Kamil Kostyn

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.

Engineering Flax with the GT Family 1 Solanum sogarandinum Glycosyltransferase SsGT1 Confers Increased Resistance to Fusarium Infection.

The aim of this study was to engineer a flax with increased resistance to pathogens. The approach was based on the recent analysis of the Solanum sogarandinum -derived glycosyltransferase (UGT) protein, designated SsGT1 (previously called 5UGT). On the basis of enzyme studies, the recombinant SsGT1 is a 7-O-glycosyltransferase, the natural substrates of which include both anthocyanidins and flavonols such as kaempferol and quercetin. Because flavonoids act as antioxidants and glycosylation increases the stability of flavonoids, it has been suggested that the accumulation of a higher quantity of flavonoid glycosides in transgenic plants might improve their resistance to pathogen infection. Flax overproducing SsGT1 showed higher resistance to Fusarium infection than wild-type plants, and this was correlated with a significant increase in the flavonoid glycoside content in the transgenic plants. Overproduction of glycosyltransferase in transgenic flax also resulted in proanthocyanin, lignan, phenolic acid, and unsaturated fatty acid accumulation in the seeds. The last is meaningful from a biotechnological point of view and might suggest the involvement of polyphenol glycosides in the protection of unsaturated fatty acids against oxidation and thus improve oil storage. It is thus suggested that introduction of SsGT1 is sufficient for engineering altered pathogen resistance in flax.

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

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

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.

Flax Engineering for Biomedical Application

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

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.

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.

Quantitative determination of the iodine values of unsaturated plant oils using infrared and Raman spectroscopy methods

ABSTRACT Thirteen edible oils: sunflower, avocado, hemp, high-linolenic flax, low-linolenic flax, safflower, walnut, roasted sesame, rice, corn, rapeseed, pumpkin seed, and hazel were studied in this work. Their fatty acid composition, iodine, acidic, peroxide, and saponification values were determined. Infrared and Raman spectra of the oils were recorded in the range 400–3200 cm−1. The integral intensities of the bands at about 1655 and 2852 cm−1 corresponding to ν(C=C) and ν(CH2) vibrations were evaluated and used to construct a relationship between the spectroscopic data and the iodine value. The following linear dependencies were obtained: Iν(C=C)/Iν(CH2) = 7.449 × 10−4 × iodine value – 0.0339 and Iν(C=C)/Iν(CH2) = 9.299 × 10−4 × iodine value – 0.023 for the infrared and Raman spectra with a correlation coefficient 0.988 and 0.976, respectively. These calibration lines can be used to determine the iodine value for oils with unknown unsaturation level, and may be applied for margarines and other fatty materials.

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.