Biosynthesis of phytosterols in cell suspension cultures of Croton Stellatopilosus

Abstract

This study aimed to use the techniques of feeding experiments to understand the biosynthetic process of terpenoids and phytosterols with respect to their origin of isoprene units in Croton stellatopilosus cell suspension cultures. Feeding experiments with labeled glucose was first optimized for feeding conditions by using radioactively labeled [1-[superscript 14]C]glucose followed by monitoring its radiolabel uptake. The preliminary results showed that the highest incorporation of the labeled glucose occurred after 4 days of the feedings. Under the optimized conditions, [1-[superscript 13]C]glucose and [2-[superscript 13]C]sodium acetate were then fed into the cell suspension cultures of C. stellatopilosus. [beta]-Sitosterol and stigmasterol, the two major phytosterols produced, were isolated and their [superscript 13]C-labeling patterns elucidated using quantitative NMR spectroscopy. Analysis of the [superscript 13]C-enrichment patterns revealed that the components of all isoprene units of the phytosterols were supplied exclusively from the mevalonate pathway. No isoprene units originated from the alternative deoxyxylulose phosphate (DXP) pathway were detected in this study. Transmission electron micrographs (TEM) showed that there were different developmental stages of chloroplasts in different C. stellatopilosus tissues. In leaf, the chloroplasts were fully developed in palisade cells, the green callus contained a few chloroplasts with partial development, whereas no chloroplasts were found in the suspension cultured cells. Molecular analysis on the transcription level of these different types of tissues by using semi-quantitative RT-PCR technique revealed that the three genes (dxs, dxr and ggpps) involved in the isoprenoid biosynthetic route of the DXP pathway were strongly expressed in the plant leaf and green callus culture but rarely expressed in the suspension culture. This study indicated that the operation of either the DXP pathway or MVA pathway for the isoprene formation in C. stellatopilosus is dependent upon types of cultures and ability of chloroplast-forming cells in the tissues of C. stellatopilosus.