Functional Analysis Of The Arabidopsis Ubiquitin-specific Protease Family

Ubiquitin-Specific Protease(UBP) genes are highly conserved in eukaryotes and play critical roles in cellular protein de-ubiquitination.Based on the presence of conserved Cys and His boxes,an in silico analysis of the Arabidopsis genome has revealed a family of 27 predicted UBP genes,and they can be further divided into 14 subfamilies.Although several of them have been studied,until now there is a lack of a systematic analysis of the expression and the developmental roles of all the members in the gene family.Further,no effort has been reported to address the functional relationship and redundancy among the gene family members.Here we report a systematic genetic and expression profiling analysis of the 27 UBP genes.We perform microarray assay to obtain detailed expression profiles of the genes. Based on the results,an analysis of 38 available T-DNA insertion lines in 25 specific UBP genes covering all 14 subfamilies shows that only loss-of-function mutants in 3 genes in 2 subfamilies exhibited noticeable phenotypes.One of the subfamilies,UBP15 subfamily,contains 5 individual genes.Besides the conserved UBP domain existing in all the family members,they also possess a single MYND zinc-finger domain.Mutations in those 5 genes lead to 2 distinct phenotypic consequences.The loss-of-function mutations in UBP15 display narrower,serrated and flat rosette leaves as well as early flowering,apical dominance losing,short or sometimes infertile siliques.Further analysis of transverse section across the leaf lamina shows that both adaxial epidermal and palisade cell numbers are decreased.In contrast,the overexpression line exhibits early rosette leaves and severely curled-down late rosette leaves,late flowering and strong apical dorminance as well as an increased leaf cell number.To assess de-ubiquitinating enzyme activity of UBP15,we perform in vitro assays by co-expressing GST-UBP15 with either substrate of UBQ1 or UBQ10.The results verify that UBP15 is a bona fide de-ubiqutinating enzyme,and Cys447 is essential for this activity which we also confirm in vivo by transforming UBP15 into wild type or ubp15-1 background.Further transcriptome analysis shows expression level of 804 genes are miss-regulated in ubp15-1,including two involved in cell cycle and another two controlling flowering.A genetic interaction analysis among members of this subfamily is performed. Phenotype of double mutant ubp15 ubp16 is more severe than that of ubp15 mutant, whereas ubp15 ubp17 is comparable to ubp15 mutant.It revealed that UBP15 and UBP16 but not UBP17 have functional redundancy,even though UBP16 and UBP17 are equally related to UBP 15.Intriguingly,mutation of another subfamily member UBP19 leads to embryo lethality while loss-of-function of its closest related member UBP18 exhibits no visible defect.Our data thus suggest that distinct UBP genes,even within a closely related subfamily, can function in different developmental pathways.Although there are clear functional redundancies among related family members,the redundancies may not be extrapolated merely based on the members' sequence homology.