Hydrogen Peroxide Positively Regulates Brassinosteroid Signaling Through Oxidation Of The BZR1 Transcription Factor

Reactive oxygen species(ROS)are initially considered to be toxic by-products that accumulated under stress conditions and cause irreversible damage to cells.Recently,ROS,especially hydrogen peroxide(H2O2),are recognized as important signaling molecules that regulate plant growth and stress responses,but the H2O2 signaling pathway and the molecular mechanism of H2O2 crosstalk with hormones are largely unclear.Brassinosteroid(BR)is an important plant steroid hormone that plays fundamental roles in numbers of plant growth,developmental processes and stress responses.In this study,we combine advantage of the biochemistry,molecular biology,genomics and genetics tools to gain a deep insight into the mechanism of H2O2 and BR crosstalk in regulation of plant cell elongation.The main results are summarized as below:1.BR induces H2O2 accumulation in plants.Exogenous BR treatment leads to H2O2 accumulated in wild type Col-0 and BR deficient mutant rot3-2,but not in BR insensitivity mutant bril-116.In addition,NADPH oxidase inhibitor Diphenylene iodonium(DPI)attenuates the BR effect on the accumulation of H2O2,suggesting that BR signaling through BRI1 triggers the production of H2O2 through a NADPH-dependent pathway.2.H2O2 is required for the BR promotion of cell elongation.To evaluate the potential roles of H2O2 in BR-mediated seedling development,we analyze BR regulation of hypocotyl elongation under the H2O2-deficient condition.Our results show that DPI treatment,which reduces the H2O2 level in plants,significantly inhibites the effects of BR on hypocotyl elongation,with high concentrations of DPI causing seedlings insensitivity to BR.H2O2 effectively restores the BR sensitivity in DPI-treated plants in a dose-dependent manner,indicating that the accumulation of H2O2 is required for BR-induced hypocotyl elongation.To corroborate these pharmacological data,we analyze the effects of H2O2 on BR-induced cell elongation in the rbohD rbohF double mutant and catalase overexpressing plants(CAT2-Ox),which are defective in normal production of H2O2.The results show that rbohD rbohF and CAT2-Ox all show BR hyposensitivity in hypocotyl elongation.Together,these results demonstrate that the BR-triggered production of H2O2 contributes to the BR-promoted cell elongation.3.H2O2 contributes to BR signaling independing on BIN2.The GSK3 kinase BIN2 is a key negative regulator in BR signal transduction,and is involved in several other signaling pathways by interacting with and/or phosphorylating additional substrates.Lithium chloride(LiCl)and bikinin inhibite GSks kinase activity.The kinase activity of BIN2 is also recently reported to be inhibited by nitric oxide(NO)in a dose-dependent manner.H2O2 and NO sometimes share common target proteins to transduce redox signals.To determine whether H2O2 regulates BR signal transduction through BIN2,we examine the effects of H2O2 on the cell elongation phenotype under LiCl,bikinin treatment and in the bin2 bill bill mutant.The results show that LiCl,bikinin and bin2 bill bil2 mutant significantly increase the cell elongation,as observed upon genetic or ligand-induced activation of the BR pathway.In contrast removal of H2O2 by DPI treatment decreases the hypocotyl length,suggesting that involvement of H2O2 in the BR signaling pathway is likely independent of BIN2 and its homologous proteins.Further confirmation of the crosstalk between H2O2 and BR downstream of BIN2 is obtained by analyzing cell elongation in BZR1?DM-Ox,bzr1-1D and bes1-D.These plants exhibit longer hypocotyl than wild-type plants,while DPI treatment inhibits the phenotype.Together,these genetic and physiological data suggest that the H2O2 regulates the BR activity downstream of BIN2,and probably through the transcription factors BZR1 and BES1.4.We carries out RNA-Seq analysis to determine whether H2O2 affects the BZR1-mediated gene expression in Arabidopsis genome.Wild-type and bzr1-1D mutant seedlings are grown in the dark for 6 days on the medium containing PPZ,and/or DPI,and/or H2O2.RNA-Seq analysis identifies 4428 genes regulaed by BZR1 in the mock condition,among these genes,the expression of 2834 genes(64%)is no longer responsive to BZR1 in the DPI-treated H2O2-deficient condition,while 1382 genes(48%)out of the 2834 genes recover responsiveness to BZR1 when H2O2 is added back to the H2O2-deficient condition.Scatter plot analysis show that the slope of the trend line in the plot comparing BZR1-regulated genes in the mock condition with that in the DPI condition is only 0.67,but is restored to 0.87 in the plot comparing the BZR1 response genes in the mock condition with that in the DPI plus H2O2 condition suggesting H2O2 plays a positive role in BZR1-regulated gene expression.To further define H2O2-dependent BZR1-regulated genes,we analyze the genes differentially expressed in bzr1-1D mutant with or without DPI treatment,among the 4428 genes affected by bzr1-1D in comparison with wild-type,2200 genes(49.7%)were also affected by DPI treatment,which we named H2O2-dependent BZR1-regulated genes.Among the 2200 H2O2-dependent BZR1-regulated genes,2053 genes(93.3%)are regulated in the opposite way by DPI and bzr1-1D,confirming that H2O2 promotes the transcriptional activity of BZR1.5.H2O2 do not affect the protein stability,phosphorylation level,and subcellular localization of BZR1.BZR1 is a key transcription factor in BR signaling pathway.Currently,the mainly activity regulation of BZR1 includes phosphorylation and dephosphorylation,protein stability and subcellular localization.To learn how H2O2 regulates the activity of BZR1,we examined whether H2O2 affects the subcellular localization,protein levels,and phosphorylation status of BZR1 by analyzing BZR1-YFP proteins in the pBZR1:BZR1-YFP transgenic plants.The results show that BR treatment induces the transport of BZR1 from the cytoplasm to the nucleus regardless of DPI presence,suggesting that reducing the content of H2O2 does not affect BR-regulated nucleo-cytoplasmic shuttling of BZR1.Treatment with BR also causes dephosphorylation of BZR1,but co-treatment with H2O2 or DPI had no obvious effects on BZR1 protein level and phosphorylation state.These results indicated that disruption of H2O2 homeostasis does not have a significant effect on BR levels or BR signaling upstream of BZR1.6.H2O2 does not affect the DNA binding ability of BZR1.BZR1,as a transcription factor,the DNA binding ability is associate with the transcriptional activity.To determine whether H2O2 affects the DNA-binding ability of BZR1,we perform DNA-protein pull-down and chromatin immunoprecipitation quantitative PCR(ChIP-qPCR)assays.The results show that BZR1 specifically binds to the promoter of SAUR15 and DWF4,which are known BZR1-binding target genes.H2O2 treatment does not alter the binding activity of BZR1 to the promoter of SAUR15 and DWF4 in vivo and in vitro,suggesting that H2O2 does not affect DNA-binding ability of BZR1.7.H2O2 induces the oxidative modification of BZR1.The direct regulatory effects exerted by H2O2 are mediated by posttranslational modification of target proteins through cysteine oxidative modification.To test whether H2O2 induces oxidation of BZR1,we perform the BIAM-labeling assay,Biotin-switch assay and liquid chromatography tandem mass spectrometry to test the oxidation level of BZR1 in vivo and in vitro.The results show that H2O2 causes oxidation of BZR1 both in vivo and in vitro.Further experiments indicate that MBP-BZR1C63S show a very weak oxidative modification,suggesting the H2O2-induced oxidation occurs mainly on Cys-63 of BZR1.Moreover,we show that mutagenesis of Cys-84 in BES1,which is equivalent to the conserved residue Cys-63 in BZR1 protein,effectively prevents the oxidation of BES1 by H2O2 treatment,suggesting that the conserved Cys-63 and Cys-84 are the major in vitro oxidized sites for BZR1 and BES1,respectively.8.Cys-63 is important for the functions of BZR1.To understand whether oxidation of Cys-63 modulates the function of BZR1 in plants,we generate transgenic Arabidopsis plants expressing BZR1P234L(bzr1-1D),BZR1P234LC63S(bzr1-1DC63S),BZR1P234LC63,73S(bzr1-1DC63,73S),BZR1P234LC91,174,240S(bzr1-1DC91,174,240S)and BZR1P234LC63,73,91,174,240S(bzrl-1D5S).Transgenic lines expressing similar protein levels of BZR1 are selected for phenotype analysis.Plants expressing bzr1-1D and bzr1-1DC91,174,240S show BR-activation phenotypes similar to the bzr1-1D mutant,with curled leaves and insensitivity to BR biosynthesis inhibitor PPZ.Transgenic plants expression bzr1-1DC63S,bzr1-1DC63,73S and bzr1-1D5S display normal leaf phenotypes and increased sensitivity to PPZ.Moreover,conversion of the cysteine-84 of besl-D to serine also suppresses the phenotypes of besl-D.Together,these results demonstrate that the conserved Cys-63 of BZR1 and Cys-84 of BES1 are essential for functions in various BR-mediated responses.Next,to test the effects of cysteine residues on BZR1 regulation of the target genes expression,we perform q-RT PCR and transient gene expression assays.The results show that the transcriptional activity of bzr1-1DC63S and bzr1-1D5S are lower than bzr1-1D.These results indicate that mutagenesis of oxidized cysteine residues decreases the transcriptional activity of BZR1.9.Oxidation promotes BZR1 binding with PIF4 and ARF6.The enrichment of PIF and ARF-bound/regulated genes among the H2O2-dependent BZR1-regulated genes suggests that H2O2 may affect the interactions between BZR1 and PIF4 or ARF6.To test this hypothesis,we perform GST pull-down,ratiometric bimolecular fluorescence complementation(rBiFC)and co-immunoprecipitation(co-IP)assays to analyze the effects of H2O2 on the binding affinity of BZR1 to PIF4 and ARF6.The results show that H2O2 clearly enhances the interactions between BZR1 and PIF4 or ARF6 both in vivo and in vitro.Together,these results demonstrate that H2O2-induced oxidation of BZR1 promotes its interactions with PIF4 and ARF6.In summary,H2O2 induces the oxidation of the BZR1 transcription factor,which functions as a master regulator of BR signaling.Oxidative modification enhances BZR1 transcriptional activity by promoting its interaction with key regulators in the auxin-signaling and light-signaling pathways,including ARF6 and P1F4.Genome-wide analysis shows that H2O2-dependent regulation of BZR1 activity plays a major role in modifying gene expression related to several BR-mediated biological processes.This study provides a new evidence for further exploring the molecular mechanism of H2O2 in regulating plant growth and adversity response.In addition,H2O2 participates in BR signal transduction process,which also greatly enriches and expands BR regulatory network.It provides a new basis for better elucidating the mechanism of plant growth and environmental adaptation.