Improving drought and cold tolerance in tobacco via EuUBC16-mediated polyubiquitination mechanisms

Ubiquitination is one of post-translational modifications of plant proteins, which is involved in the regulation of plant growth, development and various stress responses. Ubiquitin-conjugating enzymes (UBCs or E2s) are essential components of the ubiquitin cascade, with UBC13 being the only known E2 enzyme specifically responsible for catalyzing K63-linked polyubiquitination. However, research on K63-linked polyubiquitin modifications in Eucommia ulmoides remains scarce. In this study, a comprehensive identification of the UBC gene family was conducted within the E. ulmoides genome, leading to the identification of 32 UBC genes encoding conserved UBC domains. Phylogenetic analysis revealed that EuUBC1 6 and EuUBC17 are phylogenetically closely related to AtUBC13A and AtUBC13B , which are well-characterized mediators of K63-linked ubiquitination. Gene expression profiling revealed that EuUBC16 is differentially regulated under various abiotic stress conditions and in response to exogenous plant hormone treatments. Subcellular localization assays via transient expression demonstrated that EuUBC16 localizes to the nucleus, cytoplasm, and plasma membrane. Heterologous overexpression of EuUBC16 in tobacco resulted in marked inhibition of primary root elongation and enhanced tolerance to drought and cold stresses. In vitro ubiquitination assays confirmed that EuUBC16 possesses intrinsic E2 enzymatic activity, capable of forming a functional complex with EuUev1a to assemble K63-linked polyubiquitin chains, as well as mediating the formation of K48-linked polyubiquitin chains. The phenotypic alterations in root architecture and improved stress resistance observed in transgenic tobacco lines suggest a potential role for EuUBC16 in hormone signaling and stress response pathways. These findings not only elucidate the molecular function of EuUBC16 but also provide valuable genetic resources and a novel molecular basis for further investigation into the stress resistance mechanisms of E. ulmoides . • The Eucommia ulmoides genome harbors 32 UBC genes, including the K63-specific AtUBC13 homolog EuUBC16 . • EuUBC16 synthesizes K63-linked chains via EuUev1a and also catalyzes K48-linked chains in vitro. • EuUBC16 overexpression in tobacco boosts drought/cold tolerance but stunts roots, aiding stress-resistance breeding.