Functional characterization of EuSIP1 reveals its central role in trans-polyisoprene biosynthesis in Eucommia ulmoides

Eucommia ulmoides is a unique plant capable of synthesizing trans-polyisoprene (TPI), a form of natural rubber with outstanding mechanical and chemical properties. In addition to its industrial importance, the bark of E. ulmoides is a high-value traditional Chinese medicinal material used for promoting bone health, lowering blood pressure and supporting kidney function. Despite its dual utility, the molecular regulation of TPI biosynthesis in this species remains poorly understood. In this study, we performed a genome-wide analysis and identified five soluble inorganic pyrophosphatase (SIP) genes in E. ulmoides ( EuSIP1–EuSIP5 ). Among them, EuSIP1 showed strong sequence homology with SIPs previously associated with rubber production in Hevea brasiliensis . Functional characterization was conducted using Agrobacterium-mediated transformation to generate overexpression (OE) and RNA interference (RNAi) lines. Overexpression of EuSIP1 significantly reduced intracellular pyrophosphate (PPi) levels (by 40.3 %–73.1 %) and upregulated key rubber biosynthesis genes ( EuFPS1, EuSRPP1, EuIPI, EuGGPPS ), resulting in higher TPI accumulation (0.30 %–0.81 %) and increased molecular weight. In contrast, RNAi lines accumulated more PPi (1.65–2.25 × WT), exhibited suppressed biosynthetic gene expression, and showed a marked reduction in TPI content (0.05 %–0.18 %). Protein–protein docking and yeast two-hybrid assays revealed weak but reproducible interactions between EuSIP1 and rubber biosynthesis-related proteins EuFPS and EuSRPP. Subcellular localization confirmed EuSIP1 presence in both the cytoplasm and nucleus, supporting its role in intracellular PPi homeostasis. Together, these results suggest that EuSIP1 plays a dual role in regulating TPI biosynthesis, promoting TPI synthesis and elongation through the interaction of PPi inhibition by hydrolysis and rubber biosynthesis related proteins. This study advances our understanding of the molecular regulation of rubber production in E. ulmoides , a species of both economic and medicinal importance, and identifies EuSIP1 as a promising target for metabolic engineering to improve rubber yield and quality. • Genome-wide analysis identified five EuSIP genes in Eucommia ulmoides , EuSIP1 is homologous to rubber-associated SIPs. • EuSIP1 boosts rubber yield and MW by lowering PPi and interacting with EuFPS/EuSRPP, revealing SIP's role in TPI biosynthesis. • EuSIP1 interacts with core TPI enzymes (EuFPS/EuSRPP), suggesting dual roles in PPi scavenging & polymer elongation facilitation. • First evidence of SIP-mediated regulation in TPI biosynthesis.