| Brassinosteroids(BRs)are essential polyhydroxylated phytohormones that are widely identified in plant kingdom and regulate multiple biological processes during plant growth and development.BRs induce the association of their cell surface-localized receptor BRI1 and co-receptor BAK1.The subsequent transphosphorylation between BRI1 and BAK1 kinase domain fully activates BRI1.The activated BRI1 then triggers downstream signaling cascade involves protein phosphorylation and dephosphorylation.As a result,transcription factors including BES1 and BZR1 are activated and their target genes are transcriptionally regulated,showing responses to BR stimuli.As BR receptor,BRI1 has been widely studied and over 30 different bri1 mutant alleles have been isolated which have helped to elucidate detailed molecular mechanisms of BRI1 in regulating early events of BR signaling pathway.During analyzing different bri1 mutants,we accidently discovered that the defective phenotype of bri1-301 is temperature-dependent.At lower temperature,such as 18?,bri1-301 showed subtle morphological defects compared to wild-type plants.When temperature rises to 22?,a weak BR-deficient phenotype is observed in bri-301.At a higher temperature,such as 28?,bri1-301 exhibits an extremely severe phenotype reminiscent to that of a null bri1 mutant.Detailed genetic,physiological and biochemical analyses indicated that the impaired BR signaling pathway by higher temperature in bri1-301 lead the mutant severe defective phenotype when grown at 28?.The abundance of BRI1 protein in bri1-301 is significantly lower than that in wild type plants and can be further decreased by higher temperature.In an in vivo kinase assay,bri1-301 protein was found to act as a functional receptor with reduced kinase activity that is sufficient for BR perception and signaling initiation at least under a relatively low temperature condition such as 18?.This result clarifying the previously reported contradiction that bri1-301 kinase is biochemically dead in vitro but bri1-301 mutant is a waek allele.In addition,our data demonstrated that higher temperaturecaused bri1-301 degradation is independent of N-glycan-based ERQC.BAK1 plays an indispensable role during the BR signaling initiation.The loss-offunction mutant of BAK1,bak1-4,exhibits a decreased sensitivity to BRs.In order to identify new components modulating BR signaling pathway,we screened for genetic modifiers of bak1-4.ERIB1 gene was identified as a potential regulator in mediating BR signaling.ERIB1 encodes a cytoplasmic protein that is widely expressed.Loss-offunction of ERIB1 in bak1-4 can fully suppress its reduced sensitivity to BL,the most bioactive form and final biosynthetic product of BRs,in root growth inhibition assay.Compared to wild type,the root growth of erib1 single mutant is slower and is much more sensitive to BL than Auxin and Cytokinin.Consistently,induction of BES1 dephosphorylation and target-gene feedback regulation by BL is quicker in erib1 than in wild type.Disrupting the BR signaling by loss-of-function of the receptor BRI1 or activation of the negative regulator BIN2 in erib1 mutant eliminates its root hypersensitivity to exogenous BL.Careful cellular profiling of root apical meristem(RAM)indicates that in erib1,the quiescent center(QC)division is activated and the meristematic cortical cells are prematurely switched from division to differentiation and elongation.The erib1 RAM and QC phenotypes are similar to what have been reported in BL-treated Col-0 roots.The results above imply an unknown temperature-sensitive mechanism that stabilizes BRI1 conformation during the ligand perception and self-activation.bri1-301,however,may escape from or mistakenly regulated by this mechanism due to the G989 I mutation.Moreover,although the detailed molecular mechanism is still remains undiscovered,our results suggest that ERIB1 regulates root growth and development by constraining BR signaling in Arabidopsis root apical meristem. |
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