The purpose of this study was to present a chemical analysis of the metabolome of cell aggregates of the tree fern Cyathea smithii (J.D. Hooker) cell suspension culture. The LC/MS and GC/MS techniques were used for identification of metabolites. The kinetics of fresh weight, dry weight, and ash content showed 3.5-fold increases during 15-day-long culture. The analysis demonstrated high metabolic activity of cultured cells. In total, 160 metabolites from primary and secondary metabolism and almost 2000 compounds of unknown identity were identified. Three flavonoids-the chalcone isookanin [(2S)-2-(3,4-dihydroxyphenyl)-7,8-dihydroxy-2,3-dihydrochromen-4-one], a methoxy derivative of the flavone gardenin B (5-Hydroxy-2-(4-methoxyphenyl)-6,7,8-trimethoxy-4H-1-benzopyran-4-one), and the isoflavone tectoridin (4',5-Dihydro-6-methoxy-7-(O-glucoside)isoflavone)-had not been previously detected in the cell culture of C. smithii. Principal component analysis revealed five distinct groups of samples; groups 4 and 5 showed the greatest similarity and corresponded to cultures on days 12 and 15, respectively. The number of differentiating compounds was 75, indicated by a heatmap showing positive and negative correlations between the days of culture. The studies described in this paper are crucial for further identification of metabolites and establishing the relationship between the metabolic composition of tree fern cells in culture and their biological activity, assessed by physiological parameters. By determining the relationship between the chemical composition of cells and their growth from culture initiation to senescence, we will provide a more complete picture of the potential for environmental factors to regulate this relationship. Based on previous studies, environmental stimuli such as electromagnetic fields or light of different wavelengths can result in altered growth physiology and cell mass, as well as metabolite diversification and accumulation. The research results presented in this paper provide a foundation for further studies aimed at predicting and regulating the productivity of C. smithii cells in suspension culture and elucidating the significance of tree fern-derived metabolic products in human cell biology, particularly in thyroid cells.

Despite its numerous applications, flax fiber utilization is limited by time-consuming extraction largely determined by pectin content. To address this, transgenic flax plants with reduced pectin levels were generated to facilitate fiber release. We evaluated how pectin reduction affects plant growth, fiber composition, and properties. Field cultivation showed that low-pectin lines exhibited a phenotype comparable to the control. Fibers from these plants contained significantly less pectin and hemicellulose and accumulated more cellulose. These changes were accompanied by conformational and spatial reorganization of the cell wall structure. As a result, fibers with reduced pectin demonstrated improved tensile strength (by 45,6% and by 61,5% for PGI11 and RHA7 fibers, respectively) and enhanced binding capacity for therapeutic substances. Our results support the hypothesis of a compensatory mechanism between pectin and cellulose, where a decrease in one component is offset by an increase in the other. Furthermore, we show that structural reorganization induced by pectin reduction improves the mechanical properties and functional potential of flax fibers. This study highlights that targeted modification of pectin content can enhance fiber quality and functionality without adverse effects on plant growth, offering a promising strategy for optimizing flax for industrial and biomedical applications.

IntroductionFlax (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 cellsMethodsThe 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 discussionIR 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.

There is a growing demand for high-quality natural fibers, including flax fibers, which possess bioactive compounds with antioxidant, antibacterial, and anti-inflammatory properties. Traditional methods like dew retting are commonly used for flax fiber production, but alternative approaches are sought for enhanced fiber quality. This study presents a novel method involving cottonization to improve the quality of flax fibers obtained from low-quality decorticated fibers. The research evaluates the quality enhancement through various stages of the new method, comparing it with dew retting. Studies conducted on both transgenic (B14) and non-transgenic flax fibers revealed a decrease in fiber polymers and bioactive compounds after cottonization compared to dew retting. However, despite this reduction, the antioxidant potential of cottonized fibers increased significantly. Moreover, cottonized flax fibers exhibited high antimicrobial activity, particularly against Pseudomonas aeruginosa. Furthermore, extracts from both transgenic and non-transgenic cottonized flax fibers showed cytotoxic effects on hamster lung fibroblast cells, indicating potential future applications in cancer research. These findings highlight the potential of cottonization in enhancing the quality and bioactivity of flax fibers, paving the way for further exploration in various applications, including medical and textile industries.

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.

Flax fibers have bioactive properties due to their biochemical composition. The quality of the fibers is the main parameter that determines the application, and the process of obtaining fibers has a crucial impact on the composition and properties of fibers. Therefore, this study aimed to enhance the quality of flax fibers by optimizing the hydrodynamic degumming process of decorticated flax fibers. Flax fibers, both non-transgenic (Nike) and transgenic (B14) fibers with overexpression of the β-1,3-glucanase gene from potato, were obtained through nine processes that varied in the degumming temperature, degumming time, and rinsing temperature. The content of phenolic, terpene and polyamine compounds as well as antioxidant activity was determined in the fibers. Two processes, due to having the highest content of bioactive compounds, were selected for further analyses: determination of the chemical composition and structure of plant cell walls, and in vitro tests on the culture of the V79 hamster lung fibroblast cell line. Both processes led to an increase in the content of cellulose and hemicellulose, along with a decrease in the amount of pectin in B14 fibers, indicating changes in the structure of the cell wall. Water extract containing phenolic compounds obtained from flax fiber resulted in increased viability of the V79 fibroblasts and did not show changes in the level of transcripts of inflammatory genes. Based on the obtained results, process VI was identified as the most effective for obtaining flax fibers of the best quality. Additionally, the study confirmed that transgenic B14 fibers exhibited better quality than non-transgenic fibers. The findings highlight the potential of the methods used to obtain flax fibers, which may significantly impact their properties.

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.

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.

This review was designed to summarize the present state of research around the genetic and epigenetic modification of selected plant species and the potential for their application in industry. This review summarizes the activity of research groups from the University of Wrocław completed over the last 3 decades which tends to focus on potatoes and flax likely as a result of their centuries-old tradition of cultivation, processing, and use in Poland. The aims of these studies were various and included the creation of pathogen-resistant plants, increased antioxidant production, improved flax fiber quality, and improved oil properties. New plant breeds initially produced using genetic engineering technology provide an excellent basis for improving our understanding of the genes involved in potato and flax productivity and the quality of their products. These results have been published in many papers and have given rise to new methods for plant breeding and product prototypes which have been patented. However, none of the prototypes have been commercialized because of their GMO origins. In addition, later development of a novel, epigenetic method has led to the creation of more diverse products based on the newly obtained variety of flax called Silesia. These developments have facilitated the production of a range of new raw materials from these epigenetically modified plants. These include a modified oil for improved nutrition and regeneration of skin cells, seed- cake extracts that act as anti-infection agents, improved fiber production for use as bandages for chronic wounds, improved fibers for application as bio composite materials for the development of biodegradable packaging materials and scaffolds for tissue engineering, and micronized fiber for drug delivery. These modifications mean that flax has become a more useful and valuable source of a wide range of raw materials applicable in industry, allowing for the application of these materials in zero waste applications.

The aim of the work was to compare the new variety of oil flax (Silesia) with already cultivated varieties in terms of plant productivity, oil content, fatty acid composition and significant secondary metabolites. The analyzed linseed varieties are characterized by low (Linola), medium (Silesia) and high (Szafir) content of omega-3 fatty acids. Special attention was paid to the quality of the oil and the characteristics that determine its stability (reduction of susceptibility to oxidation). A number of antioxidant compounds of secondary metabolism (simple phenols, phenolic acids, flavonoids, tannins) were identified in the linseed oils. All of these compounds can affect lipid oxidation by a mechanism that attenuates initiating radicals such as hydroxyl or forms an oxidizing primary product such as peroxides. Chelation of metal ions may also be involved in lipid oxidation. We propose a mechanism that encompasses all these processes and facilitates understanding of the complex relationships between them. The general thesis is that the ratio of polyunsaturated fatty acids is associated with a better metabolic state of flaxseed, and thus with a higher nutritional value. In addition, we find a number of specialized secondary metabolites characteristic of the flax studied, which could be useful for chemotaxonomy.

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.

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.

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 β-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.

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.

Cyanogenic glucosides (CG), the monoglycosides linamarin and lotaustralin, as well as the diglucosides linustatin and neolinustatin, have been identified in flax. The roles of CG and hydrogen cyanide (HCN), specifically the product of their breakdown, differ and are understood only to a certain extent. HCN is toxic to aerobic organisms as a respiratory inhibitor and to enzymes containing heavy metals. On the other hand, CG and HCN are important factors in the plant defense system against herbivores, insects and pathogens. In this study, fluctuations in CG levels during flax growth and development (using UPLC) and the expression of genes encoding key enzymes for their metabolism (valine N-monooxygenase, linamarase, cyanoalanine nitrilase and cyanoalanine synthase) using RT-PCR were analyzed. Linola cultivar and transgenic plants characterized by increased levels of sulfur amino acids were analyzed. This enabled the demonstration of a significant relationship between the cyanide detoxification process and general metabolism. Cyanogenic glucosides are used as nitrogen-containing precursors for the synthesis of amino acids, proteins and amines. Therefore, they not only perform protective functions against herbivores but are general plant growth regulators, especially since changes in their level have been shown to be strongly correlated with significant stages of plant development.

A pathogenic strain of Fusarium culmorum caused flax disease whose first visible symptom was pigmentation and possible necrosis of the root tip. To better understand the process of infection we tried to determine the role of cell wall polymers in flax treated with F. culmorum. F. culmorum infection caused cell wall rearrangement which was associated with changes in the cell wall structure without changes in the amount of polymers. Moreover, pathogenic fungi strongly induced chitinase, β-1,3-glucanase, cellulase2, α-galactosidase and lignin-related genes, while mRNA levels of the majority of genes involved in metabolism of cell wall polysaccharide polymers decreased in time.

Previous studies showed that chalcone synthase (chs) silencing in flax (Linum usitatisimum) induces a signal transduction cascade that leads to extensive modification of plant metabolism. Result presented in the current study, performed on field grown flax plants – (across the whole vegetation period) demonstrates that, in addition to its role in tannin and lignin biosynthesis, the chs gene also participates in the regulation of flavone biosynthesis during plant growth. Apigenin and luteolin glycosides constitute the flavones, the major group of flavonoids in flax. Alterations in their levels correlate with plant growth, peaking at the flower initiation stage. Suppression of chs gene expression causes significant changes in the ratio of flavone constituents at the early stage of flax growth. A significant correlation between flavonoid 3′-hydroxylase (F3′H) gene expression and accumulation of luteolin glycosides has been found, indicating that flavone biosynthesis during flax growth and development is regulated by temporal expression of this gene. The lack of such a correlation between the flavone synthase (FNS) gene and flavone accumulation in the course of plant growth suggests that the main route of flavone biosynthesis is mediated by eriodictyol. This is the first report indicating the ratio of flavone constituents as a potent marker of flax growth stages and temporal expression of F3′H, the key gene of their biosynthesis.

INTRODUCTION:Chronic ulcers are the main cause of morbidity and mortality, and the incidence of chronic wounds is expected to increase given that people live longer and that there are civil diseases.AIM:Much attention in the treatment of wounds concerns a dressing that involves wound cleansing, bacterial balance, exudate management and local tissue in a wound environment. These important elements of the evaluation led to the development of an interactive dressing based entirely on flax raw materials.MATERIAL AND METHODS:The complete dressing for wound coverage was prepared from plant (flax) row products: seedcakes, oil, fiber. The content of bioactive compounds (qualitatively and quantitatively) was tested using chromatographic techniques, and their biological activity during tests on fibroblast cell cultures (NHDF). As a final step the clinical trial were performed.RESULTS:The dressings, which help control the microenvironment, combining with exudate using hydrophilic fibre, controlling the flow of exudate from the wound to the dressing were generated. They stimulate the activity in the healing cascade and accelerate the healing process by combining lignocellulose fibre with higher amounts of phenolic compounds, sterols, cannabidiol and unsaturated fatty acids simultaneously with the 3-hydroxybutyrate polymer. All constituents of linen dressing are natural, originate from two types of the engineered flax plant. Pre-clinical data reveal a reasonable reduction in wound size in patients with chronic leg ulcers treated with a linen dressing.CONCLUSIONS:For the first time, a successful application of the innovative interactive linen dressing in the treatment of chronic wounds was noted.

Background. The development of a new type of wound dressing material that can support skin regeneration is an important challenge to improve treatment of chronic, non-healing wounds. Objectives. The objective of this study was to compare the impact of flax fabrics from transgenic plants overexpressing phenolic acids and flavonoids (W92) and polyhydroxybutyrate (M48), as well as fabric from non-transgenic plant (Nike) on cultures of human skin cells. Material and methods. Flax fabric pieces as well as water extracts from the fabrics were co-cultured with human skin cells: keratinocytes, fibroblasts, dermal microvascular endothelial cells, and with monocytoid cell line (THP1) for 48 h. Cell viability and proliferation were assessed with the sulforhodamine B colorimetric assay. Intracellular reactive oxygen species (ROS) was estimated with the 2'7 dichlorodihydrofluorescein diacetate (DCFH-DA) oxidation method. Endothelial cell migration was measured with the scratch assay. The results were compared with the multi-criteria analysis (MCA) procedure. Results. Tested flax fabrics released flavonoids and polyhydroxybutyrate to cell culture media, as it was determined by means of the high performance liquid chromatography (HPLC) method. Fabrics from transgenic plants W92 and M48 promoted proliferation of keratinocytes and fibroblasts. Water extracts from flax fabric diminished the proliferation of monocytoid cells and decreased oxidative burst in activated THP1 cells and accelerated the velocity of dermal microvascular cell migration. The MCA proved that the sum of beneficial effects estimated in human skin cell cultures was higher (by 47% and by 34% with W92 and M48, respectively) than that of non-transgenic flax fabric (Nike). Conclusions. The W92 and M48 fabrics should be further studied as candidates for elaboration of new types of bandages, able to improve skin wound healing.

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 (β-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.