The barley UBIQUITIN-SPECIFIC PROTEASE gene family: insights into seed development and germination

UBIQUITIN-SPECIFIC PROTEASES (UBPs) are deubiquitinating enzymes that regulate protein turnover, chromatin state and signalling in plants. In Arabidopsis thaliana , UBPs have been associated with hormone responses, stress tolerance and seed traits. However, the UBP gene family in barley ( Hordeum vulgare ), a major cereal crop whose seed performance traits, such as germination, pre-harvest sprouting resistance and malting quality are of high agronomic and industrial importance, has not yet been characterised. We performed a genome-wide analysis of the barley genome and identified 26 UBP genes. Phylogenetic analysis showed that HvUBP proteins cluster into 14 clades largely shared with rice and Arabidopsis, while an expanded analysis including barley, rice, maize and wheat supported retention of major UBP lineages among the four cereal species examined. All HvUBPs contained the principal Peptidase_C19/UCH catalytic region, whereas accessory-domain composition was conserved in several orthogroups but diversified in others. In silico promoter analyses indicate enrichment for hormone- and stress-related cis -elements, particularly ABA- and jasmonate-responsive motifs. Most HvUBP genes exhibit embryo-biased expression during grain development, with peaks around 8 days after pollination, and display distinct tissue- and stage-specific transcriptional patterns during germination in radicle, scutellum and plumule. RT-qPCR validation in germinating embryos confirmed significant ABA-responsive upregulation of HvUBP26 and HvUBP5 , and ABA- and GA₃-responsive upregulation of HvUBP15 , providing experimental support for hormone-responsive regulation of selected candidates. This work provides the first genome-wide catalogue and seed-focused characterisation of the UBP gene family in barley. By integrating phylogeny, domain architecture, expression profiles and co-localisation with seed trait QTLs, among the 26 initial HvUBPs, we identify candidates with complementary lines of support. HvUBP1/2a , HvUBP3/4a and HvUBP26 co-localise with verified dormancy and germination QTL intervals, while HvUBP5 , HvUBP12/13b , HvUBP14 and HvUBP15 are supported by conserved orthology to Arabidopsis or rice UBPs implicated in seed size, embryo development and chromatin remodelling. These genes also show embryo-biased expression and significant hormone marker co-expression. RT-qPCR analysis showed that three among them, HvUBP5 , HvUBP26 and HvUBP15 respond transcriptionally to ABA and/or GA₃ during germination while two of the candidates, HvUBP1/2a and HvUBP12/13 show a non-significant directional trend towards the application of ABA. Together, our findings represent a significant step towards understanding the roles of deubiquitinases in barley seed biology, establishing a set of candidates and an experimental baseline from which targeted functional studies — including reverse genetics, hormone response assays and chromatin profiling — can now be pursued in the future.