Functional characterization of a novel bark-specific multi-product monoterpene synthase from the rubber tree (Hevea brasiliensis)

The rubber tree (Hevea brasiliensis) is highly valued for its wood and latex. Along with being a rich source of polyterpenes in the latex, rubber trees are notable for their significant emission of volatile monoterpenes that may contribute to its chemical defense. A previous in silico genome-wide analysis of Hevea brasiliensis identified 47 putative terpene synthase (HbTPS) genes, of which only one had been experimentally characterized. In this study, we report the isolation and functional characterization of HbTPS24, a new monoterpene synthase belonging to the HbTPS family, providing new insights into the molecular basis of monoterpene biosynthesis in this species. Phylogenetic analysis placed HbTPS24 in the TPS-b subfamily. In vitro enzymatic assays revealed that HbTPS24 is multi-functional, converting both neryl diphosphate and geranyl diphosphate into cyclic monoterpenes such as α-thujene, β-pinene, α-terpinene, α-terpinolene, and α-terpineol. qRT-PCR analysis showed that HbTPS24 is mainly expressed in the bark, with significantly lower levels in the leaves and undetectable levels in the latex. The predominant expression of HbTPS24 in bark tissues, the detection of specific monoterpenes such as D-limonene and α-pinene in bark extracts, along with the broader range of cyclic monoterpenes identified in both in vitro enzymatic assays and canopy-scale volatile flux measurements, strongly suggest that HbTPS24 contributes to the in planta synthesis of multiple cyclic monoterpenes. These compounds are potentially involved in mediating stress resilience in Hevea brasiliensis.