Caracterización de la producción de compuestos bioactivos y análisis de proteínas en cultivos celulares de brócoli

Abstract

The consumption of broccoli has been increased worldwide due to its high nutritive value. Moreover, broccoli contains a complex mixture of bioactive compounds showing antioxidant activity, as glucosinolates and phenolic compounds. Due to the health benefits of these secondary metabolites, the main objective of this work was the use of broccoli suspension-cultured cells to increase their production. Firstly, the biosynthesis, regulation and functions of glucosinolates in plants have been studied. In addition, different biotechnological strategies based on plant in vitro cultures used to obtain these compounds have been detailed. On the other hand, elicitation has been used to increase the production of glucosinolates in broccoli suspension-cultured cells and the expression levels of glucosinolate biosynthesis related genes in these broccoli suspension-cultured cells under elicitation conditions have been analysed. Other elicitation experiments have been carried out by modification of different culture conditions, in order to increase the glucosinolate production in broccoli suspension-cultured cells. Another objective proposed in this work was to analyse the effect of elicitors on broccoli cells. In this sense, different activity measurements of enzymes involved in antioxidant metabolism, as well as the glutathione content and lipid peroxidation levels have been carried out in elicited broccoli suspension-cultured cells. Finally, the protein pattern expressed under elicitation conditions in broccoli cells was studied by SDS-PAGE in order to identify defense-related proteins. The main conclusion obtained from this work was that elicitation of broccoli suspension-cultured cells with coronatine led to a high increase in glucosinolate production, resulting in a biotechnological production system of these compounds alternative to the whole plant extraction. Moreover, the amount of sucrose, together with the nutritional medium composition, the cell density used and the addition of tryptophan as biosynthetic pathway precursor, were factors that limited the production of glucosinolates in broccoli suspension-cultured cells. Coronatine increased the expression levels of genes involved in the biosynthetic pathway and regulation of glucosinolates. Regarding phenolic compounds, coronatine and methyl jasmonate were able to increase the production of these bioactive compounds in broccoli suspension-cultured cells. The increased bioactive compound production was correlated with alterations in the activity of enzymes involved in the antioxidative metabolism and the glutathione redox state. These results, combined with an increase in lipid peroxidation, suggested that elicitation with coronatine and methyl jasmonate led to the establishment of a mild oxidative stress in broccoli suspension-cultured cells. In addition, the oxidative stress induced the synthesis of a number of defense-related polypeptides in broccoli suspension-cultured cells.