Uncoupling brassinosteroid levels and de-etiolation in pea

The suggestion that brassinosteroids (BRs) have a negative regulatory role in de-etiolation is based largely on correlative evidence, which includes the de-etiolated phenotypes of, and increased expression of light-regulated genes in, dark-grown mutants defective in BR biosynthesis or response. However, we have obtained the first direct evidence which shows that endogenous BR levels in light-grown pea seedlings are increased, not decreased, in comparison with those grown in the dark. Similarly, we found no evidence of a decrease in castasterone (CS) levels in seedlings that were transferred from the dark to the light for 24 h. Furthermore, CS levels in the constitutively de-etiolated lip1 mutant are similar to those in wild-type plants, and are not reduced as is the case in the BR-deficient lkb plants. Unlike lip1 , the pea BR-deficient mutants lk and lkb are not de-etiolated at the morphological or molecular level, as they exhibit neither a de-etiolated phenotype or altered expression of light-regulated genes when grown in the dark. Similarly, dark-grown WT plants treated with the BR biosynthesis inhibitor, Brz, do not exhibit a de-etiolated phenotype. In addition, analysis of the lip1lkb double mutant revealed an additive phenotype indicative of the two genes acting in independent pathways. Together these results strongly suggest that BR levels do not play a negative-regulatory role in de-etiolation in pea.     

Synthesis of novel brassinosteroid biosynthesis inhibitors based on the ketoconazole scaffold

Brassinosteroids (BRs) are steroidal plant hormones that control several important agronomic traits such as plant architecture, seed yield, and stress tolerance. Inhibitors that target BR biosynthesis are candidate plant growth regulators. We synthesized novel triazole derivatives, based on the ketoconazole scaffold, that function as inhibitors of BR biosynthesis. The biological activity of the test compounds was evaluated by determining their ability to induce dwarfism in Arabidopsis seedlings grown in the dark. The chemically induced dwarfism of Arabidopsis seedlings was further evaluated by a rescue experiment using the co-application of brassinolide and/or gibberellins (GA). The structure-activity relationship studies revealed a potent BR biosynthesis inhibitor, 2RS, 4RS-1-{2-(4-chlorophenyl)-4-[2-(2-ethoxyphenyl)-ethyl]-1,3-dioxolan-2-ylmethyl}-1H-1,2,4-triazole (7m), with an IC(50) value of 0.10±0.03 μM for retardation of Arabidopsis seedling stem elongation. The compound-induced hypocotyl dwarfism was counteracted by the co-application of 10nM brassinolide, but not 1 μM GA(3), which produced seedlings that resembled BR-deficient mutants. This result suggests that 7m is a potent and specific inhibitor of BR biosynthesis.     

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.     

Synthesis and biological evaluation of novel azole derivatives as selective potent inhibitors of brassinosteroid biosynthesis

Brassinosteroids (BRs) are phytohormones that control several important agronomic traits, such as flowering, plant architecture, seed yield, and stress tolerance. To manipulate the BR levels in plant tissues using specific inhibitors of BR biosynthesis, a series of novel azole derivatives were synthesized and their inhibitory activity on BR biosynthesis was investigated. Structure–activity relationship studies revealed that 2RS, 4RS-1-[4-(2-allyloxyphenoxymethyl)-2-(4-chlorophenyl)-[1,3]dioxolan-2-ylmethyl]-1H-[1,2,4]triazole (G₂) is a highly selective inhibitor of BR biosynthesis, with an IC₅₀ value of approximately 46 ± 2 nM, which is the most potent BR biosynthesis inhibitor observed to date. Use of gibberellin (GA) biosynthesis mutants and BR signaling mutants to analyze the mechanism of action of this synthetic series indicated that the primary site of action is BR biosynthesis. Experiments feeding BR biosynthesis intermediates to chemically treated Arabidopsis seedlings suggested that the target sites of this synthetic series are CYP90s, which are responsible for the C-22 and/or C-23 hydroxylation of campesterol.     

Brz220 interacts with DWF4, a cytochrome P450 monooxygenase in brassinosteroid biosynthesis, and exerts biological activity

Arabidopsis thaliana (Arabidopsis) treated with the four stereoisomers of Brz220 (2RS, 4RS-1-[4-propyl-2-(4-trifluoromethylphenyl)-1, 3-dioxane-2-ylmethyl]-1H-1, 2, 4-triazole) showed a dwarf phenotype like brassinosteroid (BR) biosynthesis mutants that were rescued by treatment of BRs. The target sites of each Brz220 stereoisomer were investigated by treatment of Arabidopsis with BRs in the dark. The results suggest that the stereoisomers block the 22-hydroxylation step in BR biosynthesis. This step is catalyzed by DWF4, an Arabidopsis cytochrome P450 identified as a steroid 22-hydroxylase. The enzyme was expressed in E. coli, and the binding affinity of the stereoisomers to recombinant DWF4 was analyzed. The results indicate that in these stereoisomers there exists a positive correlation between binding affinity to DWF4 and inhibition of Arabidopsis hypocotyl growth. In this context, we concluded that DWF4 is the target site of Brz220 in Arabidopsis.     

Analysis of the rice mutant dwarf and gladius leaf 1. Aberrant katanin-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling

Molecular genetic studies of plant dwarf mutants have indicated that gibberellin (GA) and brassinosteroid (BR) are two major factors that determine plant height; dwarf mutants that are caused by other defects are relatively rare, especially in monocot species. Here, we report a rice (Oryza sativa) dwarf mutant, dwarf and gladius leaf 1 (dgl1), which exhibits only minimal response to GA and BR. In addition to the dwarf phenotype, dgl1 produces leaves with abnormally rounded tip regions. Positional cloning of DGL1 revealed that it encodes a 60-kD microtubule-severing katanin-like protein. The protein was found to be important in cell elongation and division, based on the observed cell phenotypes. GA biosynthetic genes are up-regulated in dgl1, but the expression of BR biosynthetic genes is not enhanced. The enhanced expression of GA biosynthetic genes in dgl1 is not caused by inappropriate GA signaling because the expression of these genes was repressed by GA3 treatment, and degradation of the rice DELLA protein SLR1 was triggered by GA3 in this mutant. Instead, aberrant microtubule organization caused by the loss of the microtubule-severing function of DGL1 may result in enhanced expression of GA biosynthetic genes in that enhanced expression was also observed in a BR-deficient mutant with aberrant microtubule organization. These results suggest that the function of DGL1 is important for cell and organ elongation in rice, and aberrant DGL1-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling.     

Nitric oxide mediates brassinosteroid-induced ABA biosynthesis involved in oxidative stress tolerance in maize leaves

The role of ABA in brassinosteroid (BR)-induced stress tolerance and the relationship between BR, nitric oxide (NO) and ABA under water stress induced by polyethylene glycol (PEG) were investigated in leaves of maize (Zea mays) plants. Water stress led to oxidative damage. Pre-treatment with the BR biosynthetic inhibitor brassinazole (Brz) aggravated the oxidative damage induced by PEG treatment, which was alleviated by the application of BR or ABA. Pre-treatment with the ABA biosynthetic inhibitor fluridone also aggravated the oxidative damage induced by PEG treatment; however, this was barely alleviated by the application of BR. BR treatment increased the content of ABA and up-regulated the expression of the ABA biosynthetic gene vp14 in maize leaves, which was blocked by pre-treatments with the NO scavenger cPTIO (2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and the nitric oxide synthase inhibitor l-NAME (N(G)-nitro-l-arginine methyl ester. Moreover, BR treatment induced increases in the generation of NO in mesophyll cells of maize leaves, and treatment with the NO donor sodium nitroprusside (SNP) up-regulated the content of ABA and the expression of vp14 in maize leaves. Our results suggest that BR-induced NO production and NO-activated ABA biosynthesis are important mechanisms for BR-enhanced water stress tolerance in leaves of maize plants.     

Growth and graviresponsiveness of primary roots of Zea mays seedlings deficient in abscisic acid and gibberellic acid

The objective of this research was to determine if gibberellic acid (GA) and/or abscisic acid (ABA) are necessary for graviresponsiveness by primary roots of Zea mays. To accomplish this objective we measured the growth and graviresponsiveness of primary roots of seedlings in which the synthesis of ABA and GA was inhibited collectively and individually by genetic and chemical means. Roots of seedlings treated with Fluridone (an inhibitor of ABA biosynthesis) and Ancymidol (an inhibitor of GA biosynthesis) were characterized by slower growth rates but not significantly different gravicultures as compared to untreated controls. Gravicurvatures of primary roots of d-5 mutants (having undetectable levels of GA) and vp-9 mutants (having undectable levels of ABA) were not significantly different from those of wild-type seedlings. Roots of seedlings in which the biosynthesis of ABA and GA was collectively inhibited were characterized by gravicurvatures not significantly different for those of controls. These results (1) indicate that drastic reductions in the amount of ABA and GA in Z. mays seedlings do not significantly alter root graviresponsiveness, (2) suggest that neither ABA nor GA is necessary for root gravicurvature, and (3) indicate that root gravicurvature is not necessarily proportional to root elongation.     

MAX2 affects multiple hormones to promote photomorphogenesis

Ubiquitin-26S proteasome system (UPS) has been shown to play central roles in light and hormone-regulated plant growth and development. Previously, we have shown that MAX2, an F-box protein, positively regulates facets of photomorphogenic development in response to light. However, how MAX2 controls these responses is still unknown. Here, we show that MAX2 oppositely regulates GA and ABA biosynthesis to optimize seed germination in response to light. Dose-response curves showed that max2 seeds are hyposensitive to GA and hypersensitive to ABA in seed germination responses. RT-PCR assays demonstrated that the expression of GA biosynthetic genes is down-regulated, while the expression of GA catabolic genes is up-regulated in the max2 seeds compared to wild-type. Interestingly, expression of both ABA biosynthetic and catabolic genes is up-regulated in the max2 seeds compared to wild-type. Treatment with an auxin transport inhibitor, NPA, showed that increased auxin transport in max2 seedlings contributes to the long hypocotyl phenotype under light. Moreover, light-signaling phenotypes are restricted to max2, as the biosynthetic mutants in the strigolactone pathway, max1, max3, and max4, did not display any defects in seed germination and seedling de-etiolation compared to wild-type. Taken together, these data suggest that MAX2 modulates multiple hormone pathways to affect photomorphogenesis.     

The alternative oxidase pathway is involved in the BR-induced salt resistance in mustard

In this study, the role of Brassinosteroids (BRs) and the relationship between the mitochondrial alternative oxidase (AOX) and ROS in the BR-induced defence response to salt stress was studied in mustard plants. Salt stress induced a significant activation of AOX. Exogenous BR significantly enhanced the capacity of the cyanide-resistant pathway, and reduced the damage of cell membrane. Pretreatment with brassinazole (Brz, an inhibitor of the BR biosynthesis pathway) significantly blocked the capacity of the cyanide-resistant pathway. BR could partly recover the AOX inactivation under salicylhydroxamic acid (SHAM, an inhibitor of the cyanide-resistant pathway) pretreatment. It was also found that BR could enhance the ROS accumulation and the antioxidant enzyme activities, while the AOX could eliminate the excessive ROS and enhance the antioxidant enzyme activities. Furthermore, the suppression of the cyanide-resistant pathway significantly increased the MDA content and the electrolyte leakage in mustard leaves, and the suppression of the BR biosynthesis had little effect on their recovering. Taken together, the cyanide-resistant pathway was involved in BR-induced salt tolerance and played an important role in maintaining the permeability of the cell membrane.     

Evidence for functional interaction between brassinosteroids and cadmium response in Arabidopsis thaliana

Plant hormones, in addition to regulating growth and development, are involved in biotic and abiotic stress responses. To investigate whether a hormone signalling pathway plays a role in the plant response to the heavy metal cadmium (Cd), gene expression data in response to eight hormone treatments were retrieved from the Genevestigator Arabidopsis thaliana database and compared with published microarray analysis performed on plants challenged with Cd. Across more than 3000 Cd-regulated genes, statistical approaches and cluster analyses highlighted that gene expression in response to Cd and brassinosteroids (BR) showed a significant similarity. Of note, over 75% of the genes showing consistent (e.g. opposite) regulation upon BR and Brz (BR biosynthesis inhibitor) exposure exhibited a BR-like response upon Cd exposure. This phenomenon was confirmed by qPCR analysis of the expression level of 10 BR-regulated genes in roots of Cd-treated wild-type (WT) plants. Although no change in BR content was observed in response to Cd in our experimental conditions, adding epibrassinolide (eBL, a synthetic brassinosteroid) to WT plants significantly enhanced Cd-induced root growth inhibition, highlighting a synergistic response between eBL and the metal. This effect was specific to this hormone treatment. On the other hand, dwarf1 seedlings, showing a reduced BR level, exhibited decreased root growth inhibition in response to Cd compared with WT, reversed by the addition of eBL. Similar results were obtained on Brz-treated WT plants. These results argue in favour of an interaction between Cd and BR signalling that modulates plant sensitivity, and opens new perspectives to understand the plant response to Cd.     

Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor

Screening for brassinosteroid (BR) biosynthesis inhibitors was performed to find chemicals that induce dwarfism in Arabidopsis, mutants that resembled BR biosynthesis mutants that can be rescued by BR. Through this screening experiment, the compound brassinazole was selected as the most potent chemical. In dark-grown Arabidopsis, brassinazole-induced morphological changes were nearly restored to those of wild type by treatment with brassinolide. The structure of brassinazole is similar to pacrobutrazol, a gibberellin biosynthesis inhibitor. However, in assays with cress (Lepidium sativum) plants, brassinazole-treated plants did not show recovery after the addition of gibberellin but showed good recovery after the addition of brassinolide. These data demonstrate that brassinazole is a specific BR biosynthesis inhibitor. Brassinazole-treated cress also showed dwarfism, with altered leaf morphology, including the downward curling and dark green color typical of Arabidopsis BR-deficient mutants, and this dwarfism was reversed by the application of 10 nM brassinolide. This result suggests that BRs are essential for plant growth, and that brassinazole can be used to clarify the function of BRs in plants as a complement to BR-deficient mutants. The brassinazole action site was also investigated by feeding BR biosynthesis intermediates to cress grown in the light.     

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.     

The Arabidopsis dwf7/ste1 mutant is defective in the delta7 sterol C-5 desaturation step leading to brassinosteroid biosynthesis.

Lesions in brassinosteroid (BR) biosynthetic genes result in characteristic dwarf phenotypes in plants. Understanding the regulation of BR biosynthesis demands continued isolation and characterization of mutants corresponding to the genes involved in BR biosynthesis. Here, we present analysis of a novel BR biosynthetic locus, dwarf7 (dwf7). Feeding studies with BR biosynthetic intermediates and analysis of endogenous levels of BR and sterol biosynthetic intermediates indicate that the defective step in dwf7-1 resides before the production of 24-methylenecholesterol in the sterol biosynthetic pathway. Furthermore, results from feeding studies with 13C-labeled mevalonic acid and compactin show that the defective step is specifically the Delta7 sterol C-5 desaturation, suggesting that dwf7 is an allele of the previously cloned STEROL1 (STE1) gene. Sequencing of the STE1 locus in two dwf7 mutants revealed premature stop codons in the first (dwf7-2) and the third (dwf7-1) exons. Thus, the reduction of BRs in dwf7 is due to a shortage of substrate sterols and is the direct cause of the dwarf phenotype in dwf7.     

The Influence of Chemical Genetics on Plant Science: Shedding Light on Functions and Mechanism of Action of Brassinosteroids Using Biosynthesis Inhibitors

When exogenous chemicals allow rapid, conditional, reversible, selective, and dose-dependent control of biological functions, they act like conditional mutations, either inducing or suppressing the formation of a specific phenotype of interest. Exploration of the small molecules that induce the brassinosteroid (BR) deficient-like phenotype in Arabidopsis led us to identify brassinazole as the first candidate for a BR biosynthesis inhibitor. Brassinazole treatment reduced BR content in plant cells. Investigation of target site(s) of brassinazole revealed that the compound directly binds to the DWF4 protein, a cytochrome P450 monooxygenase that catalyzes 22-hydroxylation of the side chain of BRs. These results suggest that brassinazole is a BR biosynthesis inhibitor. There are currently at least two BR biosynthesis inhibitors that act like conditional mutations in BR biosynthesis. They allow the investigation of the functions of BRs in a variety of plant species. Application of BR biosynthesis inhibitors to a standard genetic screen to identify mutants that confer resistance to these inhibitors allowed the identification of new components working in BR signal transduction. This method has advantages over mutant screens using BR-deficient mutants as a background. Development of chemicals that induce phenotypes of interest is now emerging as a useful way to study biological systems in plants and this would be a complement to classical biochemical and genetic methods.     

A role for brassinosteroids in germination in Arabidopsis

This paper presents evidence that plant brassinosteroid (BR) hormones play a role in promoting germination. It has long been recognized that seed dormancy and germination are regulated by the plant hormones abscisic acid (ABA) and gibberellin (GA). These two hormones act antagonistically with each other. ABA induces seed dormancy in maturing embryos and inhibits germination of seeds. GA breaks seed dormancy and promotes germination. Severe mutations in GA biosynthetic genes in Arabidopsis, such as ga1-3, result in a requirement for GA application to germinate. Whereas previous work has shown that BRs play a critical role in controlling cell elongation, cell division, and skotomorphogenesis, no germination phenotypes have been reported in BR mutants. We show that BR rescues the germination phenotype of severe GA biosynthetic mutants and of the GA-insensitive mutant sleepy1. This result shows that BR stimulates germination and raises the possibility that BR is needed for normal germination. If true, we would expect to detect a germination phenotype in BR mutants. We found that BR mutants exhibit a germination phenotype in the presence of ABA. Germination of both the BR biosynthetic mutant det2-1 and the BR-insensitive mutant bri1-1 is more strongly inhibited by ABA than is germination of wild type. Thus, the BR signal is needed to overcome inhibition of germination by ABA. Taken together, these results point to a role for BRs in stimulating germination.