A novel mitochondrial DnaJ/Hsp40 family protein BIL2 promotes plant growth and resistance against environmental stress in brassinosteroid signaling.
ABSTRACT: Plant steroid hormones, brassinosteroids, are essential for growth, development and responses to environmental stresses in plants. Although BR signaling proteins are localized in many organelles, i.e., the plasma membrane, nuclei, endoplasmic reticulum and vacuole, the details regarding the BR signaling pathway from perception at the cellular membrane receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) to nuclear events include several steps. Brz (Brz220) is a specific inhibitor of BR biosynthesis. In this study, we used Brz-mediated chemical genetics to identify Brz-insensitive-long hypocotyls 2-1D (bil2-1D). The BIL2 gene encodes a mitochondrial-localized DnaJ/Heat shock protein 40 (DnaJ/Hsp40) family, which is involved in protein folding. BIL2-overexpression plants (BIL2-OX) showed cell elongation under Brz treatment, increasing the growth of plant inflorescence and roots, the regulation of BR-responsive gene expression and suppression against the dwarfed BRI1-deficient mutant. BIL2-OX also showed resistance against the mitochondrial ATPase inhibitor oligomycin and higher levels of exogenous ATP compared with wild-type plants. BIL2 participates in resistance against salinity stress and strong light stress. Our results indicate that BIL2 induces cell elongation during BR signaling through the promotion of ATP synthesis in mitochondria.
Project description:Brassinosteroids (BRs), plant steroid hormones, play important roles in plant cell elongation and differentiation. To investigate the mechanisms of BR signaling, we previously used the BR biosynthesis inhibitor Brz as a chemical biology tool and identified the Brz-insensitive-long hypocotyl4 mutant (bil4). Although the BIL4 gene encodes a seven-transmembrane-domain protein that is evolutionarily conserved in plants and animals, the molecular function of BIL4 in BR signaling has not been elucidated. Here, we demonstrate that BIL4 is expressed in early elongating cells and regulates cell elongation in Arabidopsis. BIL4 also activates BR signaling and interacts with the BR receptor brassinosteroid insensitive 1 (BRI1) in endosomes. BIL4 deficiency increases the localization of BRI1 in the vacuoles. Our results demonstrate that BIL4 regulates cell elongation and BR signaling via the regulation of BRI1 localization.
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:Plant steroid hormones, brassinosteroids (BRs), play important roles in plants. BRs regulate the expression of several thousand genes, half of which are induced and the other half repressed by the hormone. BRs signal through plasma membrane-localized receptor kinase brassinosteroid-insensitive 1 (BRI1), BRI1-associated receptor kinase (BAK1), and several intermediates to regulate the protein levels, cellular localizations, and/or DNA binding of BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1 (BZR1) family transcription factors. Although BES1 is known to interact with other transcription factors, histone-modifying enzymes, and transcription elongation factors to activate BR-induced genes, how BES1 mediates the BR-repressed gene expression is not known. Here, we show that BES1 interacts with myeloblastosis family transcription factor-like 2 (MYBL2), a transcription repressor, to down-regulate BR-repressed gene expression. The loss-of-function mybl2 mutant enhances the phenotype of a weak allele of bri1 and suppresses the constitutive BR-response phenotype of bes1-D. The results suggest that suppression of BR-repressed gene expression is required for optimal BR response. Moreover, MYBL2 is a substrate of glycogen synthase kinase 3 (GSK3)-like kinase brassinosteroid-insensitive 2 (BIN2), which has been well established as a negative regulator in the BR pathway by phosphorylating and inhibiting the functions of BES1/BZR1. Unlike BIN2 phosphorylation of BES1/BZR1 leading to protein degradation, BIN2 phosphorylation stabilizes MYBL2. Such dual role of phosphorylation has also been reported in WNT signaling pathway in which GSK3 phosphorylation destabilizes ?-catenin and stabilizes Axin, a scaffolding protein facilitating the phosphorylation of ?-catenin by GSK3. Our results thus establish the mechanisms for BR-repressed gene expression and the integration of BR signaling and BR transcriptional network.
Project description:Brassinosteroids (BRs) are plant hormones that regulate many processes including cell elongation, leaf development, pollen tube growth and xylem differentiation. GSK3/shaggy-like kinases (GSK) are critical regulators of intracellular signalling initiated by the binding of BR to the BRI1 receptor complex. Three GSKs have already been shown to relay BR responses, including phosphorylation of the transcriptional regulator BES1. However, recent studies indicate that one or more yet unidentified protein kinases are involved in BR signalling. Here, we show that the in vivo protein kinase activity of the group-III GSK, ASKtheta, was negatively regulated by BRI1. Arabidopsis thaliana plants with enhanced ASKtheta activity displayed a bri1-like phenotype. ASKtheta overexpressors accumulated high levels of brassinolide, castasterone and typhasterol, and were insensitive to BR. ASKtheta localized to the nucleus and directly phosphorylated BES1 and BZR1. Moreover, the BES1/BZR1-like transcription factor BEH2 was isolated as an ASKtheta interaction partner in a yeast two-hybrid screen. ASKtheta phosphorylated BEH2 both in vitro and in vivo. Overall, these data provide strong evidence that ASKtheta is a novel component of the BR signalling cascade, targeting the transcription factors BES1, BZR1 and BEH2.
Project description:bri1-5 is a weak mutant of Brassinosteroid Insensitive 1 (BRI1). Suppressors by activation tagging bri1-1D, brs1-1D and bak1-1D can recover bri1-5 phenotype. We use microarray to investigate which pathways or functional categories have been transcriptionally regulated by bri1-1D, brs1-1D and bak1-1D. Whole seedlings from wild-type (WS2), bri1-5, bri1-5/brs1-1D, bri1-5/bak1-1D, bri1-5/bri1-1D. Three biological replicates for each genotype.
Project description:bri1-5 is a weak mutant of Brassinosteroid Insensitive 1 (BRI1). Suppressors by activation tagging bri1-1D, brs1-1D and bak1-1D can recover bri1-5 phenotype. We use microarray to investigate which pathways or functional categories have been transcriptionally regulated by bri1-1D, brs1-1D and bak1-1D. Overall design: Whole seedlings from wild-type (WS2), bri1-5, bri1-5/brs1-1D, bri1-5/bak1-1D, bri1-5/bri1-1D. Three biological replicates for each genotype.
Project description: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:<h4>Background</h4>N-ethyl-maleimide sensitive factor adaptor protein receptor (SNAREs) domain-containing proteins were known as key players in vesicle-associated membrane fusion. Genetic screening has revealed the function of SNAREs in different aspects of plant biology, but the role of many SNAREs are still unknown. In this study, we have characterized the role of Arabidopsis Qc-SNARE protein AtBS14b in brassinosteroids (BRs) signaling pathway.<h4>Results</h4>AtBS14b overexpression (AtBS14b ox) plants exhibited short hypocotyl and petioles lengths as well as insensitivity to exogenously supplied BR, while AtBS14b mutants did not show any visible BR-dependent morphological differences. BR biosynthesis enzyme BR6OX2 expression was slightly lower in AtBS14b ox than in wild type plants. Further BR-mediated repression of BR6OX2, CPD and DWF4 was inhibited in AtBS14b ox plants. AtBS14b-mCherry fusion protein localized in vesicular compartments surrounding plasma membrane in N. benthamiana leaves. In addition, isolation of AtBS14b-interacting BR signaling protein, which localized in plasma membrane, showed that AtBS14b directly interacted with membrane steroid binding protein 1 (MSBP1), but did not interact with BAK1 or BRI1.<h4>Conclusion</h4>These data suggested that Qc-SNARE protein AtBS14b is the first SNARE protein identified that interacts with MSBP1, and the overexpression of AtBS14b modulates BR response in Arabidopsis.
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.
Project description:Meloidogyne incognita and Meloidogyne graminicola are root-knot nematodes (RKNs) infecting rice (Oryza sativa L.) roots and severely decreasing yield, whose mechanisms of action remain unclear. We investigated RKN invasion and development in rice roots through RNA-seq transcriptome analysis. The results showed that 952 and 647 genes were differently expressed after 6 (invasion stage) and 18 (development stage) days post inoculation, respectively. Gene annotation showed that the differentially expressed genes were classified into diverse metabolic and stress response categories. Furthermore, phytohormone, transcription factor, redox signaling, and defense response pathways were enriched upon RKN infection. RNA-seq validation using qRT-PCR confirmed that CBL-interacting protein kinase (CIPK) genes (CIPK5, 8, 9, 11, 14, 23, 24, and 31) as well as brassinosteroid (BR)-related genes (OsBAK1, OsBRI1, D2, and D11) were altered by RKN infection. Analysis of the CIPK9 mutant and overexpressor indicated that the RKN populations were smaller in cipk9 and larger in CIPK9 OX, while more galls were produced in CIPK9 OX plant roots than the in wild-type roots. Significantly fewer numbers of second-stage infective juveniles (J2s) were observed in the plants expressing the BR biosynthesis gene D2 mutant and the BR receptor BRI1 activation-tagged mutant (bri1-D), and fewer galls were observed in bri1-D roots than in wild-type roots. The roots of plants expressing the regulator of ethylene signaling ERS1 (ethylene response sensor 1) mutant contained higher numbers of J2s and developed more galls compared with wild-type roots, suggesting that these signals function in RKN invasion or development. Our findings broaden our understanding of rice responses to RKN invasion and provide useful information for further research on RKN defense mechanisms.