Flax (Linum usitatissimum
L.) is an important source of natural fibres in several temperate regions of the world. Flax oil has long been used in human and animal diet and in industry as a source of oil and as the basal component or additive of various paints or polymers. Recently there has been a growing interest in the probiotic properties of flax and in its beneficial effects on coronary heart disease, some kinds of cancer and neurological and hormonal disorders [1
]. The beneficial effects are mostly due to flax lipids. Flax oil is the richest plant source of linoleic and linolenic polyunsaturated fatty acids (PUFA), which are essential for humans since they cannot be synthesized in the organism and must be ingested with food. However, essential fatty acids are highly susceptible to oxidation and therefore flax oil has a very short shelf life.
The seedcake, the residual tissues after oil pressing, has scarcely been used only for animal feeding and some beneficial effect on animal growth was observed [47
]. There is no report on seedcake biochemical composition and the effect of tissue components on accumulation and composition of fatty acids. Also there is no report on potential industrial application of biochemical components from seedcake.
Flax fibres derive from the phloem, and can be separated from the inner bark outside the cambium by the process called retting [34
] For many years, flax fibres were mainly used for textile production. Now however, they are used as constituents of composite material for high-tech applications [48
]. The strong, lightweight and low-cost flax fibres are used instead of conventional glass-fibres in reinforced plastic manufacture. Since they are biodegradable, their final composite product is easily recyclable. Very recently flax fibres application in biomedicine is also under investigation. Their beneficial role as wound dressing has been reported [7
In order to improve oil stability and to expand seedcake and fibre application, mainly in medicine, transgenic flax plant has been generated and analyzed. It was expected that overexpression of three genes coding for key enzymes of flavonoid route will result in accumulation of broad spectrum of antioxidant molecules in each part of plant and thus making their basic products (oil, fibres) biotechnologically more valuable.
The initial step in the flavonoid route of the phenylpropanoid pathway is the synthesis of naringenin chalcone through condensation of three malonyl-CoA units with p
-coumaroyl-CoA. The reaction is catalyzed by the action of chalcone synthase (CHS). The naringenin chalcone is a branch point for all flavonoid biosynthesis [49
]. Chalcones are very labile compounds and could be accumulated as dihydrochalcones or rapidly converted to the colorless flavanone naringenin by the chalcone isomerase (CHI). Flavanones act as a precursor for the synthesis of flavones and 3-OH-flavanones (dihydroflavonols). Dihydroflavonols can then be converted into flavonols by FLS (flavonol synthase) or into anthocyanins by DFR (dihydroflavonol 4-reductase).
Since our gene construction that has been introduced into transgenic flax contained cDNAs for CHS, CHI and DFR enzyme, the flavonols and anthocyanins compound content was measured. The data obtained showed an increase in these compounds level in flaxseeds, seedcakes and fibres from plants of transgenic lines. It should be pointed out that measured flavonoid compounds in seed could be than found in seedcake extract and thus suggesting that the residual tissues after oil extraction from seeds appeared to be suitable source of biomedical valuable compounds. It is also important to notice that generated transgenic flax are stable in respect to expression of exogenously introduced genes since the field grown plants of third generation contained all transgenes and they are active which was confirmed by PCR analysis of transgenic plant with total mRNA as template.
Also interesting is the identification of increased quantity of phenolic acids and lignans in seedcake from transgenic seed. It is known that these compounds like flavonoids are the constituents of phenylpropanoid pathway however they are biosynthesized on different metabolic route. In view of the presented results there should be mechanism in operation coordinating their biosynthetic coregulation [50
The core phenylpropanoid pathway converts phenylalanine to p-coumaric acid. Several branches radiate from this core reaction. Early branch point leads to the formation of simple phenylpropanoids like phenolic acids. The later incorporation of three malonyl-CoA molecules leads to generation of flavonoids. Lignans are dimers and lignins are the polymer of monolignol alcohol. Monolignol forms from coumaric acid, oxygen and methyl groups incorporated into the aromatic ring. This intermediate is then thought to be converted to respective aldehyde and then to the monolignol (coumaryl, ferulyl and sinapyl alcohols). The way in which lignans (dimer) and lignin (polymer) are formed from monolignol precursor is still poorly understood. Early suggestion was that the final stage of monolignol polymerization resulted from random coupling of monolignol units that did not require the involvement of enzymes. This opinion is now modified, it is evidenced that monolignol in the form of glycosides are transported to cell wall and then oxidized to form radicals that combine to produce polymer. There are several classes of enzymes present in cell wall that could catalyze monolignol oxidation including peroxidases, laccases, polyphenol oxidases and dirigent protein. Lignin polymer can be formed artificially by adding either peroxidase together with H2O2 or laccase with O2 which serve as oxidizing agent. This is the most substantial evidence that the same process might operating in vivo and suggests that monolignol dimers and polymers can be formed by random chemical interactions as well as by enzymatic control.
It is as yet unknown how these compounds (lignans) biosynthesis is regulated upon flavonoids overproduction [25
]. We speculate that strong activation of flavonoids synthesis in transgenic flax and compounds accumulation might be the reason for this. The high concentrations of antioxidants may result in pro-oxidative activity. Thus the increase in antioxidative compound concentration upon CHS/CHI/DFR overexpression might result in local promoting of coumaric acid oxidation and monolignol radicals generation. This suggestion is supported by the finding that the level of coumaric acid in seedcake extract from control plant is about 30% lower than this from transgenic plant.
Thus, the seedcake from transgenic seed appears as a richest source of SDG. It is important to notice the beneficial role of this compound for human health. There are several reports concluding the role of SDG in protection against different types of cancer [49
]. SDG can be metabolized by the colonic microflora to the mammalian lignans, enterodiol and enterolactone. That is the reason why flax lignans exhibit weak estrogenic and antiestrogenic properties, in a tissue-specific manner, and have potential role in the prevention and treatment of breast cancer and other hormone dependent cancers [53
]. A great number of animal model and human studies suggest that a higher intake of lignans reduces the risk of many chronic diseases including cardiovascular diseases and all types of diabetes [55
Both flavonoids and phenolic acids were shown to exhibit antioxidant properties [11
]. Since there was an increase in those compounds content detected in transgenic plants, an increase in antioxidant capacity was expected. Indeed the extract from transgenic seed and seedcake showed almost the same IC50 value and this was far lower than for control. Thus the accumulation of compounds from phenylpropanoid pathway mainly occurs in seedcake and strongly affects antioxidant potential of transgenic product.
It is known that flavonoids due to their hydrophilic nature only very partially coextract with the oil during its production. Even so the accumulation of antioxidants in seedcake was expected to affect fatty acid composition. Indeed oil produced from transgenic seeds contains more unsaturated fatty acids and the total level of fatty acids was also increased. Thus the higher quantity of phenylpropanoid compounds in seedcake indirectly affect the fatty acids stability perhaps by protection them against oxidation during technological process of oil production.
Flax retting is the process of fibre isolation and was faster in case of transgenic plant. In retting, bast fibre bundles are separated from the core, the epidermis and the cuticle [33
]. This is accomplished by the cleavage of pectins and hemicellulose in the flax cell wall, a process mainly carried out by plant pathogens like filamentous fungi [34
]. The retting efficiency depends on the degree of lignification. Since reduced lignin content in transgenic flax was observed the easier retting was expected. This was however not the case. Scanning electron microscopy does not show differences between transgenic and control fibres. Biochemical analysis of fibres suggests the increased level of catechine and acetylovanillone. Although the first resulted from flavonoid biosynthesis enhancement the second presumably derives from lignin degradation [56
] The decrease in lignin content in fibres from transgenic plant agreed with this. Also decrease in the content of syringaldehyde in transgenic fibres which is the product of lignocellulose degradation [56
] confirm that degradation process might occurs as more advanced in transgenic fibres than in control.
Infrared spectroscopy has been used for molecular characteristic of flax products for it was found to be very suitable for identification of the major chemical components and also found to provide information on the molecular changes in flax fibres caused by ageing, mechanical processing and chemical treatment [34
].Thus the main products of flax plant (oil, seedcake extract and fibres) were further analysed by this method and data compared to those from biochemical analysis.
The IR study of oil revealed that several compounds of hydrophilic nature coextract with the oil upon seed treatment with high pressure under low temperature. Among those compounds are flavonoids, phenolic acids and lignans. Although the method does not provide quantitative data on compounds content it is quite clear that their level is higher in oil from transgenic seed when compared to control. The detection of compounds of phenylpropanoid pathway in oil appears to be a good molecular background for protection of fatty acids from transgenic oil against oxidation which agrees with data from oil analyzed in TBARS method.
The observed changes in the intensity of bands characteristic for phenylpropanoids compounds in IR spectrum of seedcake extract from transgenic seed compared to control confirm the higher content of these compounds in extract from transgenic seedcake which was detected by UPLC analysis. Thus the conclusion is that the extract from transgenic seedcake contains the same phenylpropanoid compounds that were identified by UPLC analysis and they are in higher quantity when compared to control. Further the IR spectra helped to identify phenolic acids (ferulic vs. synapic) which were not resolved on UPLC column. Thus the IR spectra are suitable for detailed compounds analysis of seedcake extract.
The data from IR study confirmed the results of the biochemical analyses of the flax fibres and additionally suggests that arrangement of the cellulose polymer in the transgenic fibres differed from that in the control and a significant decrease in the number of hydrogen bonds was detected.
Thus the overall conclusion from the IR study of flax main product is that several compounds of phenylpropanoid pathway are accumulated in oil, seedcake and fibres upon three genes overexpression resulting in production of stable oil and fibres and seedcake extract ready for use in biomedicine.