Transcription profiles analysis of genes encoding 1-deoxy-D-xylulose 5-phosphate synthase and 2C-methyl-D-erythritol 4-phosphate synthase in plaunotol biosynthesis from Croton stellatopilosus.

Genes encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS; EC 2.2.1.7) and 2C-methyl-D-erythritol 4-phosphate synthase (MEPS; EC 1.1.1.267), the first two enzymes in the deoxyxylulose phosphate (DXP) pathway, were cloned from young leaves of Croton stellatopilosus, and designated as 1-deoxy-D-xylulose 5-phosphate synthase (CSDXS) and 2C-methyl-D-erythritol 4-phosphate synthase (CSMEPS), respectively. Analysis of deduced amino acid sequences of the CSDXS and the CSMEPS confirmed their nucleotide sequences as they shared high identities to other known DXSs and MEPSs in higher plants. Physiological roles of the CSDXS and the CSMEPS were determined for the mRNA expressions in leaves, twigs and roots. Transcription profiles analyses of the CSDXS and the CSMEPS genes were investigated using semi-quantitative RT-PCR technique. Relative intensities of the CSDXS and the CSMEPS expressions to house-keeping gene (18S rRNA) were calculated. The results indicated that the levels of mRNAs expressions of the CSDXS and the CSMEPS were high in leaves and twigs. This evidence was in line with the high content of plaunotol, accumulated in leaves and twigs. Neither the CSDXS nor the CSMEPS were expressed in roots, where plaunotol was not detected. From this study, it can be concluded that plaunotol is biosynthesized in the chloroplastic tissue and regulated by the CSDXS and the CSMEPS.

Establishment of Croton stellatopilosus suspension culture for geranylgeraniol production and diterpenoid biosynthesis.

Diterpenoids in higher plants are biosynthesized from isoprene units obtained from two distinct pathways: the mevalonate pathway and the deoxyxylulose phosphate pathway. The metabolic partitioning of both pathways in plant species is dependent upon the type of culture. In order to study the diterpenoid biosynthesis in Croton stellatopilosus cell culture, callus culture was firstly induced from C. stellatopilosus young leaves in Murashige and Skoog (MS) medium in the presence of 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg/l benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. The suspension culture was further induced from its callus in the same medium without gelling agent. Detection of diterpenoid accumulation by gas chromatography-mass spectrometry revealed that a cell culture could accumulate a low amount of geranylgeraniol (GGOH) and a high content of fatty acids and phytosterols. To improve the GGOH production, the culture conditions were optimized by medium manipulation in terms of hormonal factors. The growth rates of cell cultures were similar in all kinds of media. The GGOH production curve indicated that GGOH plays an important role as a primary metabolite in the cell culture. The optimum medium for GGOH production was MS medium supplemented with 2.0 mg/l 2,4-D and 2 mg/l BA that could produce GGOH with a yield of 1.14 mg/g FW.