Bioscience Methods 2024, Vol.15, No.6, 289-301 http://bioscipublisher.com/index.php/bm 291 In H. brasiliensis, gene families associated with rubber biosynthesis have undergone significant expansion. Notably, genes encoding rubber particle membrane proteins (RPMPs), including rubber elongation factor (REF) and small rubber particle protein (SRPP), are highly expressed in latex. These proteins play critical roles in the biosynthesis of cis-polyisoprene, the primary component of natural rubber. The expansion and high expression levels of these gene families underscore their importance in rubber production and stress response mechanisms (Chow et al., 2007; Chuntai et al., 2017). Comparative genomics between H. brasiliensis and other Euphorbiaceae species has revealed unique evolutionary adaptations in rubber biosynthesis. For instance, while E. ulmoides synthesizes trans-polyisoprene via farnesyl diphosphate synthases (FPSs), H. brasiliensis produces cis-polyisoprene. The independent expansion of FPS and rubber elongation factor gene families in H. brasiliensis highlights the divergent evolutionary paths taken by these species to optimize rubber production. These comparative studies provide a deeper understanding of the genetic and biochemical diversity within the Euphorbiaceae family (Wuyun et al., 2017; Chow et al., 2020). 3 Biochemical Pathways of Rubber Biosynthesis 3.1 Pathways inEucommia ulmoides In Eucommia ulmoides, the methylerythritol-phosphate (MEP) pathway is the primary route for the biosynthesis of isoprenoids, which are crucial for rubber production. This pathway operates in the plastids and is responsible for the synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the fundamental building blocks for polyisoprene synthesis. The predominance of the MEP pathway in E. ulmoides highlights its essential role in the production of trans-polyisoprene rubber, a high-molecular mass polymer of isoprene units with a trans-configuration (Kajiura et al., 2017; Wang et al., 2017). It is known that the biosynthesis pathway of trans-polyisoprene (TPI) in Eucommia ulmoides involves 47 genes, including genes from the MEP and MVA pathways, geranyl diphosphate synthase (GPS), geranylgeranyl diphosphate synthases (GGPSs), farnesyl pyrophosphate synthases (FPSs), and rubber elongation genes (Wuyun et al., 2018; Li et al., 2020). Farnesyl diphosphate synthases (FPSs) are key enzymes in the biosynthesis of prenyl precursors, which are vital for the production of various terpenoids, including rubber. In E. ulmoides, multiple FPS genes have been identified and characterized, with distinct enzymatic properties and expression patterns. For instance, EuFPS1 and EuFPS2 exhibit different substrate preferences and reaction products, which influence the synthesis of farnesyl diphosphate (FPP) and its elongation to geranylgeranyl diphosphate. These differences in FPS activity are regulated by factors such as pH, metal ion cofactors, and cofactor concentrations, ultimately affecting the biosynthesis of trans-polyisoprene (Kajiura et al., 2017; Wang et al., 2017). Eucommia rubber is a secondary metabolite of Eucommia ulmoides, but its transcriptional regulatory mechanisms for biosynthesis remain unclear. The biosynthesis of Eucommia rubber is regulated by multiple genes, such as EuFPS1 (farnesyl diphosphate synthase), a key enzyme in the biosynthetic process of Eucommia rubber. EuFPS1 is positively regulated by EuWRKY30, which plays a critical role in the synthesis of Eucommia rubber (Zhang et al., 2024). 3.2 Pathways inHevea brasiliensis In Hevea brasiliensis, the conventional mevalonate (MVA) pathway is the primary route for the synthesis of isoprenoids, including cis-polyisoprene, the main component of natural rubber. This pathway operates in the cytosol and involves the conversion of acetyl-CoA to IPP and DMAPP through a series of enzymatic reactions. The MVA pathway's role in cis-polyisoprene synthesis is crucial, as it provides the necessary precursors for the polymerization process (Chuntai et al., 2017). Although the MVA pathway is the primary route for isoprenoid biosynthesis in H. brasiliensis, there is evidence suggesting a potential role for the MEP pathway in supplying IPP. This alternative pathway, which operates in the plastids, may contribute to the overall pool of IPP and DMAPP, thereby supporting the biosynthesis of
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