Norie Watanabe

Cloning and Characterization of the 2-C-Methyl-D-erythritol 4-Phosphate (MEP) Pathway Genes of a Natural-Rubber Producing Plant, Hevea brasiliensis

Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-13C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.

Elucidation of rubber biosynthesis and accumulation in the rubber producing shrub, guayule (Parthenium argentatum Gray)

AbstractMain conclusionGuayule biosynthesizes and accumulates rubber particles predominantly in epithelial cells in the parenchyma tissue, and this biosynthesis and accumulation is accompanied by remodeling of the roles of epithelial cells. The mechanism underlying the biosynthesis and accumulation of large quantities of rubber particles and resin in the parenchyma tissue of the stem bark of guayule (Parthenium argentatum Gray) remained unanswered up to now. Here, we focused on rubber particle biosynthesis and accumulation in guayule and performed histochemical analyses using a lipophilic fluorescent dye specific for lipids and spectral confocal laser scanning microscopy. Unmixing images were constructed based on specific spectra of cis-polyisoprene and resin and showed that guayule accumulates a large amount of resin in the resin canals in parenchyma tissue and in pith. Interestingly, the fluorescence signals of rubber were predominantly detected in a specific single layer of epithelial cells around the resin canals. These epithelial cells accumulated large rubber particles and essentially no resin. Immunoblotting and immunostaining of guayule homologue of small rubber particle proteins (GHS), which contributes to the biosynthesis of rubber in guayule, showed that GHS is one of several small rubber particle proteins and is localized around rubber particles in epithelial cells. De novo sequencing of the rubber particle proteins showed the presence of all known organelle proteins, suggesting that epithelial cells biosynthesize rubber particles, followed by remodeling of the cells for the accumulation of rubber particles with subsequent decomposition of the organelles. These results indicate that epithelial cells around resin canals are bifunctional cells dedicated to the biosynthesis and accumulation of rubber particles.