BRI1 and is associated with the plasma membrane. Overexpression of the TTL gene results in a phenotype that was observed in weak bri1 mutants and null bak1 mutants. By contrast, two T-DNA insertional mutations in the TTL gene promote plant growth and enhance BR sensitivity. We hypothesized that TTL might directly regulate certain biochemical activities near the plasma membrane to control plant growth.
In Arabidopsis thaliana brassinosteroid (BR), perception is mediated by two Leu-rich repeat receptor-like kinases, BRASSINOSTEROID INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) (Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-like KINASE3 [AtSERK3]). Genetic, biochemical, and yeast (Saccharomyces cerevisiae) interaction studies suggested that the BRI1-BAK1 receptor complex initiates BR signaling, but the role of the BAK1 receptor is still not clear. Using transient expression in protoplasts of BRI1 and AtSERK3 fused to cyan and yellow fluorescent green fluorescent protein variants allowed us to localize each receptor independently in vivo. We show that BRI1, but not AtSERK3, homodimerizes in the plasma membrane, whereas BRI1 and AtSERK3 preferentially heterodimerize in the endosomes. Coexpression of BRI1 and AtSERK3 results in a change of the steady state distribution of both receptors because of accelerated endocytosis. Endocytic vesicles contain either BRI1 or AtSERK3 alone or both. We propose that the AtSERK3 protein is involved in changing the equilibrium between plasma membrane-located BRI1 homodimers and endocytosed BRI1-AtSERK3 heterodimers.
Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.
Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE 1 (BRI1) receptor serine/threonine kinase for hormone perception and signal transduction. In mammals, the transforming growth factor-beta (TGF-beta) family of polypeptides modulate numerous aspects of development and are perceived at the cell surface by a complex of type I and type II TGF-beta receptor serine/threonine kinases. TGF-beta receptor interacting protein (TRIP-1) is a cytoplasmic substrate of the TGF-beta type II receptor kinase and plays a role in TGF-beta signaling. TRIP-1 is a WD domain protein that also functions as an essential subunit of the eIF3 eukaryotic translation initiation factor in animals, yeast and plants. We previously cloned putative TRIP-1 homologs from bean and Arabidopsis and found that transgenic Arabidopsis plants expressing antisense TRIP-1 RNA exhibited a broad range of developmental defects including some morphological characteristics that resemble the phenotype of BR-deficient and -insensitive mutants. We now show that the BRI1 kinase domain phosphorylates Arabidopsis TRIP-1 on three specific sites in vitro (Thr-14, Thr-89 and either Thr-197 or Ser-198). Co-immunoprecipitation experiments using antibodies against TRIP-1, BRI1 and various fusion proteins strongly suggest that TRIP-1 and BRI1 also interact directly in vivo. These findings support a role for TRIP-1 in the molecular mechanisms of BR-regulated plant growth and development, possibly as a cytoplasmic substrate of the BRI1 receptor kinase.
SPINDLY (SPY) encodes an O-linked N-acetylglucosamine transferase that is considered to be a negative regulator of gibberellin (GA) signaling through an unknown mechanism. To understand the function of SPY in GA signaling in rice, we isolated a rice SPINDLY homolog (OsSPY) and produced knockdown transgenic plants in which OsSPY expression was reduced by introducing its antisense or RNAi construct. In knockdown plants, the enhanced elongation of lower internodes was correlated with decreased levels of OsSPY expression, similar to the spindly phenotype of Arabidopsis spy mutants, suggesting that OsSPY also functions as a negative factor in GA signaling in rice. The suppressive function of OsSPY in GA signaling was supported by the findings that the dwarfism was partially rescued and OsGA20ox2 (GA20 oxidase) expression was reduced in GA-deficient and GA-insensitive mutants by the knockdown of OsSPY function. The suppression of OsSPY function in a GA-insensitive mutant, gid2, also caused an increase in the phosphorylation of a rice DELLA protein, SLR1, but did not change the amount of SLR1. This indicates that the function of OsSPY in GA signaling is not via changes in the amount or stability of SLR1, but probably involves control of the suppressive function of SLR1. In addition to the GA-related phenotypes, OsSPY antisense and RNAi plants showed increased lamina joint bending, which is a brassinosteroid-related phenotype, indicating that OsSPY may play roles both in GA signaling and in the brassinosteroid pathway.
GSK3/SHAGGY is a highly conserved serine/threonine kinase implicated in many signaling pathways in eukaryotes. Although many GSK3/SHAGGY-like kinases have been identified in plants, little is known about their functions in plant growth and development. Here we show that the Arabidopsis BRASSINOSTEROID-INSENSITIVE 2 (BIN2) gene encodes a GSK3/SHAGGY-like kinase. Gain-of-function mutations within its coding sequence or its overexpression inhibit brassinosteroid (BR) signaling, resulting in plants that resemble BR-deficient and BR-response mutants. In contrast, reduced BIN2 expression via cosuppression partially rescues a weak BR-signaling mutation. Thus, BIN2 acts as a negative regulator to control steroid signaling in plants.
A natural brassinosteroid and a series of synthetic derivatives were found to be good inhibitors of herpes simplex virus type 1 (HSV-1) and arenavirus replication in cell culture. The synthetic compounds tested were analogues of the 24(S) ethylbrassinone. Compounds (22 R,23 R,24S)-2alpha, 3alpha,5alpha,22,23-pentahydroxy-stigmastan-6-one and (22R,23R,24S)-3beta-bromo-5alpha,22,23-trihydroxy stigmastan-6-one were cytotoxic at concentrations of 20-40 microM. (22S,23S,24S)-2alpha,3alpha,22,23-tetrahydroxy-5alpha, stigmastan-6-one, (22R,23R,24S)-3beta-acetoxy-22,23-dihydroxy-5alpha-choles tan-6-one, (22S,23S,24S)-3beta-bromo-22,23-dihydroxy-5alpha-cholestan-6 -one and (22S,23S,24S)-3beta-bromo-5alpha,22,23-trihydroxy-stigmastan -6-one were the most active of the series against HSV-1, with selectivity index (SI) values (CC50/EC50) ranging from 10.6 to 16.5. The majority of the compounds were potent inhibitors of arenaviruses, (22S,23S,24S)-3beta-bromo-5alpha,22,23-trihydroxy-stigmastan -6-one being the most active, with SI values of 307.8 and 692.5 for Tacaribe and Junin viruses, respectively. The antiviral activity of brassinosteroid derivatives was not because of direct inactivation; time-of-addition experiments suggested that a late step in HSV-1 multiplication was affected, whereas arenaviruses remained susceptible to the compounds throughout the replicative cycle.
Twenty-seven brassinosteroid derivatives were tested for antiviral activity against measles virus (MV) via a virus-yield reduction assay. Compounds 6b [(22S,235)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one], 1d [(22R,23R)-2alpha,3alpha,22,23-tetrahydroxy-beta-Homo-7-oxa-stigmastan-6-one], 8a [(22R,23R)-3beta-fluoro-22,23-dihydroxystigmastan-6-one], 9b [(22S,23S)-3beta-fluoro-5alpha,22,23-trihydroxystigmastan-6-one] and 10b [(22S,23S)-5alpha-fluor-3beta,22,23-trihydroxystigmastan-6-one], with selectivity indexes (SI) of 40, 57, 31, 37 and 53, are the derivatives with good antiviral activity against MV. These SI values are higher than those obtained with ribavirin (used as reference drug). A comparative analysis of 50% cytotoxic concentration (CC50) values, using confluent non-growing cells, gives and indication of structure-activity relationship. According to their degree of cytotoxicity the compounds were divided in three groups: low, intermediate and high cytotoxicity. By observing the chemical structures of compounds belonging to the first group we can see that less cytotoxic activities are related to the presence of a 3beta-hydroxy group on C-3 (ring A) and a double bond between C-22 and C-23 (side chain). The replacement of a 5alpha-hydroxy group by a 5alpha-fluoro group enhances cytotoxicity. Halogenated brassinosteroid derivatives in C-3 position are more cytotoxic than those with an acetoxy group in the same position. For compounds 1d, 6b, 10b and ribavirin, cytotoxicity measurements were also done with replicating cells; CC50 values were low, but they still competed favourably with ribavirin against MV.
The new brassinosteroid conjugate, teasterone-3-O-betaD-glucopyranoside, was found as a metabolite of teasterone in lily cell suspension cultures. Its structure was determined by means of FAB-MS and 1H-NMR upon comparison with the authentic compound. Furthermore, its presence in lily anthers was confirmed by FAB-MS and LC-APCI-SIM data. This is the first natural brassinosteroid conjugate glucosylated at a hydroxyl group in ring A.
After feeding of 24-epi-castasterone to the cockroach Periplaneta americana an organ-specific epimerization of the brassinosteroid to 2,24-diepi-castasterone could be detected in female insects. The metabolite being observed only in the ovaries and not in the testes of the insect was identified by GC/MS in comparison with a synthesized authentic sample. Contrary, 24-epi-brassinolide is not metabolized in the sexual organs of Periplaneta americana. This is the first evidence of a metabolic transformation of a brassinosteroid in insects.
The uptake of 86Rb+ ions by human erythrocytes as the measure of inhibition or stimulation of Na+, K(+)-ATPase by synthetic brassinolides, steroid fytohormonal promotors, was determined. No compound of the 12 tested brassinosteroids exerted higher inhibition of ATPase than digitoxin. A lower but significant inhibition of ion transport by 3 of tested derivatives, and a stimulation by 4 compounds was found. All active compounds were steroids with a 7-membered B-ring with an oxygen atom in the position 7a and with an oxogroup in position 6, i.e. they contained an lactone ring which is characteristic for natural brassinosteroids.