AtPRP17 Functions In The Regulation Of Root And Embryo Development In Arabidopsis Thaliana

The COP9 signalosome（CSN）is involved in many developmental processes in plants by interacting with different types of Cullin–RING E3 ubiquitin ligases（CRLs）,such as photomorphogenesis,flower development,auxin signaling and gibberellin signaling pathway.Nevertheless,how COP9 signalosome activity is regulated in Arabidopsis remains unknown.CSN4,a subunit of COP9 signalosome,was used as a bait protein,and its interaction protein AtPRP17 was identified by yeast two-hybrid assay.To further explore the relationship between AtPRP17 and the COP9 signalosome activity,we used atprp17 mutants as research subjects to elucidate the molecular mechanisms by which AtPRP17 regulates root apical meristem maintenance and embryo development using cellular,genetic and biochemical techniques.The main results are as follows:（1）AtPRP17 interacts with CSN4,and CSN4 is epistatic to AtPRP17.Yeast two-hybrid assay indicated that AtPRP17 interacted with CSN4,which was further confirmed by bimolecular fluorescence complementation,and protein immunoprecipitation.Genetic experiments showed that CSN4 is epistatic to AtPRP17 in regulating root elongation.（2）AtPRP17 functions in the maintenance of Arabidopsis root apical meristem.The atprp17 mutants exhibited shorter primary root and smaller root apical meristem compared with that of the wild type.In addition,the differentiation of columella initials is abnormal in atprp17 mutants,and the signal of p WOX5::GFP specifically expressed in QC（Quiescent Center）cells was significantly enhanced,and the expression scope was significantly enlarged.These indicate that AtPRP17 functions in the development of Arabidopsis root apical meristem by affecting the maintenance of QC.（3）AtPRP17 is involved in auxin signaling pathway though regulating the activity of COP9 signalosome.COP9 signalosome mediates deneddylation of CUL1 subunit of SCF^TIR1 type E3ubiquitin ligase,and affecting the activity of E3 ubiquitin ligase,which regulats AUX/IAA proteins degradation and plays a role in auxin signaling.The deneddylation activity of COP9signalosome was reduced and AUX/IAA proteins was degraded more slowly in atprp17mutants.Transcriptome analysis showed that AtPRP17 was involved in regulating the expression of auxin-related genes.Moreover,the results of high temperature treatment,external application of IAA,expression of DR5::GFP and p PIN1::PIN1-GFP in atprp17mutant indicated that AtPRP17 is involved in auxin signaling pathway.（4）AtPRP17 plays roles in the alternative splicing of CSN3 and CSN7 as a splicing factor.The results of In-GFP reporter gene assay indicated that AtPRP17 affected the splicing process of pre-m RNA in plants.Subcellular localization analysis showed that AtPRP17 was located in the nuclear speckles.All these results indicate that AtPRP17 is a splicing factor.A total of 280 genes showed abnormal alternative splicing in atprp17-1,including CSN3 and CSN7 encoding the COP9 signalosome subunits.（5）AtPRP17 plays an important role in Arabidopsis embryo development.atprp17 mutants showed shrunken seeds in mature siliques.From the 5 days after pollination,the mutant embryos showed delayed development,short cotyledon primordium,bigger cotyledon angle,and disordered cell arrangement in the embryo base region,which indicated that the embryonic pattern in the atprp17-1 mutant was changed.In summary,AtPRP17 is involved in the maintenance of Arabidopsis root apical meristem through two pathways:one pathway is the interaction between AtPRP17 and CSN4,and the other is the involvement of AtPRP17 in the alternative splicing of CSN3 and CSN7 as a splicing factor.Both of them can affect COP9 signalosome activity,which affects the degradation rate of AUX/IAA proteins and plays a role in the maintenance of the root apical meristem of Arabidopsis though auxin signaling pathway.AtPRP17 also functions in embryo development by influencing the patterning of the apical and base domains of embryos.