Quality assessment of flax advanced breeding lines varying in seed coat color and their potential use in the food and industrial applications.

BACKGROUND: With the increasing consumer awareness of the strong relationship between food and health, flax became a promising functional food due to its bioactive nutraceutical composition. Intra-specific crosses of eight contrasting flax genotypes were performed previously, and within segregating F6 progeny families, we investigated a close-up composition of phytochemicals derived from whole seeds. RESULTS: The considerable genetic variation among the flax F6 families suggested that intra-specific hybridization is essential in flax breeding to obtain and broaden genetic variability and largely affirmed the opportunity for selecting promising lines. Also, significant variations in the targeted metabolite contents and antioxidant properties were observed among brown and yellow-seeded families. Notably, brown-seeded families expressed the highest average values of saturated fatty acids, protein, fiber, tocopherol, phenolics, SDG, and SECO lignans. Yellow-seeded families represented the highest average content of unsaturated fatty acids and mucilage. The cultivation year significantly affects flaxseed's composition and functional properties, presumably due to temperature, humidity, and sunshine time differences. Interestingly, the seeds obtained in warmer conditions were more potent and had more chemical constituents. The favorable genetic correlations among all evaluated traits suggest the possibility of joint genetic selection for several nutritional and phytochemical characteristics in flax. The current study highlights the importance and utilization of 19 top families as their seeds and oil play imperative roles in the pharmaceuticals and food industries. The antioxidant capacity of the seeds showed that families 84B, 23B, 35Y, 95Y, 30B, 88B, and 78B serve as a natural source of dietary antioxidants beneficial to human health. To increase the oxidative stability of the flaxseed oil, the quality evaluation identified some families with low levels of linolenic acid. CONCLUSIONS: These findings are essential to improving flaxseed's nutritional quality and therapeutic properties through a bulk breeding program.

Investigation of the Properties of Linen Fibers and Dressings.

In antiquity, flax was used as a dressing for healing wounds. Currently, work is underway on the genetic modification of flax fibers to improve their properties. Genetic modifications have resulted in an increased content of antioxidants and more favorable mechanical properties. The works published so far have presented independent tests of fibers and dressings after appropriate technological treatments in cell cultures. This study aimed to compare the properties of the fibers and the dressing produced in cell cultures-hamster fibroblasts-V79. The research material was traditional NIKE fibers; genetically modified M, B, and MB fibers; and linen dressings obtained from these fibers. The extract from 48-h incubation of 40 mg of fiber in the culture medium, which was desolved into 10, 20, and 30 mg, was administered to the cell culture. On the other hand, a linen dressing was placed on cells with an area of 0.5 cm2, 1 cm2, 1.5 cm2, and 2 cm2. Cells with fiber or dressing were incubated for 48 h, and then, biological tests were performed, including cell viability (in propidium iodide staining), cell proliferation (in the SRB assay), evaluation of the intracellular free radical level (in the DCF-DA assay), genotoxicity (in the comet assay), assessment of the apoptotic and necrotic cells (in staining anexin-V and iodide propidium), the course of the cell cycle, and the scratch test. The correlation between apoptosis and genotoxicity and the levels of free radicals and genotoxicity were determined for the tested linen fibers and fabrics. The tests presented that the fibers are characterized by the ability to eliminate damaged cells in the elimination phase. However, the obtained fabrics gain different properties during the technological processing of the fibers into linen dressings. Linen fabrics have better regenerative properties for cells than fibers. The linseed dressing made of MB fiber has the most favorable regenerative properties.

Integument-Specific Transcriptional Regulation in the Mid-Stage of Flax Seed Development Influences the Release of Mucilage and the Seed Oil Content.

Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of this integrative biology approach suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.

Identification and Characterization of microRNAs in the Developing Seed of Linseed Flax (Linum usitatissimum L.).

Seed development plays an important role during the life cycle of plants. Linseed flax is an oil crop and the seed is a key organ for fatty acids synthesis and storage. So it is important to understand the molecular mechanism of fatty acid biosynthesis during seed development. In this study, four small RNA libraries from early seeds at 5, 10, 20 and 30 days after flowering (DAF) were constructed and used for high-throughput sequencing to identify microRNAs (miRNAs). A total of 235 miRNAs including 114 known conserved miRNAs and 121 novel miRNAs were identified. The expression patterns of these miRNAs in the four libraries were investigated by bioinformatics and quantitative real-time polymerase chain reaction (qPCR) analysis. It was found that several miRNAs, including Lus-miRNA156a was significantly correlated with seed development process. In order to confirm the actual biological function of Lus-miRNA156a, over-expression vector was constructed and transformed to Arabidopsis. The phenotypes of homozygous transgenic lines showed decreasing of oil content and most of the fatty acid content in seeds as well as late flowering time. The results provided a clue that miRNA156a participating the fatty acid biosynthesis pathway and the detailed molecular mechanism of how it regulates the pathway needs to be further investigated.

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.

Impact of fabrics from transgenic flax on cultures of skin cells.

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, 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.

Flax rhamnogalacturonan lyases: phylogeny, differential expression and modeling of protein structure.

Rhamnogalacturonan lyases (RGLs; EC 4.2.2.23) degrade the rhamnogalacturonan I (RG-I) backbone of pectins present in the plant cell wall. These enzymes belong to polysaccharide lyase family 4, members of which are mainly from plants and plant pathogens. RGLs are investigated, as a rule, as pathogen 'weapons' for plant cell wall degradation and subsequent infection. Despite the presence of genes annotated as RGLs in plant genomes and the presence of substrates for enzyme activity in plant cells, evidence supporting the involvement of this enzyme in certain processes is limited. The differential expression of some RGL genes in flax (Linum usitatissimum L.) tissues, revealed in our previous work, prompted us to carry out a total revision (phylogenetic analysis, analysis of expression and protein structure modeling) of all the sequences of flax predicted as coding for RGLs. Comparison of the expressions of LusRGL in various tissues of flax stem revealed that LusRGLs belong to distinct phylogenetic clades, which correspond to two co-expression groups. One of these groups comprised LusRGL6-A and LusRGL6-B genes and was specifically upregulated in flax fibers during deposition of the tertiary cell wall, which has complex RG-I as a key noncellulosic component. The results of homology modeling and docking demonstrated that the topology of the LusRGL6-A catalytic site allowed binding to the RG-I ligand. These findings lead us to suggest the presence of RGL activity in planta and the involvement of special isoforms of RGLs in the modification of RG-I of the tertiary cell wall in plant fibers.

Insight into the Influence of Cultivar Type, Cultivation Year, and Site on the Lignans and Related Phenolic Profiles, and the Health-Promoting Antioxidant Potential of Flax (Linum usitatissimum L.) Seeds.

Flaxseeds are a functional food representing, by far, the richest natural grain source of lignans, and accumulate substantial amounts of other health beneficial phenolic compounds (i.e., flavonols, hydroxycinnamic acids). This specific accumulation pattern is related to their numerous beneficial effects on human health. However, to date, little data is available concerning the relative impact of genetic and geographic parameters on the phytochemical yield and composition. Here, the major influence of the cultivar over geographic parameters on the flaxseed phytochemical accumulation yield and composition is evidenced. The importance of genetic parameters on the lignan accumulation was further confirmed by gene expression analysis monitored by RT-qPCR. The corresponding antioxidant activity of these flaxseed extracts was evaluated, both in vitro, using ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and iron chelating assays, as well as in vivo, by monitoring the impact of UV-induced oxidative stress on the lipid membrane peroxidation of yeast cells. Our results, both the in vitro and in vivo studies, confirm that flaxseed extracts are an effective protector against oxidative stress. The results point out that secoisolariciresinol diglucoside, caffeic acid glucoside, and p-coumaric acid glucoside are the main contributors to the antioxidant capacity. Considering the health benefits of these compounds, the present study demonstrates that the flaxseed cultivar type could greatly influence the phytochemical intakes and, therefore, the associated biological activities. We recommend that this crucial parameter be considered in epidemiological studies dealing with flaxseeds.

A new genotype of flax (Linum usitatissimum L.) with decreased susceptibility to fat oxidation: consequences to hematological and biochemical profiles of blood indices.

BACKGROUND: Flaxseed is an alternative to marine products that provide the traditional dietary sources of ω-fatty acids. A new genotype of flax, W92, is rich in natural antioxidants as well as having a reduced content of α-linolenic acid and therefore shows decreased susceptibility to fat oxidation. The objective of this study was to evaluate the effect of a diet supplemented with W92 flaxseed on hematological and biochemical blood indices. RESULTS: A positive impact of diet with the addition of flaxseed was observed on erythrocyte indices, including red blood cell (RBC), hematocrit (HCT), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) values. There were no significant differences for white blood cell (WBC), total protein and glucose values. Aspartate aminotransferase and alanine aminotransferase estimations in serum were also carried out and no obvious toxicity to the liver was shown. Moreover, a lipid profile was performed in serum samples and a decrease in total cholesterol and low-density lipoprotein cholesterol (LDL-C), accompanied by an increase in high-density lipoprotein cholesterol (HDL-C), was observed in rabbits fed flaxseed diets. CONCLUSION: Based on the results obtained, it appeared that the inclusion of a new genetically modified type of flaxseed in the diet altered cholesterol metabolism and could reduce the possibility of cardiovascular diseases. Diet enrichment with W92 flaxseed may be a solution to the health issues that are a result of improper diet in humans and animals. © 2016 Society of Chemical Industry.

Transcriptome portrait of cellulose-enriched flax fibres at advanced stage of specialization.

Functional specialization of cells is among the most fundamental processes of higher organism ontogenesis. The major obstacle to studying this phenomenon in plants is the difficulty of isolating certain types of cells at defined stages of in planta development for in-depth analysis. A rare opportunity is given by the developed model system of flax (Linum usitatissimum L.) phloem fibres that can be purified from the surrounding tissues at the stage of the tertiary cell wall deposition. The performed comparison of the whole transcriptome profile in isolated fibres and other portions of the flax stem, together with fibre metabolism characterization, helped to elucidate the general picture of the advanced stage of plant cell specialization and to reveal novel participants potentially involved in fibre metabolism regulation and cell wall formation. Down-regulation of all genes encoding proteins involved in xylan and lignin synthesis and up-regulation of genes for the specific set of transcription factors transcribed during tertiary cell wall formation were revealed. The increased abundance of transcripts for several glycosyltransferases indicated the enzymes that may be involved in synthesis of fibre-specific version of rhamnogalacturonan I.

IMPACT OF FABRICS FROM TRANSGENIC FLAX PLANT ON HUMAN DERMAL FIBROBLASTS IN VITRO PROLIFERATION.

Previously it was documented that transgenic flax plants, which contained an increased level of polyphenolic compounds, significantly improved healing of skin wounds lesions. In order to recognize mechanisms of beneficial action of transgenic flax fabrics on wound healing, in the present study the impact of flax fabric pieces/cuts from three types of transgenic flax on normal human dermal fibroblasts primary culture (NHDF) was investigated. NHDF cell cultures were exposed for 48 h to specific area of flax fabric cuts, made from M50, B 14 and M50+B14 (intertwined fibers of M and B), or parallely, extracts from fibers of the tested flax materials to cell culture medium. Cultures were inspected for cell viability, proliferation, cell cycle changes and for their resistance to oxidative stress (consecutive addition of H2,O2, to harvested cell cultures). None of the tested flax fabrics were cytotoxic to fibroblast cultures and also did not increase significantly a frequency of apoptotic cells in cultures. In the comet assay, the tested flax fabrics revealed significant protective effect on DNA damage ciused by addition of H202 to the cultures at the end of incubation time. Fabrics from transgenic flax significantly enhanced fibroblasts proliferation in vitro estimated with the SRB test. Flow cytometric analysis revealed higher frequency of cells in the S phase, in the presence of transgenic flax fabrics. Fabrics from B14 and M50+B14 flax are the most potent activators of NHDF cells in applied in vityo tests, hence they could be recommended for elaboration of new type bandage, able to improve skin wound healing.

FLAX OIL FROM TRANSGENIC LINUM USITATISSIMUM SELECTIVELY INHIBITS IN VITRO PROLIFERATION OF HUMAN CANCER CELL LINES.

Emulsions made of oils from transgenic flaxseeds significantly decreased in vitro proliferation of six tested human cancer cell lines in 48-h cultures, as assessed with the standard sulforhodamine assay. However, the emulsions also increased proliferation rate of normal human dermal fibroblasts and, to a lower extend, of keratinocytes. Both inhibition of in vitro proliferation of human cancer cell lines and stimulation of proliferation of normal dermal fibroblasts and keratinocytes were especially strong with the emulsion type B and with emulsion type M. Oils from seeds of transgenic flax type B and M should be considered as valuable adjunct to standard cytostatic therapy of human cancers and also could be applied to improve the treatment of skin lesions in wound healing.

Impact of CAD-deficiency in flax on biogas production.

Global warming and the reduction in our fossil fuel reservoir have forced humanity to look for new means of energy production. Agricultural waste remains a large source for biofuel and bioenergy production. Flax shives are a waste product obtained during the processing of flax fibers. We investigated the possibility of using low-lignin flax shives for biogas production, specifically by assessing the impact of CAD deficiency on the biochemical and structural properties of shives. The study used genetically modified flax plants with a silenced CAD gene, which encodes the key enzyme for lignin synthesis. Reducing the lignin content modified cellulose crystallinity, improved flax shive fermentation and optimized biogas production. Chemical pretreatment of the shive biomass further increased biogas production efficiency.

QTL for fatty acid composition and yield in linseed (Linum usitatissimum L.).

KEY MESSAGE: The combined SSR-SNP map and 20 QTL for agronomic and quality traits will assist in marker assisted breeding as well as map-based cloning of key genes in linseed. Flax is an important nutraceutical crop mostly because it is a rich source of omega-3 fatty acids and antioxidant compounds. Canada is the largest producer and exporter of oilseed flax (or linseed), creating a growing need to improve crop productivity and quality. In this study, a genetic map was constructed based on selected 329 single nucleotide polymorphic markers and 362 simple sequence repeat markers using a recombinant inbred line population of 243 individuals from a cross between the Canadian varieties CDC Bethune and Macbeth. The genetic map consisted of 15 linkage groups comprising 691 markers with an average marker density of one marker every 1.9 cM. A total of 20 quantitative trait loci (QTL) were identified corresponding to 14 traits. Three QTL each for oleic acid and stearic acid, two QTL each for linoleic acid and iodine value and one each for palmitic acid, linolenic acid, oil content, seed protein, cell wall, straw weight, thousand seed weight, seeds per boll, yield and days to maturity were identified. The QTL for cell wall, straw weight, seeds per boll, yield and days to maturity all co-located on linkage group 4. Analysis of the candidate gene regions underlying the QTL identified proteins involved in cell wall and fibre synthesis, fatty acid biosynthesis as well as their metabolism and yield component traits. This study provides the foundation for assisting in map-based cloning of the QTL and marker assisted selection of a wide range of quality and agronomic traits in linseed and potentially fibre flax.

Kinetics of glucosylated and non-glucosylated aryltetralin lignans in Linum hairy root cultures.

Due to their pronounced cytotoxic activity, a number of aryltetralin lignans (ATLs), such as podophyllotoxin (PTOX), are used as antitumor compounds. The production of such molecules from entire plants or plant cell-tissue-organ cultures is thus of interest to the pharmaceutical industry. Hairy root cultures constitute a good tool not only for phytochemical production but also for investigating plant secondary metabolism. This work reports on the growth and ATL biosynthesis in two hairy root cultures of Linum album Kotschy ex Boiss. and Linum flavum. The kinetics of accumulation of the intermediates of MPTOX biosynthesis and of their glucosylated forms are described over a 21-day period of growth. An accumulation of non-glucosylated forms of the ATLs during the exponential phase of the cultures is followed by an accumulation of the glucosylated forms during the stationary phase. Our results show a strong coordination of the biosynthetic paths derived from deoxypodophyllotoxin via deoxypodophyllotoxin 6-hydroxylase and deoxypodophyllotoxin 7-hydroxylase, and a coordinated glucosylation of podophyllotoxin, methoxypodophyllotoxin, and 5'-demethoxymethoxypodophyllotoxin. Furthermore, our results suggest an important role of ß-peltatin-6-glucoside formation in the control of ATL accumulation in Linum hairy root cultures.

Plant phosphorus acquisition in a common mycorrhizal network: regulation of phosphate transporter genes of the Pht1 family in sorghum and flax.

In a preceding microcosm study, we found huge differences in phosphorus (P) acquisition in sorghum (Sorghum bicolor) and flax (Linum usitatissimum) sharing a common mycorrhizal network (CMN). Is the transcriptional regulation of arbuscular mycorrhizal (AM)-induced inorganic orthophosphate (Pi) transporters responsible for these differences? We characterized and analyzed the expression of Pi transporters of the Pht1 family in both plant species, and identified two new AM-inducible Pi transporters in flax. Mycorrhizal Pi acquisition was strongly affected by the combination of plant and AM fungal species. A corresponding change in the expression of two AM-inducible Pht1 transporters was noticed in both plants (SbPT9, SbPT10, LuPT5 and LuPT8), but the effect was very weak. Overall, the expression level of these genes did not explain why flax took up more Pi from the CMN than did sorghum. The post-transcriptional regulation of the transporters and their biochemical properties may be more important for their function than the fine-tuning of their gene expression.

Genetically modified flax expressing NAP-SsGT1 transgene: examination of anti-inflammatory action.

The aim of the work was to define the influence of dietary supplementation with GM (genetically modified) GT#4 flaxseed cake enriched in polyphenols on inflammation development in mice liver. Mice were given ad libitum isoprotein diets: (1) standard diet; (2) high-fat diet rich in lard, high-fat diet enriched with 30% of (3) isogenic flax Linola seed cake; and (4) GM GT#4 flaxseed cake; for 96 days. Administration of transgenic and isogenic seed cake lowered body weight gain, of transgenic to the standard diet level. Serum total antioxidant status was statistically significantly improved in GT#4 flaxseed cake group and did not differ from Linola. Serum thiobarbituric acid reactive substances, lipid profile and the liver concentration of pro-inflammatory cytokine tumor necrosis factor-α were ameliorated by GM and isogenic flaxseed cake consumption. The level of pro-inflammatory cytokine interferon-γ did not differ between mice obtaining GM GT#4 and non-GM flaxseed cakes. The C-reactive protein concentration was reduced in animals fed GT#4 flaxseed cake and did not differ from those fed non-GM flaxseed cake-based diet. Similarly, the liver structure of mice consuming diets enriched in flaxseed cake was improved. Dietetic enrichment with GM GT#4 and non-GM flaxseed cakes may be a promising solution for health problems resulting from improper diet.

Functional characterization of flax fatty acid desaturase FAD2 and FAD3 isoforms expressed in yeast reveals a broad diversity in activity.

With 45 % or more oil content that contains more than 55 % alpha linolenic (LIN) acid, linseed (Linum usitatissimum L.) is one of the richest plant sources of this essential fatty acid. Fatty acid desaturases 2 (FAD2) and 3 (FAD3) are the main enzymes responsible for the Δ12 and Δ15 desaturation in planta. In linseed, the oilseed morphotype of flax, two paralogous copies, and several alleles exist for each gene. Here, we cloned three alleles of FAD2A, four of FAD2B, six of FAD3A, and seven of FAD3B into a pYES vector and transformed all 20 constructs and an empty construct in yeast. The transformants were induced in the presence of oleic (OLE) acid substrate for FAD2 constructs and linoleic (LIO) acid for FAD3. Conversion rates of OLE acid into LIO acid and LIO acid into LIN acid were measured by gas chromatography. Conversion rate of FAD2 exceeded that of FAD3 enzymes with FAD2B having a conversion rate approximately 10 % higher than FAD2A. All FAD2 isoforms were active, but significant differences existed between isoforms of both FAD2 enzymes. Two FAD3A and three FAD3B isoforms were not functional. Some nonfunctional enzymes resulted from the presence of nonsense mutations causing premature stop codons, but FAD3B-C and FAD3B-F seem to be associated with single amino acid changes. The activity of FAD3A-C was more than fivefold greater than the most common isoform FAD3A-A, while FAD3A-F was fourfold greater. Such isoforms could be incorporated into breeding lines to possibly further increase the proportion of LIN acid in linseed.

Radioactive Chernobyl environment has produced high-oil flax seeds that show proteome alterations related to carbon metabolism during seed development.

Starting in 2007, we have grown soybean (Glycine max [L.] Merr. variety Soniachna) and flax (Linum usitatissimum, L. variety Kyivskyi) in the radio-contaminated Chernobyl area and analyzed the seed proteomes. In the second-generation flax seeds, we detected a 12% increase in oil content. To characterize the bases for this increase, seed development has been studied. Flax seeds were harvested in biological triplicate at 2, 4, and 6 weeks after flowering and at maturity from plants grown in nonradioactive and radio-contaminated plots in the Chernobyl area for two generations. Quantitative proteomic analyses based on 2-D gel electrophoresis (2-DE) allowed us to establish developmental profiles for 199 2-DE spots in both plots, out of which 79 were reliably identified by tandem mass spectrometry. The data suggest a statistically significant increased abundance of proteins associated with pyruvate biosynthesis via cytoplasmic glycolysis, L-malate decarboxylation, isocitrate dehydrogenation, and ethanol oxidation to acetaldehyde in early stages of seed development. This was followed by statistically significant increased abundance of ketoacyl-[acylcarrier protein] synthase I related to condensation of malonyl-ACP with elongating fatty acid chains. On the basis of these and previous data, we propose a preliminary model for plant adaptation to growth in a radio-contaminated environment. One aspect of the model suggests that changes in carbon assimilation and fatty acid biosynthesis are an integral part of plant adaptation.

Identification of a pair of phospholipid:diacylglycerol acyltransferases from developing flax (Linum usitatissimum L.) seed catalyzing the selective production of trilinolenin.

The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3(cis)(Δ9,12,15)) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.