Biosynthesis and bioactivity of glucosinolates and their production in plant in vitro cultures.

MAIN CONCLUSION: Glucosinolates are biologically active compounds which are involved in plant defense reaction. The use of plant in vitro cultures and genetic engineering is a promising strategy for their sustainable production. Glucosinolates are a class of secondary metabolites found mainly in Brassicaceae, which contain nitrogen and sulfur in their structures. Glucosinolates are divided into three groups depending on the amino acid from which they are biosynthesized. Aliphatic glucosinolates are generally derived from leucine, valine, methionine, isoleucine and alanine while indole and aromatic glucosinolates are derived from tryptophan and phenylalanine or tyrosine, respectively. These compounds are hydrolyzed by the enzyme myrosinase when plants are stressed by biotic and abiotic factors, obtaining different degradation products. Glucosinolates and their hydrolysis products play an important role in plant defense responses against different types of stresses. In addition, these compounds have beneficial effect on human health because they are strong antioxidants and they have potent cardiovascular, antidiabetic, antimicrobial and antitumoral activities. Due to all the properties described above, the demand for glucosinolates and their hydrolysis products has enormously increased, and therefore, new strategies that allow the production of these compounds to be improved are needed. The use of plant in vitro cultures is emerging as a biotechnological strategy to obtain glucosinolates and their derivatives. This work is focused on the biosynthesis of glucosinolates and the bioactivity of these compounds in plants. In addition, a detailed study on the strategies used to increase the production of several glucosinolates, in particular those synthesized in Brassicaceae, using in vitro plant cultures has been made. Special attention has been paid for increasing the production of glucosinolates and their derivatives using metabolic engineering.

Enhanced bioactive compound production in broccoli cells due to coronatine and methyl jasmonate is linked to antioxidative metabolism.

Elicited broccoli suspension-cultured cells (SCC) provide a useful system for obtaining bioactive compounds, including glucosinolates (GS) and phenolic compounds (PCs). In this work, coronatine (Cor) and methyl jasmonate (MJ) were used to increase the bioactive compound production in broccoli SCC. Although the use of Cor and MJ in secondary metabolite production has already been described, information concerning how elicitors affect cell metabolism is scarce. It has been suggested that Cor and MJ trigger defence reactions affecting the antioxidative metabolism. In the current study, the concentration of 0.5 µM Cor was the most effective treatment for increasing both the total antioxidant capacity (measured as ferulic acid equivalents) and glucosinolate content in broccoli SCC. The elicited broccoli SCC also showed higher polyphenol oxidase activity than the control cells. Elicitation altered the antioxidative metabolism of broccoli SCC, which displayed biochemical changes in antioxidant enzymes, a decrease in the glutathione redox state and an increase in lipid peroxidation levels. Furthermore, we studied the effect of elicitation on the protein profile and observed an induction of defence-related proteins. All of these findings suggest that elicitation not only increases bioactive compound production, but it also leads to mild oxidative stress in broccoli SCC that could be an important factor triggering the production of these compounds.

New strategies for the use of Linum usitatissimum cell factories for the production of bioactive compounds.

In this work, suspension-cultured cells of Linum usitatissimum L. were used to evaluate the effect of two types of cyclodextrins, ß-glucan and (Z)-3-hexenol separately or in combination on phytosterol and tocopherol production. Suspension-cultured cells of L. usitatissimum were able to produce high levels of phytosterols in the presence of 50 mM methylated-ß-cyclodextrins (1325.96 ± 107.06 µg g dry weight(-1)) separately or in combination with ß-glucan (1278.57 ± 190.10 µg g dry weight(-1)) or (Z)-3-hexenol (1507.88 ± 173.02 µg g dry weight(-1)), being cyclodextrins able to increase both the secretion and accumulation of phytosterols in the spent medium, whereas ß-glucan and (Z)-3-hexenol themselves only increased its intracellular accumulation. Moreover, the phytosterol values found in the presence of hydroxypropylated-ß-cyclodextrins were lower than those found in the presence of methylated-ß-cyclodextrins in all cases studied. However, the results showed that the presence of methylated-ß-cyclodextrins did not increase the tocopherols production and only an increase in tocopherol levels was observed when cells were elicited with 50 mM hydroxypropylated-ß-cyclodextrins in combination with ß-glucan (174 µg g dry weight(-1)) or (Z)-3-hexenol (257 µg g dry weight(-1)). Since the levels of tocopherol produced in the combined treatment were higher than the sum of the individual treatments, a synergistic effect between both elicitors was assumed. To sum up, flax cell cultures elicited with cyclodextrins alone or in combination with ß-glucan or (Z)-3-hexenol were able produce phytosterols and tocopherols, and therefore, these elicited suspension-cultured cells of L. usitatissimum can provide an alternative system, which is at the same time more sustainable, economical and ecological for their production.