EuPPPDE8, a deubiquitinating enzyme, promotes rubber biosynthesis by interacting with EuFPS1 and EuMGRN1 in Eucommia...

The plant Peptidase Domain Containing Enzymes (PPPDE) gene family plays crucial roles in plant growth and development, stress response, and signal transduction. Building upon previous work where a deubiquitinating enzyme was identified as an interacting protein of Eucommia ulmoides farnesyl pyrophosphate synthase 1 (EuFPS1), a key enzyme in Eucommia ulmoides rubber biosynthesis, this study systematically identified 9 PPPDE family members encoding deubiquitinating isopeptidases with the peptidase domain C97 from the Eucommia ulmoides genome. The analysis of collinearity and phylogenetic relationships among 86 PPPDE and PPPDE-like proteins from five species, including Eucommia ulmoides, demonstrated a high degree of homology for PPPDEs in rubber-producing plants. The gene encoding the EuFPS1-interacting protein, Eucommia ulmoides peptidase domain-containing enzyme 8 (EuPPPDE8), was cloned. Yeast two-hybrid (Y2H) and Bimolecular Fluorescence Complementation (BiFC) assays confirmed the interaction between EuFPS1 and EuPPPDE8. It was found that under low-temperature conditions, the expression of the EuPPPDE8 was downregulated, while farnesyl pyrophosphate synthase enzyme activity and rubber content in Eucommia ulmoides also decreased significantly. Using EuPPPDE8 as bait, 23 potential interacting proteins were screened, including Eucommia ulmoides mahogunin ring finger 1 (EuMGRN1). Experiments confirmed the interaction between EuMGRN1 and EuPPPDE8. EuPPPDE8 was localized to the cytoplasm. It is hypothesized that EuPPPDE8 may also participate in the EuMGRN1-mediated proteolytic pathway, antagonizing EuMGRN1-regulated proteolysis through its deubiquitinating activity. By overexpressing and silencing the EuPPPDE8 gene, it was discovered that overexpression of EuPPPDE8 led to a significant 51.51% increase in Eucommia rubber compared to the wild type, whereas silencing the gene resulted in reduced rubber synthesis, reaching only 0.82 times the rubber content of the wild type. This proves that the EuPPPDE8 gene functions to promote Eucommia rubber biosynthesis. The research findings provide important foundational data for further elucidating the functional role of deubiquitination regulation in Eucommia ulmoides rubber biosynthesis.