Molecular Mechanism Of Ubiquitin-conjugating Enzyme OsUBC11 On Root Development Of Rice

The root system is the main organ for plants to absorb nutrients and water.Its development is an important process during plant growth.The functions of root system include absorbing nutrients and water from the soil,taking roots in the soil to prevent lodging,and serving as the main acceptor interface between plants and various biotic and abiotic factors in the soil environment.Crop root development ultimately determines crop yield.Therefore,it has been one of the focuses of rice breeders to identify the genes regulating root development and verify their functions.Auxin plays a role in inducing and regulating plant root growth and development during plant growth and development,and its signal regulation pathway is significantly associated with ubiquitination pathway.The ubiquitination pathway is mainly involved in biological processes such as hormone signal transduction,flowering development,and photomorphogenesis.Ubiquitin-conjugating enzymes are important components of plant ubiquitination.Their main functions are to recruit downstream E3 ubiquitin ligases and form complexes with them to make ubiquitin molecules closer to substrate proteins,so that target proteins can be labeled and finally complete the ubiquitination process.Among them,Ubiquitin complex SCF^TIR1E3 disaggregates auxin signal repressor protein AUX/IAA,and eventually disaggregates the AUX/IAA-ARF heterodimer,and releases ARF and activating auxin regulatory signals.The regulatory relationship between ubiquitination pathway and auxin has been well studied,but the relationship between ubiquitin-conjugating enzymes and root development was rarely reported.In this study,a short root mutant,R164,was identified by a large scale screening of T-DNA insertion mutant libraries.Self-formed adaptor PCR（SEFA-PCR）and sequencing alignment identified four tandem 35S enhancer elements of T-DNA inserted in the promoter region of a ubiquitin-conjugating enzyme OsUBC11 gene,which activated the transcription of this gene.Further studies showed that OsUBC11 was involved in the auxin pathway.It also affected the expression of other root development genes to regulate root development.\We got the following results:1.OsUBC11 protein has a conserved UBC domain and is a key component of ubiquitin-binding enzymes.There is a homologous gene Os UBC12 in rice and three homologous genes in Arabidopsis,namely At UBC7,At UBC13 and At UBC14,but the specific functions of these homologous genes have not been reported.2.The results of tissue expression analysis showed that OsUBC11 was mainly expressed in roots.A stable transgenic line was constructed by fusing the promoter of OsUBC11 and GUS reporter.The expression of OsUBC11 was the highest in rice roots,suggesting that OsUBC11 may be a key gene regulating root development.3.OsUBC11 was found to be a nuclear localization gene in rice protoplasts by constructing OsUBC11-GFP and transforming it into rice protoplasts.4.Ubiquitin-conjugating enzymes mainly bind ubiquitin molecules through their conserved cysteine sites and complete their ubiquitination function.In this study,OsUBC11-His and mutant recombinant proteins were obtained by point mutation and prokaryotic expression.Western blot results showed that the deletion of the conservative cysteine site of OsUBC11 lost its ubiquitination activity,indicating that OsUBC11 had ubiquitin conjugating enzyme activity.At the same time,the K48amino acid mutation of ubiquitin molecule also prevented OsUBC11 from forming ubiquitin cascade.These results indicated that OsUBC11 was ubiquitinated by degradation of downstream target proteins through K48 polyubiquitination pathway.5.Overexpression of OsUBC11 inhibited primary and lateral root development,similar to the phenotype of activating mutant R164.Exogenous auxin treatment could partially restore the phenotype.The auxin concentration in roots was significantly decreased in the overexpression line and the activating mutant R164.These results indicated that overexpression of OsUBC11 inhibited root development by reducing auxin accumulation in roots.These results indicated that OsUBC11 was involved in auxin pathway to regulate root development.6.Overexpression of OsUBC11 inhibited the transcription expression of auxin synthesis genes（family）Os TAA1,Os YUCCA4,Os YUCCA6 and Os YUCCA9,and the auxin intracellular transport regulatory genes Os AUX1 and Os AUX4,and auxin signal regulator Os IAA31,Os ARF16 and Os ARF25.These results suggest that OsUBC11may regulate rice root development through auxin synthesis,transport,or regulating the transcription level of auxin regulators.In addition,the transcription level of OsUBC11 was significantly inhibited under the transient treatment of 1μM naphthoacetic acid,and the transcriptional level measurements were consistent with the results of GUS staining,indicating that high concentration of auxin may feedback inhibit the expression of OsUBC11.The relationship between OsUBC11 and auxin was further confirmed.7.Furthermore,the transcriptional levels of Os CRL1,Os CRL4,Os CRL5,Os CKX4 and Os WOX11,which have been reported to be involved in root development,were significantly down-regulated in OsUBC11-overexpressing lines.These results suggested that OsUBC11 affected or cooperated with other root development key genes to control root development in rice.8.In addition,overexpression or activation of OsUBC11 not only regulated root development,but also significantly reduced plant height by affecting the length of the top internode,reduced fertile pollen content and set rate,and ultimately reduced grain yield.These results indicate that OsUBC11 is a conserved ubiquitin-binding enzyme.Overexpression of OsUBC11 inhibits root development,plant height and seed setting rate of rice seedlings by affecting the auxin signaling pathway,with significant effects on rice yield.