Nowadays, the challenge for biotechnology is to develop tools for agriculture and industry to provide plants characterized by productivity and quality that will satisfy the growing demand for different kinds of natural products. To meet the challenge, the generation and application of genetically modified plants is justified. However, the strong social resistance to genetically modified organisms and restrictive regulations in European Union countries necessitated the development of a new technology for new plant types generation which uses the knowledge resulting from analysis of genetically modified plants to generate favourably altered plants while omitting the introduction of heterologous genes to their genome. Four-year experiments led to the development of a technology inducing heritable epigenetic gene activation without transgenesis.
The method comprises the induction of changes in methylation/demethylation of the endogenous gene by the plant’s treatment with short oligodeoxynucleotides antisense to the coding region. In vitro cultured plants and F3 generation flax plants overproducing the β-1,3-glucanase gene (EMO-βGlu flax) were characterized by up-regulation of β-glucanase and chitinase genes, decreases in the methylation of CCGG sequences in the β-glucanase gene and in total DNA methylation and, more importantly, reasonable resistance against Fusarium infection. In addition, EMO-βGlu flax obtained by this technology showed similar features as those obtained by genetic engineering.
To our best knowledge, this is the first report on plant gene activation by treatment with oligodeoxynucleotides homologous to the coding region of the gene. Apart from the evident effectiveness, the most important issue is that the EMO method allows generation of favourably altered plants, whose cultivation makes the plant producer independent from the complicated procedure of obtaining an agreement on GMO release into the environment and whose products might be more easily introduced to the global market.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0261-z) contains supplementary material, which is available to authorized users.
Flax resistance; Fusarium infection; Epigenetically modified organism; DNA methylation/demethylation; OLIGO technology; β-1,3-glucanase
The aim of the work was to define the influence of dietary supplementation with GM (genetically modified) GT#4 flaxseed cake enriched in polyphenols on inflammation development in mice liver. Mice were given ad libitum isoprotein diets: (1) standard diet; (2) high-fat diet rich in lard, high-fat diet enriched with 30% of (3) isogenic flax Linola seed cake; and (4) GM GT#4 flaxseed cake; for 96 days. Administration of transgenic and isogenic seed cake lowered body weight gain, of transgenic to the standard diet level. Serum total antioxidant status was statistically significantly improved in GT#4 flaxseed cake group and did not differ from Linola. Serum thiobarbituric acid reactive substances, lipid profile and the liver concentration of pro-inflammatory cytokine tumor necrosis factor-α were ameliorated by GM and isogenic flaxseed cake consumption. The level of pro-inflammatory cytokine interferon-γ did not differ between mice obtaining GM GT#4 and non-GM flaxseed cakes. The C-reactive protein concentration was reduced in animals fed GT#4 flaxseed cake and did not differ from those fed non-GM flaxseed cake-based diet. Similarly, the liver structure of mice consuming diets enriched in flaxseed cake was improved. Dietetic enrichment with GM GT#4 and non-GM flaxseed cakes may be a promising solution for health problems resulting from improper diet.
genetically modified flax; seed cake; flavonoid glycosides; inflammation; food safety; mouse
Flavonoids are a large group of secondary plant metabolites with many important functions; they play a role in fruit, flower and seed pigmentation and are involved in multiple protective mechanisms. They are very active natural antioxidants, acting as antimicrobial compounds in defense against pathogens, and they protect the plant against various stress factors, including excessive solar radiation and temperature. They are also an animal deterrent. Flax is already a very useful crop plant with nutritional and biomedical applications. With increased phenylpropanoid content, flax plants could be used in the production of improved dietary supplements and antimicrobial agents. The main aim of this study was to engineer a flax variety with increased flavonoid content by crossing two transgenic flax varieties that have heightened flavonoid levels. A mother plant that over expresses genes encoding the flavonoid biosynthesis pathway enzymes chalcone synthase, chalcone isomerase and dihydroflavonol reductase was crossed with plants overexpressing the glucosyltransferase (GT) gene. It was expected that the progeny would display better properties thanks to the simultaneous increases in flavonoid synthesis and stability. In comparison to the control and parental plants, plants of the selected flax lines were found to have increased contents of flavonoids and other phenylpropanoids, including phenolic acids, in their stems and seeds. A significant increase in the secoisolariciresinol diglucoside content was found in the seeds. The antioxidative properties of extracts from W92 × GT crossbreed plants were higher than the control (non-transgenic) and parental plants. These results correlated with the increase in the susceptibility of the crossbreeds to Fusarium infection. The increased flavonoid content did not cause any negative phenotypic changes or reduce the yield of seeds.
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The online version of this article (doi:10.1007/s11032-014-0149-5) contains supplementary material, which is available to authorized users.
Flax; Flavonoids; Fusarium; Cross breeding; Antioxidants; Antifungal activity
The antibiotic resistance of pathogenic microorganisms is a worldwide problem. Each year several million people across the world acquire infections with bacteria that are antibiotic-resistant, which is costly in terms of human health. New antibiotics are extremely needed to overcome the current resistance problem.
Transgenic flax plants overproducing compounds from phenylpropanoid pathway accumulate phenolic derivatives of potential antioxidative, and thus, antimicrobial activity. Alkali hydrolyzed seedcake extract containing coumaric acid, ferulic acid, caffeic acid, and lignan in high quantities was used as an assayed against pathogenic bacteria (commonly used model organisms and clinical strains). It was shown that the extract components had antibacterial activity, which might be useful as a prophylactic against bacterial infection. Bacteria topoisomerase II (gyrase) inhibition and genomic DNA disintegration are suggested to be the main reason for rendering antibacterial action.
The data obtained strongly suggest that the seedcake extract preparation is a suitable candidate for antimicrobial action with a broad spectrum and partial selectivity. Such preparation can be applied in cases where there is a risk of multibacterial infection and excellent answer on global increase in multidrug resistance in pathogenic bacteria.
Antimicrobial compound; Phenolic acid; Flax; Alternative antibiotic; Flax seedcake
In recent decades cultivation of flax and its application have dramatically decreased. One of the reasons for this is unpredictable quality and properties of flax fibre, because they depend on environmental factors, retting duration and growing conditions. These factors have contribution to the fibre composition, which consists of cellulose, hemicelluloses, lignin and pectin. By far, it is largely established that in flax, lignin reduces an accessibility of enzymes either to pectin, hemicelluloses or cellulose (during retting or in biofuel synthesis and paper production).
Therefore, in this study we evaluated composition and properties of flax fibre from plants with silenced CAD (cinnamyl alcohol dehydrogenase) gene, which is key in the lignin biosynthesis. There is evidence that CAD is a useful tool to improve lignin digestibility and/or to lower the lignin levels in plants.
Two studied lines responded differentially to the introduced modification due to the efficiency of the CAD silencing. Phylogenetic analysis revealed that flax CAD belongs to the “bona-fide” CAD family. CAD down-regulation had an effect in the reduced lignin amount in the flax fibre cell wall and as FT-IR results suggests, disturbed lignin composition and structure. Moreover introduced modification activated a compensatory mechanism which was manifested in the accumulation of cellulose and/or pectin. These changes had putative correlation with observed improved fiber’s tensile strength. Moreover, CAD down-regulation did not disturb at all or has only slight effect on flax plants’ development in vivo, however, the resistance against flax major pathogen Fusarium oxysporum decreased slightly. The modification positively affected fibre possessing; it resulted in more uniform retting.
The major finding of our paper is that the modification targeted directly to block lignin synthesis caused not only reduced lignin level in fibre, but also affected amount and organization of cellulose and pectin. However, to conclude that all observed changes are trustworthy and correlated exclusively to CAD repression, further analysis of the modified plants genome is necessary. Secondly, this is one of the first studies on the crop from the low-lignin plants from the field trail which demonstrates that such plants could be successfully cultivated in a field.
Cinnamyl alcohol dehydrogenase (CAD); Lignin; Cell wall; Flax fibre; Linum usitatissimum; L
Recently, in order to improve the resistance of flax plants to pathogen infection, transgenic flax that overproduces β-1,3-glucanase was created. β-1,3-glucanase is a PR protein that hydrolyses the β-glucans, which are a major component of the cell wall in many groups of fungi. For this study, we used fourth-generation field-cultivated plants of the Fusarium -resistant transgenic line B14 to evaluate how overexpression of the β-1,3-glucanase gene influences the quantity, quality and composition of flax fibres, which are the main product obtained from flax straw.
Overproduction of β-1,3-glucanase did not affect the quantity of the fibre obtained from the flax straw and did not significantly alter the essential mechanical characteristics of the retted fibres. However, changes in the contents of the major components of the cell wall (cellulose, hemicellulose, pectin and lignin) were revealed. Overexpression of the β-1,3-glucanase gene resulted in higher cellulose, hemicellulose and pectin contents and a lower lignin content in the fibres. Increases in the uronic acid content in particular fractions (with the exception of the 1 M KOH-soluble fraction of hemicelluloses) and changes in the sugar composition of the cell wall were detected in the fibres of the transgenic flax when compared to the contents for the control plants. The callose content was lower in the fibres of the transgenic flax. Additionally, the analysis of phenolic compound contents in five fractions of the cell wall revealed important changes, which were reflected in the antioxidant potential of these fractions.
Overexpression of the β-1,3-glucanase gene has a significant influence on the biochemical composition of flax fibres. The constitutive overproduction of β-1,3-glucanase causes a decrease in the callose content, and the resulting excess glucose serves as a substrate for the production of other polysaccharides. The monosaccharide excess redirects the phenolic compounds to bind with polysaccharides instead of to partake in lignin synthesis. The mechanical properties of the transgenic fibres are strengthened by their improved biochemical composition, and the increased antioxidant potential of the fibres supports the potential use of transgenic flax fibres for biomedical applications.
Biopolymers; Fibres; Flax; Linum usitatissimum
FlaxAid is a newly developed type of dressing enriched in particular flavonoids through genetic engineering of flax plants that exhibit health-promoting activities due to their strong antioxidant properties. The purpose of the current study was to assess the clinical efficacy of the FlaxAid bandage therapy for a patient affected with a diabetic foot ulcer which was unresponsive to previous treatments. The patient was treated with FlaxAid bandages for 12 weeks and the size and properties of the wound were routinely observed and recorded. Due to the the clinical picture of the wound study design was adopted whereby the comparative treatment was cotton gauze wetted with isotonic salt solution.
Following therapy, the foot ulcer decreased in size, despite the decompensation of advanced diabetes. It is believed that the beneficial nature of FlaxAid is derived from its high level and broad spectrum of antioxidants.
The FlaxAid dressing provides a novel and effective method for the treatment of diabetic foot ulcers. This study presents a preliminary pilot investigation and a larger number of subjects need to be included within the study in order to draw firm clinical conclusions. Efforts to this effect are currently under way.
Leg ulcer; Diabetes; Linen dressing; Therapy; Genetic engineering
Flax (Linum usitatissimum) is a potential source of many bioactive components that can be found in its oil and fibers, but also in the seedcake, which is rich in antioxidants. To increase the levels of medically beneficial compounds, a genetically modified flax type (named GT) with an elevated level of phenylopropanoids and their glycoside derivatives was generated. In this study, we investigated the influence of GT seedcake extract preparations on human fibroblast proliferation and migration, and looked at the effect on a human skin model. Moreover, we verified its activity against bacteria of clinical relevance.
The GT flax used in this study is characterized by overexpression of the glucosyltransferase gene derived from Solanum sogarandinum. Five GT seedcake preparations were generated. Their composition was assessed using ultra pressure liquid chromatography and confirmed using the UPLC-QTOF method. For the in vitro evaluation, the influence of the GT seedcake preparations on normal human dermal fibroblast proliferation was assessed using the MTT test and the wound scratch assay. A human skin model was used to evaluate the potential for skin irritation. To assess the antimicrobial properties of GT preparations, the percentage of inhibition of bacterial growth was calculated.
The GT seedcake extract had elevated levels of phenylopropanoid compounds in comparison to the control, non-transformed plants. Significant increases in the content of ferulic acid, p-coumaric acid and caffeic acid, and their glucoside derivatives, kaempferol, quercitin and secoisolariciresinol diglucoside (SDG) were observed in the seeds of the modified plants. The GT seedcake preparations were shown to promote the proliferation of normal human dermal fibroblasts and the migration of fibroblasts in the wound scratch assay. The superior effect of GT seedcake extract on fibroblast migration was observed after a 24-hour treatment. The skin irritation test indicated that GT seedcake preparations have no harmful effect on human skin. Moreover, GT seedcake preparations exhibited inhibitory properties toward two bacterial strains: Staphylococcus aureus and Escherichia coli.
We suggest that preparations derived from the new GT flax are an effective source of phenylopropanoids and that their glycoside derivatives and might be promising natural products with both healing and bacteriostatic effects. This flax-derived product is a good candidate for application in the repair and regeneration of human skin and might also be an alternative to antibiotic therapy for infected wounds.
Flax; Seedcake; Phenylpropanoids; Fibroblasts
Although arbuscular mycorrhizal fungi (AMF) are known for their positive effect on flax growth, the impact of genetic manipulation in this crop on arbuscular mycorrhiza and plant performance was assessed for the first time. Five types of transgenic flax that were generated to improve fiber quality and resistance to pathogens, through increased levels of either phenylpropanoids (W92.40), glycosyltransferase (GT4, GT5), or PR2 beta-1,3-glucanase (B14) or produce polyhydroxybutyrate (M50), were used. Introduced genetic modifications did not change the degree of mycorrhizal colonization as compared to parent cultivars Linola and Nike. Arbuscules were well developed in each tested transgenic type (except M50). In two lines (W92.40 and B14), a higher abundance of arbuscules was observed when compared to control, untransformed flax plants. However, in some cases (W92.40, GT4, GT5, and B14 Md), the mycorrhizal dependency for biomass production of transgenic plants was slightly lower when compared to the original cultivars. No significant influence of mycorrhiza on the photosynthetic activity of transformed lines was found, but in most cases P concentration in mycorrhizal plants remained higher than in nonmycorrhizal ones. The transformed flax lines meet the demands for better quality of fiber and higher resistance to pathogens, without significantly influencing the interaction with AMF.
Mycorrhiza; Linum usitatissimum; Transgenic flax; Plant performance
Flavonoids 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.
Flax 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.
All 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.
In a previous study, anthocyanin levels in potato plants were increased by manipulating genes connected with the flavonoid biosynthesis pathway. However, starch content and tuber yield were dramatically reduced in the transgenic plants, which over-expressed dihydroflavonol reductase (DFR).
Transgenic plants over-expressing dihydroflavonol reductase (DFR) were subsequently transformed with the cDNA coding for the glycosyltransferase (UGT) of Solanum sogarandinum in order to obtain plants with a high anthocyanin content without reducing tuber yield and quality. Based on enzyme studies, the recombinant UGT is a 7-O-glycosyltransferase whose natural substrates include both anthocyanidins and flavonols such as kaempferol and quercetin. In the super-transformed plants, tuber production was much higher than in the original transgenic plants bearing only the transgene coding for DFR, and was almost the same as in the control plants. The anthocyanin level was lower than in the initial plants, but still higher than in the control plants. Unexpectedly, the super-transformed plants also produced large amounts of kaempferol, chlorogenic acid, isochlorogenic acid, sinapic acid and proanthocyanins.
In plants over-expressing both the transgene for DFR and the transgene for UGT, the synthesis of phenolic acids was diverted away from the anthocyanin branch. This represents a novel approach to manipulating phenolic acids synthesis in plants.
Even though the catecholamines (dopamine, norepinephrine and epinephrine) have been detected in plants their role is poorly documented. Correlations between norepinephrine, soluble sugars and starch concentration have been recently reported for potato plants over-expressing tyrosine decarboxylase, the enzyme mediating the first step of catecholamine synthesis. More recently norepinephrine level was shown to significantly increase after osmotic stress, abscisic acid treatment and wounding. Therefore, it is possible that catecholamines might play a role in plant stress responses by modulating primary carbon metabolism, possibly by a mechanism similar to that in animal cells. Since to date no catecholamine receptor has been identified in plants we transformed potato plants with a cDNA encoding human dopamine receptor (HD1).
Tuber analysis of transgenic plants revealed changes in the activities of key enzymes mediating sucrose to starch conversion (ADP-glucose phosphorylase and sucrose synthase) and sucrose synthesis (sucrose phosphate synthase) leading to altered content of both soluble sugars and starch. Surprisingly the catecholamine level measured in transgenic plants was significantly increased; the reason for this is as yet unknown. However the presence of the receptor affected a broader range of enzyme activities than those affected by the massive accumulation of norepinephrine reported for plants over-expressing tyrosine decarboxylase. Therefore, it is suggested that the presence of the exogenous receptor activates catecholamine cAMP signalling in plants.
Our data support the possible involvement of catecholamines in regulating plant carbon metabolism via cAMP signalling pathway.