Endophytic non-pathogenic Fusarium oxysporum reorganizes the cell wall in flax seedlings.

Introduction: Flax (Linum usitatissimum) is a crop producing valuable products like seeds and fiber. However, its cultivation faces challenges from environmental stress factors and significant yield losses due to fungal infections. The major threat is Fusarium oxysporum f.sp lini, causing fusarium wilt of flax. Interestingly, within the Fusarium family, there are non-pathogenic strains known as biocontrols, which protect plants from infections caused by pathogenic strains. When exposed to a non-pathogenic strain, flax exhibits defense responses similar to those seen during pathogenic infections. This sensitization process activates immune reactions, preparing the plant to better combat potential pathogenic strains. The plant cell wall is crucial for defending against pathogens. It serves as the primary barrier, blocking pathogen entry into plant cells. Methods: The aim of the study was to investigate the effects of treating flax with a non-pathogenic Fusarium oxysporum strain, focusing on cell wall remodeling. The infection's progress was monitored by determining the fungal DNA content and microscopic observation. The plant defense response was confirmed by an increase in the level of Pathogenesis-Related (PR) genes transcripts. The reorganization of flax cell wall during non-pathogenic Fusarium oxysporum strain infection was examined using Infrared spectroscopy (IR), determination of cell wall polymer content, and analysis of mRNA level of genes involved in their metabolism. Results and discussion: IR analysis revealed reduced cellulose content in flax seedlings after treatment with Fo47 and that the cellulose chains were shorter and more loosely bound. Hemicellulose content was also reduced but only after 12h and 36h. The total pectin content remained unchanged, while the relative share of simple sugars and uronic acids in the pectin fractions changed over time. In addition, a dynamic change in the level of methylesterification of carboxyl groups of pectin was observed in flax seedlings treated with Fo47 compared to untreated seedlings. The increase in lignin content was observed only 48 hours after the treatment with non-pathogenic Fusarium oxysporum. Analysis of mRNA levels of cell wall polymer metabolism genes showed significant changes over time in all analyzed genes. In conclusion, the research suggests that the rearrangement of the cell wall is likely one of the mechanisms behind flax sensitization by the non-pathogenic Fusarium oxysporum strain. Understanding these processes could help in developing strategies to enhance flax's resistance to fusarium wilt and improve its overall yield and quality.

Epigenetic weapons of plants against fungal pathogens.

In the natural environment, plants face constant exposure to biotic stress caused by fungal attacks. The plant's response to various biotic stresses relies heavily on its ability to rapidly adjust the transcriptome. External signals are transmitted to the nucleus, leading to activation of transcription factors that subsequently enhance the expression of specific defense-related genes. Epigenetic mechanisms, including histone modifications and DNA methylation, which are closely linked to chromatin states, regulate gene expression associated with defense against biotic stress. Additionally, chromatin remodelers and non-coding RNA play a significant role in plant defense against stressors. These molecular modifications enable plants to exhibit enhanced resistance and productivity under diverse environmental conditions. Epigenetic mechanisms also contribute to stress-induced environmental epigenetic memory and priming in plants, enabling them to recall past molecular experiences and utilize this stored information for adaptation to new conditions. In the arms race between fungi and plants, a significant aspect is the cross-kingdom RNAi mechanism, whereby sRNAs can traverse organismal boundaries. Fungi utilize sRNA as an effector molecule to silence plant resistance genes, while plants transport sRNA, primarily through extracellular vesicles, to pathogens in order to suppress virulence-related genes. In this review, we summarize contemporary knowledge on epigenetic mechanisms of plant defense against attack by pathogenic fungi. The role of epigenetic mechanisms during plant-fungus symbiotic interactions is also considered.

Overexpression of Bacterial Beta-Ketothiolase Improves Flax (Linum usitatissimum L.) Retting and Changes the Fibre Properties.

Beta-ketothiolases are involved in the beta-oxidation of fatty acids and the metabolism of hormones, benzenoids, and hydroxybutyrate. The expression of bacterial beta-ketothiolase in flax (Linum usitatissimum L.) results in an increase in endogenous beta-ketothiolase mRNA levels and beta-hydroxybutyrate content. In the present work, the effect of overexpression of beta-ketothiolase on retting and stem and fibre composition of flax plants is presented. The content of the components was evaluated by high-performance liquid chromatography, gas chromatography-mass spectrometry, Fourier-transform infrared spectroscopy, and biochemical methods. Changes in the stem cell walls, especially in the lower lignin and pectin content, resulted in more efficient retting. The overexpression of beta-ketothiolase reduced the fatty acid and carotenoid contents in flax and affected the distribution of phenolic compounds between free and cell wall-bound components. The obtained fibres were characterized by a slightly lower content of phenolic compounds and changes in the composition of the cell wall. Based on the IR analysis, we concluded that the production of hydroxybutyrate reduced the cellulose crystallinity and led to the formation of shorter but more flexible cellulose chains, while not changing the content of the cell wall components. We speculate that the changes in chemical composition of the stems and fibres are the result of the regulatory properties of hydroxybutyrate. This provides us with a novel way to influence metabolic composition in agriculturally important crops.

Abscisic Acid-Defensive Player in Flax Response to Fusarium culmorum Infection.

Fusarium culmorum is a ubiquitous soil pathogen with a wide host range. In flax (Linum ussitatissimum), it causes foot and root rot and accumulation of mycotoxins in flax products. Fungal infections lead to huge losses in the flax industry. Moreover, due to mycotoxin accumulation, flax products constitute a potential threat to the consumers. We discovered that the defense against this pathogen in flax is based on early oxidative burst among others. In flax plants infected with F. culmorum, the most affected genes are connected with ROS production and processing, callose synthesis and ABA production. We hypothesize that ABA triggers defense mechanism in flax and is a significant player in a successful response to infection.

Composition and Antimicrobial Activity of Ilex Leaves Water Extracts.

Plants from the Ilex genus are known for properties such as antimicrobial and anti-inflammatory activity, can act as antiobesity agents and thus can be helpful in medicine. Some holly species, such as Ilex paraguariensis (widely known in the form of popular beverage: yerba mate), have been investigated, while others have been partially researched or remain unknown. Therefore, we performed qualitative and quantitative phytochemical analyses and screened antimicrobial properties of lesser-studied species (I. aquifolium L., I. aquifolium 'Argentea Marginata' and I. × meserveae 'Blue Angel'). I. paraguariensis was used as a standard species for comparison purposes. Investigations were performed on water extracts due to their expected activity and composition. Antimicrobial research included evaluating minimal inhibitory, bactericidal (Staphylococcus aureus and Escherichia coli) and fungicidal concentration (Candida albicans, Alternaria alternata, Fusarium oxysporum, and Aspergillus niger) of extracts. The influence of the extracts on the production, eradication, and viability of bacterial biofilms was also analysed. It was established that Ilex paraguariensis possesses the richest profile of hydroxycinnamic acids derivatives in terms of component concentration and diversity. Ilex spp., especially I. × meserveae, contain a slightly higher amount of flavonoids and more different flavonoid derivatives than I. paraguariensis. However, the strongest antibacterial activity was shown by I. aquifolium L. and its cultivar 'Argentea Marginata' in terms of minimal inhibitory, bactericidal and fungicidal concentration, and biofilm assays. Extracts from both species significantly reduced the biofilm viability of S. aureus as well, which may be of use in the production of multicomponent lavaseptics, antiseptics, diuretics (supporting urinary tract infection therapy) and, due to their action on fungi, additives to growth media for specific fungi. The significant content of saponins enables Ilex extracts to be used as natural emulsifiers, for example, in cosmetics. Moreover, relatively high chlorogenic acid and rutin content may suggest use of Ilex spp. to treat obesity, digestive problems, in chemoprevention, and as preservatives in the food industry.

Influence of the Bioactive Diet Components on the Gene Expression Regulation.

Diet bioactive components, in the concept of nutrigenetics and nutrigenomics, consist of food constituents, which can transfer information from the external environment and influence gene expression in the cell and thus the function of the whole organism. It is crucial to regard food not only as the source of energy and basic nutriments, crucial for living and organism development, but also as the factor influencing health/disease, biochemical mechanisms, and activation of biochemical pathways. Bioactive components of the diet regulate gene expression through changes in the chromatin structure (including DNA methylation and histone modification), non-coding RNA, activation of transcription factors by signalling cascades, or direct ligand binding to the nuclear receptors. Analysis of interactions between diet components and human genome structure and gene activity is a modern approach that will help to better understand these relations and will allow designing dietary guidances, which can help maintain good health.

3-Hydroxybutyrate as a Metabolite and a Signal Molecule Regulating Processes of Living Organisms.

3-hydroxybutyrate (3-HB) as a very important metabolite occurs in animals, bacteria and plants. It is well known that in animals, 3-HB is formed as a product of the normal metabolism of fatty acid oxidation and can therefore be used as an energy source in the absence of sufficient blood glucose. In microorganisms, 3-HB mainly serves as a substrate for the synthesis of polyhydroxybutyrate, which is a reserve material. Recent studies show that in plants, 3-HB acts as a regulatory molecule that most likely influences the expression of genes involved in DNA methylation, thereby altering DNA methylation levels. Additionally, in animals, 3-HB is not only an intermediate metabolite, but also an important regulatory molecule that can influence gene expression, lipid metabolism, neuronal function, and overall metabolic rate. Some of these effects are the direct effects of 3-HB itself, while others are indirect effects, regulated by the metabolites into which 3-HB is converted. One of the most important regulatory functions of 3-HB is the inhibition of the activity of histone deacetylases and thus the epigenetic regulation of many genes. Due to the number of functions of this compound, it also shows promising therapeutic properties.

3-Hydroxybutyrate Is Active Compound in Flax That Upregulates Genes Involved in DNA Methylation.

In mammalian cells, 3-hydroxybutyrate (3-HB) is not only an intermediate metabolite during the oxidation of fatty acids, but also an important signaling molecule. On the other hand, the information about the metabolism or function of this compound in plants is scarce. In our study, we show for the first time that this compound naturally occurs in flax. The expression of bacterial ß-ketothiolase in flax affects expression of endogenous genes of the 3-HB biosynthesis pathway and the compound content. The increase in 3-HB content in transgenic plants or after control plants treatment with 3-HB resulted in upregulation of genes involved in chromatin remodeling. The observation that 3-HB is an endogenous activator of methyltransferase 3 (CMT3), decreased DNA methylation I (DDM1), DEMETER DNA glycosylase (DME), and an inhibitor of sirtuin 1 (SRT1) provides an example of integration of different genes in chromatin remodeling. The changes in chromatin remodeling gene expression concomitant with those involved in phenolics and the lignin biosynthesis pathway suggest potential integration of secondary metabolic status with epigenetic changes.

Fusarium oxysporum infection activates the plastidial branch of the terpenoid biosynthesis pathway in flax, leading to increased ABA synthesis.

MAIN CONCLUSION: Upregulation of the terpenoid pathway and increased ABA content in flax upon Fusarium infection leads to activation of the early plant's response (PR genes, cell wall remodeling, and redox status). Plants have developed a number of defense strategies against the adverse effects of fungi such as Fusarium oxysporum. One such defense is the production of antioxidant secondary metabolites, which fall into two main groups: the phenylpropanoids and the terpenoids. While functions and biosynthesis of phenylpropanoids have been extensively studied, very little is known about the genes controlling the terpenoid synthesis pathway in flax. They can serve as antioxidants, but are also substrates for a plethora of different compounds, including those of regulatory functions, like ABA. ABA's function during pathogen attack remains obscure and often depends on the specific plant-pathogen interactions. In our study we showed that in flax the non-mevalonate pathway is strongly activated in the early hours of pathogen infection and that there is a redirection of metabolites towards ABA synthesis. The elevated synthesis of ABA correlates with flax resistance to F. oxysporum, thus we suggest ABA to be a positive regulator of the plant's early response to the infection.

DNA Methylation Profile of ß-1,3-Glucanase and Chitinase Genes in Flax Shows Specificity Towards Fusarium Oxysporum Strains Differing in Pathogenicity.

Most losses in flax (Linum usitatissimum L.) crops are caused by fungal infections. The new epigenetic approach to improve plant resistance requires broadening the knowledge about the influence of pathogenic and non-pathogenic Fusarium oxysporum strains on changes in the profile of DNA methylation. Two contrasting effects on the levels of methylation in flax have been detected for both types of Fusarium strain infection: Genome-wide hypermethylation and hypomethylation of resistance-related genes (ß-1,3-glucanase and chitinase). Despite the differences in methylation profile, the expression of these genes increased. Plants pretreated with the non-pathogenic strain memorize the hypomethylation pattern and then react more efficiently upon pathogen infection. The peak of demethylation correlates with the alteration in gene expression induced by the non-pathogenic strain. In the case of pathogen infection, the expression peak lags behind the gene demethylation. Dynamic changes in tetramer methylation induced by both pathogenic and non-pathogenic Fusarium strains are dependent on the ratio between the level of methyltransferase and demethylase gene expression. Infection with both Fusarium strains suppressed methyltransferase expression and increased the demethylase (demeter) transcript level. The obtained results provide important new information about changes in methylation profile and thus expression regulation of pathogenesis-related genes in the flax plant response to stressors.

Expression of heterologous lycopene ß-cyclase gene in flax can cause silencing of its endogenous counterpart by changes in gene-body methylation and in ABA homeostasis mechanism.

Previously we described flax plants with expression of Arabidopsis lycopene ß-cyclase (lcb) gene in which decreased expression of the endogenous lcb and increased resistance to fungal pathogen was observed. We suggested that co-suppression was responsible for the change. In this study we investigated the molecular basis of the observed effect in detail. We found that methylation changes in the Lulcb gene body might be responsible for repression of the gene. Treatment with azacitidine (DNA methylation inhibitor) confirmed the results. Moreover, we studied how the manipulation of carotenoid biosynthesis pathway increased ABA level in these plants. We suggest that elevated ABA levels may be responsible for the increased resistance of the flax plants to pathogen infection through activation of chitinase (PR gene).

The cinnamyl alcohol dehydrogenase family in flax: Differentiation during plant growth and under stress conditions.

Cinnamyl alcohol dehydrogenase (CAD), which catalyzes the reduction of cinnamaldehydes to their alcohol derivatives, is represented by a large family of proteins. The aim of the study was to identify the CAD isoforms in flax (Linum usitatissimum L.) - LuCADs - and to determine their specificity to enhance knowledge of the mechanisms controlling cell wall lignification in flax under environmental stresses. On the basis of genome-wide analysis, we identified 15 isoforms (one in two copies) belonging to three major classes of the CAD protein family. Their specificity was determined at the transcriptomic level in different tissues/organs, under Fusarium infection and abiotic stresses. Considering the function of particular LuCADs, it was established that LuCAD1 and 2 belong to Class I and they take part in the lignification of maturing stem and in the response to cold and drought stress. The Class II members LuCAD3, LuCAD4, LuCAD5 and LuCAD6 play various roles in flax being putatively responsible for lignin synthesis in different organs or under certain conditions. The obtained results indicate that within Class II, LuCAD6 was the most abundant in seedlings and maturing stems, LuCAD3 in leaves, and LuCAD4 in stems. Comparative analysis showed that expression of LuCAD genes in roots after F. oxysporum infection had the greatest contribution to differentiation of LuCAD expression patterns. Surprisingly, most of the analyzed LuCAD isoforms had reduced expression after pathogen infection. The decrease in mRNA level was primarily observed for LuCAD6 and LuCAD4, but also LuCAD1 and 8. However, the induction of LuCAD expression was mostly characteristic for Class I LuCAD1 and 2 in leaves. For cold stress, a clear correlation with phylogenic class membership was observed. Low temperatures caused induction of CAD isoforms belonging to Class I and repression of LuCADs from Class III.

V79 Fibroblasts Are Protected Against Reactive Oxygen Species by Flax Fabric.

Chinese hamster pulmonary fibroblasts (V79 cells) pre-treated with flax fabrics derived from non-modified or genetically engineered flax fibres and treated with H2O2 revealed a markedly lower level of intracellular reactive oxygen species (ROS) than control, non-pre-treated cells. The fabrics were prepared from fibres derived from two kinds of transgenic plants: W92 plants, which overproduce flavonoids, and M type plants, which produce hydroxybutyrate polymer in their vascular bundles and thus in fibres. Incubating the V79 cells with the flax fabrics prior to H2O2 treatment also reduced the amount of DNA damage, as established using the comet assay (also known as alkaline single-cell gel electrophoresis) and pulsed-field electrophoresis of intact cellular DNA. Selected gene expression analysis revealed the activator impact of fabrics on the apoptotic (BCL2 family, caspases) gene expression. This promoting activity was also detected for histone acetyltransferase (HAT; MYST2) gene expression. The flax fabric derived from both GM flax plants exhibited a protective effect against oxidative stress and ROS-mediated genotoxic damage, but the W92 fabric was the strongest. It is thus suggested that these fabrics might be useful as a basis for new biomedical products (e.g. wound dressings) that actively protect cells against inflammation and degeneration.

Evaluation of the significance of cell wall polymers in flax infected with a pathogenic strain of Fusarium oxysporum.

BACKGROUND: Fusarium oxysporum infection leads to Fusarium-derived wilt, which is responsible for the greatest losses in flax (Linum usitatissimum) crop yield. Plants infected by Fusarium oxysporum show severe symptoms of dehydration due to the growth of the fungus in vascular tissues. As the disease develops, vascular browning and leaf yellowing can be observed. In the case of more virulent strains, plants die. The pathogen's attack starts with secretion of enzymes degrading the host cell wall. The main aim of the study was to evaluate the role of the cell wall polymers in the flax plant response to the infection in order to better understand the process of resistance and develop new ways to protect plants against infection. For this purpose, the expression of genes involved in cell wall polymer metabolism and corresponding polymer levels were investigated in flax seedlings after incubation with Fusarium oxysporum. RESULTS: This analysis was facilitated by selecting two groups of genes responding differently to the infection. The first group comprised genes strongly affected by the infection and activated later (phenylalanine ammonia lyase and glucosyltransferase). The second group comprised genes which are slightly affected (up to five times) and their expression vary as the infection progresses. Fusarium oxysporum infection did not affect the contents of cell wall polymers, but changed their structure. CONCLUSION: The results suggest that the role of the cell wall polymers in the plant response to Fusarium oxysporum infection is manifested through changes in expression of their genes and rearrangement of the cell wall polymers. Our studies provided new information about the role of cellulose and hemicelluloses in the infection process, the change of their structure and the expression of genes participating in their metabolism during the pathogen infection. We also confirmed the role of pectin and lignin in this process, indicating the major changes at the mRNA level of lignin metabolism genes and the loosening of the pectin structure.

Emulsions Made of Oils from Seeds of GM Flax Protect V79 Cells against Oxidative Stress.

Polyunsaturated fatty acids, sterols, and hydrophilic phenolic compounds are components of flax oil that act as antioxidants. We investigated the impact of flax oil from transgenic flax in the form of emulsions on stressed Chinese hamster pulmonary fibroblasts. We found that the emulsions protect V79 cells against the H2O2 and the effect is dose dependent. They reduced the level of intracellular reactive oxygen species and protected genomic DNA against damage. The rate of cell proliferation increased upon treatment with the emulsions at a low concentration, while at a high concentration it decreased significantly, accompanied by increased frequency of apoptotic cell death. Expression analysis of selected genes revealed the upregulatory impact of the emulsions on the histones, acetylases, and deacetylases. Expression of apoptotic, proinflammatory, and anti-inflammatory genes was also altered. It is thus suggested that flax oil emulsions might be useful as a basis for biomedical products that actively protect cells against inflammation and degeneration. The beneficial effect on fibroblast resistance to oxidative damage was superior in the emulsion made of oil from transgenic plants which was correlated with the quantity of antioxidants and squalene. The emulsions from transgenic flax are promising candidates for skin protection against oxidative damage.

Methyl Salicylate Level Increase in Flax after Fusarium oxysporum Infection Is Associated with Phenylpropanoid Pathway Activation.

Flax (Linum usitatissimum) is a crop plant valued for its oil and fiber. Unfortunately, large losses in cultivation of this plant are caused by fungal infections, with Fusarium oxysporum being one of its most dangerous pathogens. Among the plant's defense strategies, changes in the expression of genes of the shikimate/phenylpropanoid/benzoate pathway and thus in phenolic contents occur. Among the benzoates, salicylic acid, and its methylated form methyl salicylate play an important role in regulating plants' response to stress conditions. Upon treatment of flax plants with the fungus we found that methyl salicylate content increased (4.8-fold of the control) and the expression profiles of the analyzed genes suggest that it is produced most likely from cinnamic acid, through the ß-oxidative route. At the same time activation of some genes involved in lignin and flavonoid biosynthesis was observed. We suggest that increased methyl salicylate biosynthesis during flax response to F. oxysporum infection may be associated with phenylpropanoid pathway activation.

Polyamine metabolism in flax in response to treatment with pathogenic and non-pathogenic Fusarium strains.

Flax crop yield is limited by various environmental stress factors, but the largest crop losses worldwide are caused by Fusarium infection. Polyamines are one of the many plant metabolites possibly involved in the plant response to infection. However, in flax plants the polyamine composition, genes involved in polyamine synthesis, and in particular their regulation, were previously unknown. The aim of this study was to investigate the polyamine synthesis pathway in flax and its involvement in response to pathogen infection. It is well established that polyamines are essential for the growth and development of both plants and fungi, but their role in pathogen infection still remains unknown. In our study we correlated the expression of genes involved in polyamine metabolism with the polyamine levels in plant tissues and compared the results for flax seedlings treated with two pathogenic and one non-pathogenic strains of Fusarium. We observed an increase in the expression of genes participating in polyamine synthesis after fungal infection, and it was reflected in an increase of polyamine content in the plant tissues. The highest level of mRNA was characteristic for ornithine decarboxylase during infection with all tested, pathogenic and non-pathogenic, Fusarium strains and the arginine decarboxylase gene during infection with the pathogenic strain of Fusarium culmorum. The main polyamine identified in the flax seedlings was putrescine, and its level changed the most during infection. Moreover, the considerable increase in the contents of cell wall-bound polyamines compared to the levels of free and conjugated polyamines may indicate that their main role during pathogen infection lies in strengthening of the cell wall. In vitro experiments showed that the polyamines inhibit Fusarium growth, which suggests that they play an important role in plant defense mechanisms. Furthermore, changes in metabolism and content of polyamines indicate different defense mechanisms activated in flax in response to infection by pathogenic and non-pathogenic Fusarium strains.

[The development, differentiation and composition of flax fiber cells]. / Rozwój, róznicowanie sie i sklad komórek wlókna lnianego.

Having vascular origin, flax fiber belongs to the sclerenchyma (steroids) and its structure is limited to the cell wall. What determines fiber properties is its composition, which in practice means the composition of the secondary cell wall. It consists of four main polymers which constitute approximately 90% of the fiber: cellulose, hemicellulose, pectin, lignin, and a variety of secondary metabolites, proteins, waxes and inorganic compounds. The cell wall is a structure with a high complexity of both the composition and interactions of the particular elements between themselves. It is determined by differentiation and cell growth as well as environmental factors, biotic and abiotic stresses. The molecular background of these processes and mechanisms regulating the synthesis and rearrangement of secondary cell walls components are being intensively studied. In this work we described the latest news about the development, composition and metabolism of flax fiber cell wall components together with the molecular explanation of these processes.

Crossbreeding of transgenic flax plants overproducing flavonoids and glucosyltransferase results in progeny with improved antifungal and antioxidative properties.

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.

Oligonucleotide treatment causes flax ß-glucanase up-regulation via changes in gene-body methylation.

BACKGROUND: 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. RESULTS: 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 ß-1,3-glucanase gene (EMO-ßGlu flax) were characterized by up-regulation of ß-glucanase and chitinase genes, decreases in the methylation of CCGG sequences in the ß-glucanase gene and in total DNA methylation and, more importantly, reasonable resistance against Fusarium infection. In addition, EMO-ßGlu flax obtained by this technology showed similar features as those obtained by genetic engineering. CONCLUSION: 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.