Propiconazole is a specific and accessible brassinosteroid (BR) biosynthesis inhibitor for Arabidopsis and maize.
ABSTRACT: Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA(3) co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species.
Project description:Brassinosteroids (BRs) are steroidal phytohormones that are involved in diverse physiological processes and affect many important traits, such as plant stature, stress tolerance, leaf angle, fertility, and grain filling. BR signaling and biosynthetic pathways have been studied in various plants, such as the model dicot Arabidopsis thaliana; however, relatively little is known about these pathways in monocots.To characterize BR-related processes in the model grass Brachypodium distachyon, we studied the response of these plants to the specific BR biosynthesis inhibitor, propiconazole (Pcz). We found that treatments with Pcz produced a dwarf phenotype in B. distachyon seedlings, similar to that observed in Pcz-treated Arabidopsis plants and in characterized BR-deficient mutants. Through bioinformatics analysis, we identified a list of putative homologs of genes known to be involved in BR biosynthesis and signaling in Arabidopsis, such as DWF4, BR6OX2, CPD, BRI1, and BIN2. Evaluating the response of these genes to Pcz treatments revealed that candidates for BdDWF4, BR6OX2 and, CPD were under feedback regulation. In addition, Arabidopsis plants heterologously expressing BdDWF4 displayed tall statures and elongated petioles, as would be expected in plants with elevated levels of BRs. Moreover, heterologous expression of BdBIN2 in Arabidopsis resulted in dwarfism, suggesting that BdBIN2 functions as a negative regulator of BR signaling. However, the dwarf phenotypes of Arabidopsis bri1-5, a weak BRI1 mutant allele, were not complemented by overexpression of BdBRI1, indicating that BdBRI1 and BRI1 are not functionally equivalent.We identified components of the BR biosynthetic and signaling pathways in Brachypodium, and provided examples of both similarities and differences in the BR biology of these two plants. Our results suggest a framework for understanding BR biology in monocot crop plants such as Zea mays (maize) and Oryza sativa (rice).
Project description:The isolation of propiconazole (PCZ) degrading bacterium BBK_9 strain was done from paddy soil, and it was identified as Burkholderia sp. based on the morphological characteristics and biochemical properties combined with 16S rRNA gene sequencing analysis. It has been seen that the factors such as temperature and pH influence the biodegradation process. The role of plasmid was studied in the degradation process by plasmid curing method. The PCZ acts as the sole carbon source and as energy substrate which can be utilized by the strain for its growth in Mineral salt medium and degraded 8.89 µg ml-1 of PCZ at 30 °C and pH 7 within 4 days. During the bioconversion process of PCZ, three metabolite were formed such as 1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl) ethanone, 1-[2-(4-chlorophenyl) ethyl]-1H-1,2,4-triazole and 1-ethyl-1H-1,2,4-triazole. The LD50 value of BBK_9 strain was determined with acridine orange which resulted in 40 µg ml-1 at cell density of 0.243 at 660 nm. Furthermore, plasmid curing was done using LD50 concentration and from that three plasmids got cured in the sixth generation. It was found that, cured strain was able to degrade 7.37 µg ml-1 of PCZ, indicating the plasmid encoded gene were not responsible for the PCZ degradation. On the source of these outcomes, strain BBK_9 can be used as potential strain for bioremediation of contaminated sites.
Project description:Melatonin functions as a plant hormone/regulator in the regulation of growth and development. However, the underlying mechanisms are still unclear. In this study, we found that a high dose of melatonin inhibited hypocotyl elongation in a dose-dependent manner in Arabidopsis. An expression profile analysis showed that hypocotyl growth inhibition by melatonin was involved in reprograming the expression of cell elongation genes and brassinosteroid (BRs) biosynthetic genes. Furthermore, similar to BR biosynthetic inhibitor brassinazole (BRZ), a high concentration of melatonin upregulated BR-biosynthetic genes and downregulated BR-induced genes involved in cell elongation, while melatonin was inefficient in brassinazole-resistant mutants like the bzr1-1D and bes1-D in hypocotyl inhibition. The comparative expression profile analysis showed an opposite expression mode in the co-regulated genes between melatonin and BZR1 or melatonin and brassinolide (BL). Additionally, exogenous BL rescued the repressive phenotype of BR biosynthesis-deficient mutant like det2-1 even in the presence of high-dose melatonin, but not BR receptor mutant bri1-5 or signal transduction mutant bin2-1. A biochemical analysis further confirmed that melatonin reduced endogenous BR levels in a dose-dependent manner in Arabidopsis. Taken together, these results indicate that melatonin inhibits BR biosynthesis but does not block BR signaling in the inhibition of hypocotyl elongation and extends insights on the role of melatonin in cross-talking with plant hormone signaling.
Project description:Interactions between signaling pathways help guide plant development. In this study, we found that brassinosteroid (BR) signaling converges with SUPPRESSOR OF PHYTOCHROME B4-#3 (SOB3) to influence both the transcription of genes involved in cell elongation and hypocotyl growth. Specifically, SOB3 mutant hypocotyl phenotypes, which are readily apparent when the seedlings are grown in dim white light, were attenuated by treatment with either brassinolide (BL) or the BR biosynthesis inhibitor brassinazole (BRZ). Hypocotyls of SOB3 mutant seedlings grown in white light with a higher fluence rate also exhibited altered sensitivities to BL, further suggesting a connection to BR signaling. However, the impact of BL treatment on SOB3 mutants grown in moderate-intensity white light was reduced when polar auxin transport was inhibited. BL treatment enhanced transcript accumulation for all six members of the SMALL AUXIN UP RNA19 (SAUR19) subfamily, which promote cell expansion, are repressed by SOB3 and light, and are induced by auxin. Conversely, BRZ inhibited the expression of SAUR19 and its homologs. Expression of these SAURs was also enhanced in lines expressing a constitutively active form of the BR signaling component BZR1, further indicating that the transcription of SAUR19 subfamily members are influenced by this hormone signaling pathway. Taken together, these results indicate that SOB3 and BR signaling converge to influence the transcription of hypocotyl growth-promoting SAUR19 subfamily members.
Project description:BACKGROUND:Yeasts, which are ubiquitous in agroecosystems, are known to degrade various xenobiotics. The aim of this study was to analyze the effect of fungicides on the abundance of natural yeast communities colonizing winter wheat leaves, to evaluate the sensitivity of yeast isolates to fungicides in vivo, and to select yeasts that degrade propiconazole. RESULTS:Fungicides applied during the growing season generally did not affect the counts of endophytic yeasts colonizing wheat leaves. Propiconazole and a commercial mixture of flusilazole and carbendazim decreased the counts of epiphytic yeasts, but the size of the yeast community was restored after 10?days. Epoxiconazole and a commercial mixture of fluoxastrobin and prothioconazole clearly stimulated epiphyte growth. The predominant species isolated from leaves were Aureobasidium pullulans and Rhodotorula glutinis. In the disk diffusion test, 14 out of 75 yeast isolates were not sensitive to any of the tested fungicides. After 48?h of incubation in an aqueous solution of propiconazole, the Rhodotorula glutinis Rg 55 isolate degraded the fungicide in 75%. Isolates Rh. glutinis Rg 92 and Rg 55 minimized the phytotoxic effects of propiconazole under greenhouse conditions. The first isolate contributed to an increase in the dry matter content of wheat seedlings, whereas the other reduced the severity of chlorosis. CONCLUSION:Not sensitivity of many yeast colonizing wheat leaves on the fungicides and the potential of isolate Rhodotorula glutinis Rg 55 to degrade of propiconazole was established. Yeast may partially eliminate the ecologically negative effect of fungicides.
Project description:The plant steroid hormone brassinosteroids (BRs) are important signal mediators that regulate broad aspects of plant growth and development. With the discovery of brassinoazole (Brz), the first specific inhibitor of BR biosynthesis, several triazole-type BR biosynthesis inhibitors have been developed. In this article, we report that fenarimol (FM), a pyrimidine-type fungicide, exhibits potent inhibitory activity against BR biosynthesis. FM induces dwarfism and the open cotyledon phenotype of Arabidopsis seedlings in the dark. The IC50 value for FM to inhibit stem elongation of Arabidopsis seedlings grown in the dark was approximately 1.8 ± 0.2 ?M. FM-induced dwarfism of Arabidopsis seedlings could be restored by brassinolide (BL) but not by gibberellin (GA). Assessment of the target site of FM in BR biosynthesis by feeding BR biosynthesis intermediates indicated that FM interferes with the side chain hydroxylation of BR biosynthesis from campestanol to teasterone. Determination of the binding affinity of FM to purified recombinant CYP90D1 indicated that FM induced a typical type II binding spectrum with a Kd value of approximately 0.79 ?M. Quantitative real-time PCR analysis of the expression level of the BR responsive gene in Arabidopsis seedlings indicated that FM induces the BR deficiency in Arabidopsis.
Project description:Brassinosteroids (BRs) are a group of plant steroid hormones that play important roles in regulating plant development. In addition, BRs show considerable functional redundancy with other plant hormones such as gibberellins (GAs). BRASSINAZOLE RESISTANT1 (BZR1) and BRI1-EMS-SUPPRESSOR1 (BES1) transcription factors are negative feedback regulators of BR biosynthesis. This study provides evidence for the roles of MdBZR1 and MdBZR1-2like in promoting GA production. These results also show that BRs regulate GA biosynthesis to improve salt tolerance in apple calli. Moreover, this research proposes a regulatory model, in which MdBZR1 and MdBZR1-2like bind to the promoters of GA biosynthetic genes to regulate their expression in a BR-dependent manner. The expression of key GA biosynthetic genes, MdGA20ox1, MdGA20ox2, and MdGA3ox1 in yeast helps to maintain normal growth even under intense salt stress. In summary, this study underscores the roles of MdBZR1 and MdBZR1-2like in improving salt tolerance by regulating GA biosynthesis in apple calli.
Project description:Small increases in temperature result in enhanced elongation of the hypocotyl and petioles and hyponastic growth, in an adaptive response directed to the cooling of the leaves and to protect the shoot meristem from the warm soil. This response, collectively termed as thermomorphogenesis, relies on the faster reversion of phyB Pfr at warmer temperatures, which leads to enhanced activity of the basic-helix-loop-helix PHYTOCHROME INTERACTING FACTOR 4 (PIF4). PIF4 acts as a molecular hub integrating light and temperature cues with endogenous hormonal signaling, and drives thermoresponsive growth by directly activating auxin synthesis and signaling genes. Growth promotion by PIF4 depends on brassinosteroid (BR) signaling, as indicated by the impaired thermoresponse of BR-defective mutants and the partial restoration of pifq thermoresponsive defects by brassinolide (BL) application. Also, phyB limits thermomorphogenic elongation through negative regulation of the E3 ubiquitin ligase COP1 that triggers nuclear degradation of multiple photomorphogenesis-promoting factors acting antagonistically to PIF4. COP1 is indeed observed to accumulate in the nucleus in darkness, or in response to warm temperatures, with constitutive photomorphogenic cop1 mutants failing to respond to temperature. Here we explored the role of BR signaling on COP1 function, by growing cop1 seedlings on BL or the inhibitor brassinazole (BRZ), under different light and temperature regimes. We show that weak cop1 alleles exhibit a hyposensitive response to BL. Furthermore, while cop1-6 mutants display as described a wild-type response to temperature in continuous darkness, this response is abolished by BRZ. Application of this inhibitor likewise suppressed temperature-induced COP1 nuclear accumulation in N. benthamiana leaves. Overall these results demonstrate that cop1-6 is not a temperature-conditional allele, but this mutation allows for a partially active protein which unveils a pivotal role of active BR signaling in the control of COP1 activity.
Project description:Brassinosteroids (BRs) are essential for many biological processes in plants, however, little is known about their roles in early fruit development. To address this, BR levels were manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz) and their effects on early fruit development, cell division, and expression of cyclin and cyclin-dependent kinases (CDKs) genes were examined in two cucumber cultivars that differ in parthenocarpic capacity. The application of EBR induced parthenocarpic growth accompanied by active cell division in Jinchun No. 4, a cultivar without parthenocarpic capacity, whereas Brz treatment inhibited fruit set and, subsequently, fruit growth in Jinchun No. 2, a cultivar with natural parthenocarpic capacity, and this inhibitory effect could be rescued by the application of EBR. RT-PCR analysis showed both pollination and EBR induced expression of cell cycle-related genes (CycA, CycB, CycD3;1, CycD3;2, and CDKB) after anthesis. cDNA sequences for CsCycD3;1 and CsCycD3;2 were isolated through PCR amplification. Both CsCycD3;1 and CsCycD3;2 transcripts were up-regulated by EBR treatment and pollination but strongly repressed by Brz treatment. Meanwhile, BR6ox1 and SMT transcripts, two genes involved in BR synthesis, exhibited feedback regulation. These results strongly suggest that BRs play an important role during early fruit development in cucumber.
Project description:The expression of DWARF4 (DWF4), which encodes a C-22 hydroxylase, is crucial for brassinosteroid (BR) biosynthesis and for the feedback control of endogenous BR levels. To advance our knowledge of BRs, we examined the effects of different plant hormones on DWF4 transcription in Arabidopsis thaliana. Semi-quantitative reverse-transcriptase PCR showed that the amount of the DWF4 mRNA precursor either decreased or increased, similarly with its mature form, in response to an exogenously applied bioactive BR, brassinolide (BL), and a BR biosynthesis inhibitor, brassinazole (Brz), respectively. The response to these chemicals in the levels of ?-glucuronidase (GUS) mRNA and its enzymatic activity is similar to the response of native DWF4 mRNA in DWF4::GUS plants. Contrary to the effects of BL, exogenous auxin induced GUS activity, but this enhancement was suppressed by anti-auxins, such as ?-(phenylethyl-2-one)-IAA and ?-tert-butoxycarbonylaminohexyl-IAA, suggesting the involvement of SCF(TIR1)-mediated auxin signaling in auxin-induced DWF4 transcription. Auxin-enhanced GUS activity was observed exclusively in roots; it was the most prominent in the elongation zones of both primary and lateral roots. Furthermore, auxin-induced lateral root elongation was suppressed by both Brz application and the dwf4 mutation, and this suppression was rescued by BL, suggesting that BRs act positively on root elongation under the control of auxin. Altogether, our results indicate that DWF4 transcription plays a novel role in the BR-auxin crosstalk associated with root elongation, in addition to its role in BR homeostasis.