Transcriptome profiling of flax plants exposed to a low-frequency alternating electromagnetic field.

All living organisms on Earth evolved in the presence of an electromagnetic field (EMF), adapted to the environment of EMF, and even learned to utilize it for their purposes. However, during the last century, the Earth's core lost its exclusivity, and many EMF sources appeared due to the development of electricity and electronics. Previous research suggested that the EMF led to changes in intercellular free radical homeostasis and further altered the expression of genes involved in plant response to environmental stresses, inorganic ion transport, and cell wall constituent biosynthesis. Later, CTCT sequence motifs in gene promoters were proposed to be responsible for the response to EMF. How these motifs or different mechanisms are involved in the plant reaction to external EMF remains unknown. Moreover, as many genes activated under EMF treatment do not have the CTCT repeats in their promoters, we aimed to determine the transcription profile of a plant exposed to an EMF and identify the genes that are directly involved in response to the treatment to find the common denominator of the observed changes in the plant transcriptome.

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.

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.

Transcriptomic profiling of susceptible and resistant flax seedlings after Fusarium oxysporum lini infection.

In this study transcriptome was analyzed on two fibrous varieties of flax: the susceptible Regina and the resistant Nike. The experiment was carried out on 2-week-old seedlings, because in this phase of development flax is the most susceptible to infection. We analyzed the whole seedlings, which allowed us to recognize the systemic response of the plants to the infection. We decided to analyze two time points: 24h and 48h, because our goal was to learn the mechanisms activated in the initial stages of infection, these points were selected based on the previous analysis of chitinase gene expression, whose increase in time of Fusarium oxysporum lini infection has been repeatedly confirmed both in the case of flax and other plant species. The results show that although qualitatively the responses of the two varieties are similar, it is the degree of the response that plays the role in the differences of their resistance to F. oxysporum.

Expression of the Tyrosine Hydroxylase Gene from Rat Leads to Oxidative Stress in Potato Plants.

Catecholamines are biogenic aromatic amines common among both animals and plants. In animals, they are synthesized via tyrosine hydroxylation, while both hydroxylation or decarboxylation of tyrosine are possible in plants, depending on the species, though no tyrosine hydroxylase-a counterpart of the animal enzyme-has been identified yet. It is known that in potato plants, it is the decarboxylation of tyrosine that leads to catecholamine production. In this paper, we present the effects of the induction of an alternative route of catecholamine production by introducing the tyrosine hydroxylase gene from rat. We demonstrate that an animal system can be used by the plant. However, it does not function to synthesize catecholamines. Instead, it leads to elevated reactive oxygen species content and a constant stress condition in the plant, which responds with elevated antioxidant levels and improved resistance to infection.

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.

Electromagnetic Field Seems to Not Influence Transcription via CTCT Motif in Three Plant Promoters.

It was proposed that magnetic fields (MFs) can influence gene transcription via CTCT motif located in human HSP70 promoter. To check the universality of this mechanism, we estimated the potential role of this motif on plant gene transcription in response to MFs using both bioinformatics and experimental studies. We searched potential promoter sequences (1000 bp upstream) in the potato Solanum tuberosum and thale cress Arabidopsis thaliana genomes for the CTCT sequence. The motif was found, on average, 3.6 and 4.3 times per promoter (148,487 and 134,361 motifs in total) in these two species, respectively; however, the CTCT sequences were not randomly distributed in the promoter regions but were preferentially located near the transcription initiation site and were closely packed. The closer these CTCT sequences to the transcription initiation site, the smaller distance between them in both plants. One can assume that genes with many CTCT motifs in their promoter regions can be potentially regulated by MFs. To check this assumption, we tested the influence of MFs on gene expression in a transgenic potato with three promoters (16R, 20R, and 5UGT) containing from 3 to 12 CTCT sequences and starting expression of ß-glucuronidase as a reported gene. The potatoes were exposed to a 50 Hz 60-70 A/m MF for 30 min and the reporter gene activity was measured for up to 24 h. Although other factors induced the reporter gene activity, the MF did not. It implies the CTCT motif does not mediate in response to MF in the tested plant promoters.

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.

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.

Flavonoids as important molecules of plant interactions with the environment.

Flavonoids are small molecular secondary metabolites synthesized by plants with various biological activities. Due to their physical and biochemical properties, they are capable of participating in plants' interactions with other organisms (microorganisms, animals and other plants) and their reactions to environmental stresses. The majority of their functions result from their strong antioxidative properties. Although an increasing number of studies focus on the application of flavonoids in medicine or the food industry, their relevance for the plants themselves also deserves extensive investigations. This review summarizes the current knowledge on the functions of flavonoids in the physiology of plants and their relations with the environment.

Transgenic potato plants with overexpression of dihydroflavonol reductase can serve as efficient nutrition sources.

Potato (Solanum tuberosum) is considered to be one of the most important crops cultivated in Europe and the entire world. The tubers of the potato are characterized by rich starch and protein contents and high concentrations of antioxidants, such as vitamin C and flavonoids. Notably, the presence of the phenolic antioxidants is of high importance as they have health-related properties. They are known to reduce the incidence of atherosclerosis, prevent certain kinds of cancer, and aid with many other kinds of diseases. The aim of this study was to find the most efficient way to increase the content of phenolic antioxidants in potato tubers through transgenesis. The results showed that the most efficacious way to achieve this goal was the overexpression of the dihydroflavonol reductase gene (DFR). The produced transgenic potato plants served as a nutrition source for laboratory rats; the study has confirmed their nontoxicity and nutritional benefits on the tested animals.

Flax terpenoid pathway as a source of health promoting compounds.

Flax is an important crop plant grown mainly for its fiber and seeds, which are also rich in omega-3 fatty acids and valuable antioxidants derived from the terpenoid pathways including carotenoids, tocochromanols and sterols. Many of those components found in flax have been recently shown to positively influence human health. Although terpenes vary greatly in their chemical structure, mainly two mechanisms of their biological activity can be considered: direct antioxidation, and a recently explored one, connected to specific receptor and cell signaling pathway activation. Recent studies show that many of the health promoting agents derived from flax act through both of the mentioned mechanisms, resulting in synergistic physiological effects. The work summarizes the two mechanisms, focusing mainly on the one involving cell signaling, as a promising target for medicine and pharmacotherapy.

Genes of phenylpropanoid pathway are activated in early response to Fusarium attack in flax plants.

Fusarium is the most common flax pathogen causing serious plant diseases and in most cases leading to plant death. To protect itself, the plant activates a number of genes and metabolic pathways, both to counteract the effects of the pathogen, and to eliminate the threat. The identification of the plant genes which respond to infection is the approach, that has been used in this study. Forty-seven flax genes have been identified by means of cDNA subtraction method as those, which respond to pathogen infection. Subtracted genes were classified into several classes and the prevalence of the genes involved in the broad spectrum of antioxidants biosynthesis has been noticed. By means of semi-quantitative RT-PCR and metabolite profiling, the involvement of subtracted genes controlling phenylpropanoid pathway in flax upon infection was positively verified. We identified the key genes of the synthesis of these compounds. At the same time we determined the level of the metabolites produced in the phenylpropanoid pathway (flavonoids, phenolic acids) in early response to Fusarium attack by means of GC-MS technique. To the best of our knowledge this is the first report to describe genes and metabolites of early flax response to pathogens studied in a comprehensive way.

The changes in pectin metabolism in flax infected with Fusarium.

Fusarium culmorum and Fusarium oxysporum are the most common fungal pathogens of flax (Linum usitatissimum L.), thus leading to the greatest losses in crop yield. A subtractive cDNA library was constructed from flax seedlings exposed for two days to F. oxysporum. This revealed a set of genes that are potentially involved in the flax defense responses. Two of those genes directly participate in cell wall sugar polymer metabolism: UDP-D-glucuronate 4-epimerase (GAE; EC 5.1.3.6) and formate dehydrogenase (FDH; EC 1.2.1.2). GAE delivers the main substrate for pectin biosynthesis, and decreases were detected in its mRNA level after Fusarium infection. FDH participates in the metabolism of formic acid, and the expression level of its gene increased after Fusarium infection. However, metabolite profiling analysis disclosed that the pectin content in the infected plants remained unchanged, but that there were reductions in both the levels of the soluble sugars that serve as pectin precursors, and in the level of formic acid. Since formic acid is the product of pectin demethylesterification, the level of mRNAs coding for pectin methylesterase (EC 3.1.1.11) in the infected flax was measured, revealing a decrease in its expression upon plant infection. Transgenic flax plants overexpressing fungal polygalacturonase (EC 3.2.1.15) and rhamnogalacturonase (EC 3.2.1.-) showed a decrease in the pectin content and an elevated level of formic acid, but the level of expression of the FDH gene remained unchanged. It is suspected that the expression of the formate dehydrogenase gene is directly controlled by the pathogen in the early stage of infection, and additionally by pectin degradation in the later stages.