Magdalena Żuk

Flavonoid engineering of flax potentiate its biotechnological application

BackgroundFlavonoids are a group of secondary plant metabolites important for plant growth and development. They show also a protective effect against colon and breast cancer, diabetes, hypercholesterolemic atherosclerosis, lupus nephritis, and immune and inflammatory reactions. Thus, overproduction of these compounds in flax by genetic engineering method might potentiate biotechnological application of these plant products.ResultsFlax plants of third generation overexpressing key genes of flavonoid pathway cultivated in field were used as plant material throughout this study. The biochemical properties of seed, oil and seedcake extracts and fibre from natural and transgenic flax plants were compared. The data obtained suggests that the introduced genes were stably inherited and expressed through plant generations.Overproduction of flavonoid compounds resulted in increase of fatty acids accumulation in oil from transgenic seeds due to protection from oxidation offered during synthesis and seed maturation. The biochemical analysis of seedcake extracts from seeds of transgenic flax revealed significant increase in flavonoids (kaempferol), phenolic acids (coumaric, ferulic, synapic acids) and lignan content. Fibres, another product of flax plant showed increase in the level of catechine and acetylvanillone and decrease in phenolic acids upon flax modification.Biochemical analysis results were confirmed using IR spectroscopy. The integral intensities of IR bands have been used for identification of the component of phenylpropanoid pathway in oil, seedcake extract and fibre from control and transgenic flax. It was shown that levels of flavonoids, phenolic acids and lignans in oil and seedcake extract was higher in transgenic flax products compared to control. An FT-IR study of fibres confirmed the biochemical data and revealed that the arrangement of the cellulose polymer in the transgenic fibres differs from the control; in particular a significant decrease in the number of hydrogen bonds was detected.ConclusionsAll analysed products from generated transgenic plants were enriched with antioxidant compounds derived from phenylopropanoid pathway Thus the products provide valuable source of flavonoids, phenolic acids and lignan for biomedical application. The compounds composition and quantity from transgenic plants was confirmed by IR spectroscopy. Thus the infrared spectroscopy appeared to be suitable method for characterization of flax products.

New dressing materials derived from transgenic flax products to treat long-standing venous ulcers - a pilot study.

A new flax dressing product was developed based on three components (fibers, oil emulsion, and seedcake extract) from genetically engineered flax plants that were obtained by plant transformation using three genes controlling the synthesis of antioxidative compounds from the phenylpropanoid pathway. Simultaneous flax explant transformation with three genes coding for chalcone synthase, chalcone isomerase, and dihydroflavonol reductase resulted in an accumulation of phenolic acids in the fibers, polyunsaturated fatty acids in the oil, and lignans in the seedcake. The fibers, oil, and seedcake from transgenic flax contained a broad spectrum of antioxidative compounds. They were tested for cytotoxicity, and none were found to have a negative effect on the growth and morphology of Balb/3T3 cells. In this preliminary report, we present pilot data on the effects of using linen dressing treatment on its own or in combination with oil emulsion and/or seedcake extract on chronic wound healing. After a 12‐week study, we concluded that an application of a modified flax‐dressing (linen) bandage might yield a more rapid rate of healing and reduce the wound exudes and wound size. In several cases, wound healing was completed during the period of investigation. Interestingly and importantly, the patients reported that the new bandage made from modified flax diminished the pain accompanying chronic venous ulceration. Further study is required to determine any definitive effects of flax bandage on wound healing. This is the first pilot study report suggesting the benefits of a flax‐based dressing on wound healing.

Poly-3-hydroxy butyric acid interaction with the transgenic flax fibers: FT-IR and Raman spectra of the composite extracted from a GM flax.

The FT-IR and FT-Raman studies have been performed on commercial 3-hydroxy-butyric acid, commercial poly-3-hydroxy butyric acid as well as poly-3-hydroxy butyric acid (PHB) produced by bacteria. The data were compared to those obtained for poly-3-hydroxy butyric acid extracted from natural and genetically modified flax. Genetically modified flax was generated by expression of three bacterial genes coding for synthesis of poly-3-hydroxy butyric acid. Thus transgenic flaxes were enhanced with different amount of the PHB. The discussion of polymer structure and vibrational properties has been done in order to get insight into differences among these materials. The interaction between the cellulose of flax fibers and embedded poly-3-hydroxybutyric acid has been also discussed. The spectroscopic data provide evidences for structural changes in cellulose and in PHB when synthesized in fibers. Based on this data it is suggesting that cellulose and PHB interact by hydrogen and ester bonds.

IR and Raman studies of oil and seedcake extracts from natural and genetically modified flax seeds

Flax plant of the third generation (F3) overexpressing key genes of flavonoid pathway cultivated in field in 2008 season was used as the plant material throughout this study. The biochemical properties of seed, oil and seedcake extracts from natural and transgenic flax plants were compared. Overproduction of flavonoids (kaempferol), phenolic acids (coumaric, ferulic/synapic) and lignan-secoisolariciresinol diglucoside (SDG) in oil and extracts from transgenic seeds has been revealed providing a valuable source of these compounds for biotechnological application. The changes in fatty acids composition and increase in their stability against oxidation along three plant generations were also detected. The analysis of oil and seedcake extracts was performed using Raman and IR spectroscopy. The wavenumbers and integral intensities of Raman and IR bands were used to identify the components of phenylpropanoid pathway in oil and seedcake extracts from control and transgenic flax seeds. The spectroscopic data were compared to those obtained from biochemical analysis.

Flax Engineering for Biomedical Application

Flax (Linum usitatissimum) is an important crop plant that is widely distributed in the Mediterranean and temperate climate zones. It has great significance for industry as a valuable source of oil and fibres. A unique feature of flax is the possibility of whole plant exploitation with almost no waste products. For this reason, flax has quite significant potential for biotechnological application. To increase the valuable qualities of flax products, the flax genome has been genetically modified, with the specific aims to improve the plant’s pathogen resistance, taste and nutritional properties, and to produce pharmaceuticals and other compounds. In this chapter, we describe the plant characteristics that show the biochemical and industrial importance of flax oil and fibres and their various possible applications and the relevant genetic modifications. Since ancient times, flax has been known to be a source of oil and fibres, and it has been cultivated as a dual-purpose plant for a long time. Nowadays, it is a multi-purpose plant and its exploitation is not restricted to the production of linen fibre and oil. Actually, whole plant exploitation is possible, which justifies the name given to it by Linnaeus: L. usitatissimum, meaning “useful flax”. There is a wide range of possible applications of flax (Fig.1). The long fibres are used in the textile industry, and the short fibres in paper production, isolation materials and biocomposite production. The wooden shives released during flax scutching can serve as an energy source. Flax seeds also have many important applications, and due to its high nutritional value, it is used in the food, pharmaceutical and health care industries. The seedcake, which is rich in antioxidants, is used in the pharmaceutical and cosmetic industries. The development of molecular biology emerged as an important tool for the genetic modification of plants, and enabled the improvement of many different features of wild type plants. These modifications broadened the range of practical applications for flax, making the plant more valuable and more significant for the innovative biotechnological industry.

New biocomposites based on bioplastic flax fibers and biodegradable polymers

A new generation of entirely biodegradable and bioactive composites with polylactic acid (PLA) or poly‐ε‐caprolactone (PCL) as the matrix and bioplastic flax fibers as reinforcement were analyzed. Bioplastic fibers contain polyhydroxybutyrate and were obtained from transgenic flax. Biochemical analysis of fibers revealed presence of several antioxidative compounds of hydrophilic (phenolics) and hydrophobic [cannabidiol (CBD), lutein] nature, indicating their high antioxidant potential. The presence of CBD and lutein in flax fibers is reported for the first time. FTIR analysis showed intermolecular hydrogen bonds between the constituents in composite PLA+flax fibers which were not detected in PCL‐based composite. Mechanical analysis of prepared composites revealed improved stiffness and a decrease in tensile strength. The viability of human dermal fibroblasts on the surface of composites made of PLA and transgenic flax fibers was the same as for cells cultured without composites and only slightly lower (to 9%) for PCL‐based composites. The amount of platelets and Escherichia coli cells aggregated on the surface of the PLA based composites was significantly lower than for pure polymer. Thus, composites made of PLA and transgenic flax fibers seem to have bacteriostatic, platelet anti‐aggregated, and non‐cytotoxic effect.

Improved properties of micronized genetically modified flax fibers.

The aim of this study was to investigate the effect of micronization on the compound content, crystalline structure and physicochemical properties of fiber from genetically modified (GM) flax. The GM flax was transformed with three bacterial (Ralstonia eutropha) genes coding for enzymes of polyhydroxybutyrate (PHB) synthesis and under the control of the vascular bundle promoter. The modification resulted in fibers containing the 3-hydroxybutyrate polymer bound to cellulose via hydrogen and ester bonds and antioxidant compounds (phenolic acids, vanillin, vitexin, etc.). The fibers appeared to have a significantly decreased particle size after 20h of ball-milling treatment. Micronized fibers showed reduced phenolic contents and antioxidant capacity compared to the results for untreated fibers. An increased level of PHB was also detected. Micronization introduces structural changes in fiber constituents (cellulose, hemicellulose, pectin, lignin, PHB) and micronized fibers exhibit more functional groups (hydroxyl, carboxyl) derived from those constituents. It is thus concluded that micronization treatments improve the functional properties of the fiber components.

The influence of modified 14-3-3 protein synthesis in potato plants on the nutritional value of the tubers

Abstract The recently created six transgenic potato genotypes with overexpressed or underexpressed P14-3-3a (29G) and P14-3-3c (20R) isoforms of 14-3-3 protein were field-trialled (1998–2001). The contents of protein, starch, reducing sugars, sucrose and lipids were determined in the transgenic and control tubers harvested from the field. The obtained results showed a significant increase in crude protein content in potatoes with repression of P14-3-3c isoform and in potatoes with blocked P14-3-3a synthesis in comparison to the control line. A stable increase in lipid content of potatoes with overexpression of 14-3-3 protein from Cucurbita pepo in the field trials was observed. The variability of the investigated genotypes, in respect to the nutritional components, was statistically analysed using discriminant function and cluster analyses. The dominant influence of the variability of the genotypes exerted significant differentiation of protein, lipid and starch contents. These components showed the greatest discriminant power in the variability of genotypes. These results confirm the suggestion that 14-3-3 protein co-ordinates primary metabolite synthesis in plants.

Natural phenolics greatly increase flax (Linum usitatissimum) oil stability.

BackgroundFlaxseed oil is characterized by high content of essential polyunsaturated fatty acids (PUFA) promoted as a human dietary supplement protecting against atherosclerosis. The disadvantage of the high PUFA content in flax oil is high susceptibility to oxidation, which can result in carcinogenic compound formation. Linola flax cultivar is characterized by high linoleic acid content in comparison to traditional flax cultivars rich in linolenic acid. The changes in fatty acid proportions increase oxidative stability of Linola oil and broaden its use as an edible oil for cooking. However one of investigated transgenic lines has high ALA content making it suitable as omega-3 source. Protection of PUFA oxidation is a critical factor in oil quality. The aim of this study was to investigate the impact of phenylpropanoid contents on the oil properties important during the whole technological process from seed storage to grinding and oil pressing, which may influence health benefits as well as shelf-life, and to establish guidelines for the selection of new cultivars.MethodsThe composition of oils was determined by chromatographic (GS-FID and LC-PDA-MS) methods. Antioxidant properties of secondary metabolites were analyzed by DPPH method. The stability of oils was investigated: a) during regular storage by measuring acid value peroxide value p-anisidine value malondialdehyde, conjugated dienes and trienes; b) by using accelerated rancidity tests by TBARS reaction; c) by thermoanalytical - differential scanning calorimetry (DSC).ResultsIn one approach, in order to increase oil stability, exogenous substances added are mainly lipid soluble antioxidants from the isoprenoid pathway, such as tocopherol and carotene. The other approach is based on transgenic plant generation that accumulates water soluble compounds. Increased accumulation of phenolic compounds in flax seeds was achieved by three different strategies that modify genes coding for enzymes from the phenylpropanoid pathway. The three types of transgenic flax had different phenylpropanoid profiles detected in oil, highly increasing its stability.ConclusionsWe found that hydrophilic phenylpropanoids more than lipophilic isoprenoid compounds determine oil stability however they can work synergistically. Among phenolics the caffeic acid was most effective in increasing oil stability.

Repression of six 14-3-3 protein isoforms resulting in the activation of nitrate and carbon fixation key enzymes from transgenic potato plants

Six 14-3-3 full-size cDNAs from a potato plant were cloned and sequenced, and their high-sequence homology was established. The expression profile revealed developmental regulation and lack of organ specificity of 14-3-3 isoform synthesis in potato leaves. In order to analyse their function, transgenic plants with repression of all found isoforms were created. Since, 14-3-3 regulates in vitro assay sucrose phosphate synthase (SPS) and nitrate reductase (NR) activities, both enzyme activities and products of the interacting enzyme were analysed for transgenic plants. It was found that all transgenic lines showed significant increase in SPS and NR activities. The increase in enzyme activities was complemented by the addition of recombinant 14-3-3 protein either from potato or C. pepo. Thus, it is suggested that 14-3-3 is the main regulator of NR and SPS activities in in vivo trials and the regulatory function is isoform-independent. Sucrose and glutamate contents corresponded with the enzyme activity increase especially in case of two transgenic lines, G3.40 and G3.49, respectively. Four out of five analysed transgenic lines showed significant increase in tuber starch accumulation.