Análisis metabolómico y genómico de cultivos celulares de zanahoria

Abstract

The main objective was to characterize secondary metabolites produced in two carrot cell lines. Thus, one of the specific objectives was to characterize the production of isoprenoid and phenolic compounds in cell suspensions of a Daucus carota green cell line under control conditions and under elicitation conditions with cyclodextrins, methyl jasmonate, Hexenol and beta-glucan separately or in combination. Moreover, the production of isoprenoids and phenolic compounds was characterized in cell suspensions of orange D. carota cell line under control conditions and under conditions of elicitation with cyclodextrins and methyl jasmonate. It was also studied the production of phytosterols and carotenoids and the expression of genes involved in their biosynthetic pathway in cell suspensions of orange D. carota cell line in the presence of inhibitors of the biosynthesis pathway of carotenoids and phytosterols. The results derived from this study refer to the green carrot cell line constitutively biosynthesized the following isoprenoids: carotenoids, alfa-tocopherol, chlorophyll a and b, and phytosterols. The main isoprenoid found in this green carrot line was lutein, a yellow xanthophyll. The characterization of the green carrot cell line showed that phytosterols and phenolic compounds wew mainly accumulated in the extracellular medium (15100 microg/L and 477.5 microg/L, respectively) in the presence of cyclodextrins. Unlike the aforementioned compounds, beta-carotene (1138.1 microg/L), lutein (25949.5 microg/L), alfa-tocopherol (8063.8 microg/L) and chlorophyll 1625.1 microg/L) and b (9958.3 microg / L) were mainly accumulated within the cells. Therefore, cyclodextrins were able to induce the biosynthetic pathway of mevalonate, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulating them outside the cells. However, in the absence of cyclodextrins, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate biosynthetic pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of phytosterols and phenolic compounds by increasing the flow of carbon towards the mevalonate cytosolic pathway and towards the phenylpropanoid pathway. In addition, beta-glucan was able to induce the intracellular accumulation of alfa-tocopherol in the green carrot cell line. On the other hand, the orange carrot cell line constitutively biosynthesized carotenoids and phytosterols. The major isoprenoids accumulated in this cell line were beta-carotene and lutein. Analysis of this orange line under elicitation conditions (50 mM cyclodextrins) showed that phytosterols accumulated mainly in the extracellular medium. However, beta-carotene and lutein were essentially accumulated within the cells. Combined treatment of cyclodextrins and methyl jasmonate improved the extracellular accumulation of phenolic compounds. Therefore, the orange carrot cell line under elicitation condition provides an efficient biotechnological system to produce bioactive compounds. The addition of Terbinafine and diflufenican, which inhibited the biosynthetic pathways of phytosterols and carotenoids respectively, caused a high accumulation of squalene and phytoene. These bioactive compounds are not usually accumulated and, because of this, the orange carrot cell line treated with these inhibitors is an alternative source to produce them. The highest expression levels of the square gene were found after 24h of treatment with the inhibitor terbinafine. This fact may explain the high intracellular accumulation of squalene with no negative effect on phytosterol levels. In addition, the cyclodextrins did not increase the expression levels of squalene synthase. This fact revealed that cyclodextrins do not function as induction molecules in the biosynthesis of phytosterols, but are capable of extracting them from cell membranes. Finally, gene expression levels of squalene synthase suggest that the accumulation of total carotene content could be linked to the expression of this gene. However, treatment with diflufenican decreased expression levels of phytoene synthase 1 and phytoene desaturase, suggesting that high phytoene accumulation did not correlate with a high level of expression of these genes.