The Molecular Genetical And Biochemical Study And Function Investigation Of 26S Proteasome Subunit RPN1a In Arabidopsis

The plant growth and development have to synthesize functional protein accurately and timely,removing proteins that unneeded in time.The proteasomal degradation pathway is essential for many cellular processes,widely involve in hormone signal transduction,cell cycle regulation,stress responses,and almost every process by degradating protein selectively.The 26S proteasome is an ATP dependent,and multiple-subunits assembled huge complex,which is constructed by 19S particles(the regulatory particle,the RP)and 20S core particles(core protease,CP),that widespread in plant cells.The RP accomplish substrate recognition,remove ubiquitin from substrate proteins,unfold the substrates,open the blocks in the 20S,and guide substrates into the degradation chamber for degradation in the presence of ATP.The degradation of RP is specific,that brings finely and precisely proteasomal degradationr during plant growth and development.The specific functions of most RP is not yet clear at present,only a fewer reports show that some RP subunit responsible for plant stress adaption,related mechanisms are not very clear.The functional study of RP subunit is of great importance to reveal the detailed mechanism.Use the RPN1a for bait to screen the Arabidopsis whole genome plasmid library by yeast two hybrid method,we found that the key ABA(Abscisic acid)signaling protein ABI1 interacts with RPN1a.We identified the RPN1A T-DNA insertion homozygotes,proved that RPNla negatively regulate the ABA signaling pathway,and found that the insertion mutants have more trichomes,and the branch number were increased.Further research show that RPN1a involved in trichomes development through the GA and CK signaling pathways.This study focus on the functional study of RPN1a in ABA signaling pathway and in trichomes development,detailed results are as follows:(1)Searched the RPN family members in the Arabidopsis thaliana database(Tair)for bioinformatics analysis.The results showed that the RPN family members distribute varying in chromosome,share low sequence similarity in amino acid,constructed by uncertain number of exons and introns.The RPN family members were evolutionary diversified.There was no signal peptide,no obvious hydrophobic area,and no transmembrane domain.RPNla is not a membrane protein.In accordance with the bioinformatics results,the RPN1a fusion protein with green fluorescent protein label driven by its own promoter expressed everywhere.(2)Characterization of transcriptional regulatory elements in the promoter region of RPNla show that there were number of stress related and plant hormone related transcriptional regulatory elements,implying RPN1a involve in plant stress response.After screen a cDNA library by the yeast two-hybrid system,using RPN1a as the bait,we found that ABI1 interact with RPN1a,further confirmed by yeast two-hybrid system and bimolecular fluorescence complementation(BiFC).Further research show that RPNla could not interact with PYR1 and PYL1,RCAR1,ABI2,HAB1,PP2CA,SnRK2.2,SnRK2.3 and OST1,suggesting that RPN1a might involved in ABA signaling pathways through the interactions with ABI1.(3)The mRNA and protein level of RPN1a were supressed by ABA treatment.The rpnla mutants were hypersensitive to ABA,the seed germinate,post-germinate development and root elongation were more suppressed by ABA treatment in rpn1a mutants.The stomata close is more rapid in rpn1a than in WT,RPN1a fusion protein driven by its own promoter expresses much more in guard cells than the surrounding cells,and the transcript levels of key ABA signaling pathways genes increased in rpn1a mutants,suggesting that RPN1a negatively regulate the ABA signaling pathways.Further study show that ABI5 accumulated more in rpn1a mutants than in WT,but RPN1a can not interact with ABI5 directly,which suggest that RPN1a interact with ABI1 to regulate the abundance of ABI5 to participate in regulating the ABA signaling pathways.(4)Exogenous GA(Gibberellic acid)and CK(Cytokinin)induce the epidermal cells formation into trichomes in Arabidopsis thaliana,ZFP6(zinc finger protein 6)integrates the gibberellin and cytokinin signaling to regulate the initiation of trichomes.Driven by its own promoter,RPN1a-GFP fusion protein accumulated more in trichomes initiating differentiation stage cells,and in branch developing stage cells in the nucleus and cytoplasm than in surrounding cells.When treated by exogenous GA and 6-BA the transcripts of RPN1a were supressed,the transcription level of the trichomes initiation and trichomes branch formation key genes altered in rpn1a.Together proving that:RPN1a participate in trichomes initiating by suppressing the expression of ZFP6 and involved in trichomes branching by regulating the expression of ZFP6,PYM and KAK.