Flax phenylpropanoids: characterization and biotechnological approach for production enhancement

Flax (Linum usitatissimum L.) is a common fiber and oilseed crop considered as a valuable functional food since it constitutes one of the key sources of ?-3 fatty acids, phenylpropanoids (lignans) and mucilage [1]. During the last years there has been an increasing interest in dietary human consumption of flaxseeds in order to improve the nutritional and health status. In our laboratory we are interested in the study of phenylpropanoids metabolism. The main class of phenylpropanoids found in flax are lignans, diphenolic compounds displaying a broad range of biological activities such as antioxidant, cytotoxic, antifungal, antiviral, and phytoestrogenic [2; 3]. These phenolic compounds are of great interest for pharmaceutical, cosmetic and food industry. With the aim to identify and select the cultivar with the highest production of metabolites, the phenylpropanoid content of 7 flax cultivars (Linoal, Valoal, Natural, Festival, Merlin, Solal, Kaolin) and the expression level of key genes (PAL, Phenylalanine ammonia-lyase and 4CL, 4-Coumarate:CoA ligase) involved in their biosynthesis were compared. Moreover, the cultivar showing either the highest level of phenylpropanoids production and the highest level of expression of two key biosynthetic genes were chosen to start the production of flax cell cultures in order to improve the accumulation of bioactive compounds. In fact, it has been reported that plant cell cultures represent a promising system to optimize the production of valuable secondary metabolites [4]. Results. The 1H-NMR metabolite profiling of leaves (Fig.1) showed the presence of flavonoids, isoorientin, luteolin, apigenin and isovitexin, whereas seeds showed mainly lignans compounds. Real-Time PCR approach was used to evaluate the relative expression level of PAL and 4CL, representing two genes in the first part of phenylpropanoid biosynthetic pathway. Among the cultivars analysed, we found that Valoal and Solal are those with the highest expression level of PAL and 4CL both in leaves and in seeds (Fig.2). The determination of the specific activity of PAL and 4CL enzymes was in agreement with gene expression data, highlighting Valoal and Solal as the cultivars with the highest phenylpropanoids production. On the basis of these results Valoal was selected to start the production of in vitro cell cultures. To verify if in this system the ability to produce bioactive compounds was maintained we performed both biochemical and 1H-NMR analysis on the cultured cells. Preliminary analysis showed that flax cell cultures mainly produce compounds belonging to lignans, resembling the secondary metabolites identified in flax seeds. To increase the performance of flax cell cultures two strategies were used: a) the optimization of the medium composition and b) the use of some elicitors such as yeast extract, salicylic acid and methyl-jasmonate. Conclusion. The results obtained from flax leaves and seeds highlighted the presence of flavonoids and lignans with demonstrated antioxidant, antinflamatory, chemopreventive properties on human. The molecular analysis performed on the general phenylpropanoid pathway could help to better understand the biosynthesis of these compounds. Moreover, the preliminary results from the in vitro cultured cells suggest that this system is able to produce lignans as secondary metabolites resembling the flax seeds tissue. This finding is particularly encouraging since lignans have phytoestrogenic and cancer chemopreventive properties [5].