| In eukaryotic cell,a bilayered nuclear envelope(NE)sequesters all genetic material and separate transcriptional machinery of the nucleus from the translational machinery and metabolic events of the cytoplasm.This segregation facilitates the precise regulation of cellular processes and necessitates the intercommunication between these two compartments.Nuclear pore complexes(NPCs)Studded throughout the NE and provide permselective,bidirectional gateways for the exchange of molecules.Metal Ions and metabolites as well as small neutral proteins are allowed through the NPCs via pasive diffusion.In contrast,macromolecules greater than about 40 kDa usually complexd with nuclear transport receptors(NTRs)and then be actively transported through the NPCs.Two large evolutionarily conserved families of NTRs,karyopherin/importin(IMP)? and IMP? are required for most of the active transport pathways.In the well-characterized classical import pathway,IMP? recognizes the NLS containing substrates and subsequently forms a trimeric complex with IMP?.The interaction of IMP? with nucleoporins initiates the nuclear pore translocation.The functions of IMP? acts as nuclear import carrier adaptor that have been repeatedly reported in animal and fungal cells,little is known about it in plants.Recently,studies reported that the nucleocytoplasmic partitioning plays an important role in various biological processes,however,until now,the cellular and developmental processes directly associate with arabidopsis IMP? receptors,have been limitedly reported.In this study,we found that IMP?1,IMP?2 and IMP?3 play transport indepent roles,which function as transcriptional repressors involving in JA signaling pathway.The main results are as follows:(1)The IMP?1,IMP?2 and IMP?3 Function Redundantly to Negatively Regulate JA-induced Anthocyanin Accumulation.By phenotype screening of the single,double and triple mutants of imp?1,imp?2 and imp?3,we found that imp?1imp?2imp?3 triple mutant(TM)exhibited obvious increase in JA-induced anthocyanin accumulation and in expression of JA-regulated anthocyanin biosynthetic genes.However,there is no significant difference between WT and TM in sucrose or cytokinin(6-BA)induced anthhocyanin accumulation,respectively.(2)The Triple Mutant Exhibited increased JA-mediated Systemic Wound Reponse.Detailed analysis the systemic wound response showed that the wound-induced expression of the primary response genes in wild-type increased and peaked at 2h,which then declined gradually during the remainder of the time course.Whereas we observed that wound caused a sharp increase and peaked at 1h with significant higher expression level in the triple mutant.These results suggest that TM is more sensitive to simulated wound treatment than wild-type.(3)IMP?1,IMP?2 and IMP?3 P hysically Interact with MYC2.By yeast two hybrid screening,we found that besides karyopherins,the key b HLH transcription factor of JA signaling,MYC2,specifically interact with IMP?1,IMP?2,IMP?3,but not IMP?9.However,two closest homologs of MYC2,MYC3 and MYC4 did not interact with IMP?1,IMP?2 and IMP?3 in yeast.In addition,our results showed that IMP?2 wan able to interact with MYC2 in nucleus of N.benthamiana leaves using bimolecular fluorescence complementation(BiFC)assays.Moreover,intrac ellular distridution of MYC2 was not changed in TM.Using deletion assays,we identified that both the JAZ Interacting Domain(JID)and bHLH motif were able to interact with IMP?1,IMP?2,IMP?3,and Co-immunoprecipitation(Co-IP)assay confirmed that MYC2 N-terminal fragment(MYC2NT1-364)interact with IMP?2 in vivo,suggesting that the IMP?-MYC2 interaction is independent of the MYC2 NLS KRPKKRGRK433-441.IMP?296-535 containing 9 ARM-repeats,which did not interact with the empty vector,was responsible fo r binding to MYC2 and deletion mutants,further indicate that IMP ?-MYC2 interaction has no relation with the nuclear import.(4)IMP?1,IMP?2 and IMP?3 Interact with the MYB Transcription Factors of WD-Repeat/bHLH/MYB complexs.O ur results showed that IMP?1,IMP?2 and IMP?3 interact with MYB75 and MYB90 using Y2 H assays.IMP?2-MYB75 YFP signals were also detected in nucleus of N.benthamiana leaves.In addition,both the N-terminal fragments and C-terminal fragments of MYB75 were able to interact with IMP?1,IMP?2 and IMP?3.Further more the JAZ-MYB75 interactions were inhibited by IMP?1,IMP?2 and IMP?3.Taken together,we proposed that IMP?1,IMP?2 and IMP?3 may play a transport independent role in the interactions with MYB75 and MYB90.(5)IMP?1,IMP?2 and IMP?3 Inhibit Transcriptional Activity of MYC2 and MYB75.MYC2 acts as transcription activator to significantly induce expression of PTAT1-LUC and PVSP1-LUC,which was obviously enhanced or repressed by coexpression of MED25 and JAZ9,respectively.Coexpression of IMP?1,IMP?2 and IMP?3 repressed MYC2-activated PTAT1-LUC and PVSP1-LUC expression,respectively.Moreover,we found that IMP?296-535 was sufficient to inhibit the MYC2-activated PTAT1-LUC and PVSP1-LUC expression.MYB75,as the important transcription activator of JA-induced anthocyanin accumulation,significantly induced the expression of PDRF-LUC,which was obviously repressed by JAZ9,but not MED25.Similar to the repression effect of IMP? on MYC2,coexpression of IMP?1,IMP?2,IMP?3 and IMP?296-535 effectively inhibited the expression of PDFR-LUC.These results suggested that the IMP?1,IMP?2 and IMP?3 directly interact with these transcription factors to repress the transcriptional activity.(6)IMP?1,IMP?2 and IMP?3 interfer with the DNA binding activity of MYC2 and MYB75.Our results showed that interaction with IMP? proteins inhibit the G-box binding activity of MYC2 via Y1 H and EMSA assays.Similarly,we found that that binding of MYB75 to the PAP1 cis-regulatory element(PC E)was also inhibited by expression of IMP?2 and IMP?3,but not IMP?1.Thus IMP?1,IMP?2 and IMP?3 inhibit the transcriptional activity of MYC2 and MYB75 through interfering with their DNA binding activity.(7)IMP?1,IMP?2 and IMP?3 inhibit MYC2-MED25 and WD-Repeat/bHLH/MYB complex formation.Using Y3 H and pull-down assays,the results showed that the binding of MED25 to MYC2 was clearly disrupted by IMP?1,IMP?2 and IMP?3.Similarly,and IMP?1,IMP?2 and IMP?3 strongly inhibited MYB75 binding to TT8.Taken together,we proposed that IMP?1,IMP?2 and IMP?3 repress transcription activators through preventing the transcription initiation complex formation,as well.(8)IMP?1,IMP?2 and IMP?3 proteins negatively regulate the JA-induced Anthocyanin accumulation through d isruption the WD-Repeat/bHLH/MYB complex.We generated quadruple mutant by genetic cross the imp?1 imp?2 imp?3 with ttg1-lm.The increased JA-induced anthocyanin accumulation was completely disrupted in the quadruple mutant ttg1-lm imp?1 imp?2 imp?3(QM)under MeJA treament.The expression of the ‘late' anthocyanin biosynthetic genes(UF3GT,DFR)in the quadruple mutant decreased to a similar level with the ttg1-lm mutant.These results suggest that the increased JA-induced anthocyanin accumulation in TM requires the integrated WD-Repeat/bHLH/MYB complex. |
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