Comprehensive genome-wide analysis of PUB (Plant U-box) genes in rye and its closely related species and functional characterization of ScPUB6

Plant U-box (PUB) proteins are a type of E3 ligases that are major components of the ubiquitination cascade. PUBs can act as positive or negative regulators in diverse biological processes, including plant responses to biotic and abiotic stresses such as cold stress. However, PUBs in rye (Secale cereale L.), the most cold-resistant winter crop, remain poorly studied. In this study, 100 PUBs in the rye genome and 214 genes in the oat genome were identified. The rye PUBs were classified into seven classes based on protein motifs, and dispersed throughout the whole rye chromosomes with 30 duplication events. Cis-acting regulatory element analysis indicated that all the ScPUB genes possess abiotic stress-responsive elements in their promoter. Further, synteny analysis revealed that the PUBs in rye had higher similarity with wheat and barley, indicating a closer evolutionary distance than that with oat. Rye tissue-specific and abiotic stress-responsive ScPUBs gene expression were examined, and ScPUB6, which is responsive to the cold treatment, was chosen for further analysis. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays revealed three PUB6-interacting proteins (HSP70, 70-kDa heat shock protein; TIG, Trigger factor-like protein; MYB, Myb transcription factor), which might interact in the plasma membrane. Overall, this analysis of U-box E3 ligases could provide valuable information for an improved understanding of the function of PUBs in the cold resistance mechanism of winter crops.