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

ABSTRACT: 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.

SUBMITTER: Mierziak J

PROVIDER: S-EPMC4257994 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Flax (Linum usitatissimum) is a potential source of many bioactive components that can be found in its oil and fibers, but also in the seedcake, which is rich in antioxidants. To increase the levels of medically beneficial compounds, a genetically modified flax type (named GT) with an elevated level of phenylopropanoids and their glycoside derivatives was generated. In this study, we investigated the influence of GT seedcake extract preparations on human fibroblast proliferation and migration, and looked at the effect on a human skin model. Moreover, we verified its activity against bacteria of clinical relevance. METHODS: The GT flax used in this study is characterized by overexpression of the glucosyltransferase gene derived from Solanum sogarandinum. Five GT seedcake preparations were generated. Their composition was assessed using ultra pressure liquid chromatography and confirmed using the UPLC-QTOF method. For the in vitro evaluation, the influence of the GT seedcake preparations on normal human dermal fibroblast proliferation was assessed using the MTT test and the wound scratch assay. A human skin model was used to evaluate the potential for skin irritation. To assess the antimicrobial properties of GT preparations, the percentage of inhibition of bacterial growth was calculated. RESULTS: The GT seedcake extract had elevated levels of phenylopropanoid compounds in comparison to the control, non-transformed plants. Significant increases in the content of ferulic acid, p-coumaric acid and caffeic acid, and their glucoside derivatives, kaempferol, quercitin and secoisolariciresinol diglucoside (SDG) were observed in the seeds of the modified plants. The GT seedcake preparations were shown to promote the proliferation of normal human dermal fibroblasts and the migration of fibroblasts in the wound scratch assay. The superior effect of GT seedcake extract on fibroblast migration was observed after a 24-hour treatment. The skin irritation test indicated that GT seedcake preparations have no harmful effect on human skin. Moreover, GT seedcake preparations exhibited inhibitory properties toward two bacterial strains: Staphylococcus aureus and Escherichia coli. CONCLUSIONS: We suggest that preparations derived from the new GT flax are an effective source of phenylopropanoids and that their glycoside derivatives and might be promising natural products with both healing and bacteriostatic effects. This flax-derived product is a good candidate for application in the repair and regeneration of human skin and might also be an alternative to antibiotic therapy for infected wounds.

Project description:Circadian clock genes holds tremendous potential for breeding crops better adapted to environmental fluctuations inherent to climate change. Endogenous TOC1 promoter and CCA1 gene from rice were isolated, cloned and mobilized into pCAMBIA1300 vectors and RNAi constructs A, B and C. Embryogenic calli of varying ages derived from mature seeds of Taipei 309 were employed for Agrobacterium-mediated genetic transformation generating T0, T1 and T2 independent transgenic lines were analyzed for over-expression and repression of CCA1 gene along with various morphological traits. Six hundred and thirty two T0 transgenic plants were generated from rice calli using constructs A, B and C. T0 progeny plants derived from constructs A, B and C did not show any considerable difference in morphological traits. T1and T2 progeny plants derived from construct A exhibited over-expression of CCA1 gene, on the contrary, progeny plants derived from RNAi constructs B and C exhibited repression of CCA1 gene in qRT-PCR analysis at different time points and showed rhythmicity peaking at dawn (6:00 AM) and lowest expression at 12:00 Noon. T1 and T2 progeny plants derived from construct A, namely, A-17 and A-45 exhibited reduced number of tillers/panicles (6-8), reduced thousand seed weight (10.1-16.6g), decreased seed length (4.98 to 6.58mm), decreased seed width (1.1-1.8mm) as compared to wild type plants. T1 and T2 progeny plants of construct B and C showed increased number of tillers/panicles (8-19), better seed yield (4.98-28.9g), increased thousand seed weight (15.6-29.03g), slightly increased seed length (5.7-7.43mm) and slightly increased seed width (1.7-2.98mm) as compared to wild type plants. Chlorophyll content in T1 and T2 progeny plants did not show any significant difference among the three constructs, however, rhythmicity was observed over the period of time in conjunction to CCA1 gene expression. Evidence has been presented which demonstrates that endogenous repression of CCA1 gene resulted in improved morphological traits: increased number of tillers/panicle, thousand seed weight, seed size; whereas, over-expression leads to diminution in morphological traits: decreased number of tillers/panicle, thousand seed weight, seed size as compared to the wild type in T1 and T2 progeny plants. This is first report of successful regulation of endogenous CCA1 gene under control of TOC1 promoter and its effect on improved growth vigor in Japonica rice.

Project description:Recently, in order to improve the resistance of flax plants to pathogen infection, transgenic flax that overproduces ?-1,3-glucanase was created. ?-1,3-glucanase is a PR protein that hydrolyses the ?-glucans, which are a major component of the cell wall in many groups of fungi. For this study, we used fourth-generation field-cultivated plants of the Fusarium -resistant transgenic line B14 to evaluate how overexpression of the ?-1,3-glucanase gene influences the quantity, quality and composition of flax fibres, which are the main product obtained from flax straw.Overproduction of ?-1,3-glucanase did not affect the quantity of the fibre obtained from the flax straw and did not significantly alter the essential mechanical characteristics of the retted fibres. However, changes in the contents of the major components of the cell wall (cellulose, hemicellulose, pectin and lignin) were revealed. Overexpression of the ?-1,3-glucanase gene resulted in higher cellulose, hemicellulose and pectin contents and a lower lignin content in the fibres. Increases in the uronic acid content in particular fractions (with the exception of the 1 M KOH-soluble fraction of hemicelluloses) and changes in the sugar composition of the cell wall were detected in the fibres of the transgenic flax when compared to the contents for the control plants. The callose content was lower in the fibres of the transgenic flax. Additionally, the analysis of phenolic compound contents in five fractions of the cell wall revealed important changes, which were reflected in the antioxidant potential of these fractions.Overexpression of the ?-1,3-glucanase gene has a significant influence on the biochemical composition of flax fibres. The constitutive overproduction of ?-1,3-glucanase causes a decrease in the callose content, and the resulting excess glucose serves as a substrate for the production of other polysaccharides. The monosaccharide excess redirects the phenolic compounds to bind with polysaccharides instead of to partake in lignin synthesis. The mechanical properties of the transgenic fibres are strengthened by their improved biochemical composition, and the increased antioxidant potential of the fibres supports the potential use of transgenic flax fibres for biomedical applications.

Project description:In a last few decades mushrooms are increasingly attracting attention as functional food and sources of biologically active compounds. Several Trametes species have been used for centuries in traditional medicine of East Asia cultures, but only T. versicolor was studied sufficiently while there are less substantial data about medicinal properties of other species. Trametes versicolor, T. hirsuta and T. gibbosa were the species tested for biological activities. Antifungal potentials of extracts were assessed for clinical strains of selected Candida and Aspergillus species. ABTS and FRAP assays were used to evaluate antioxidant capacities of studied extracts. Cytotoxic activity was determined against human cervix and lung adenocarcinoma and colon carcinoma cell lines. Antineurodegenerative activity was assessed by determining the rate of acetylcholinesterase and tyrosinase activity. The presence of metabolites in extracts of mycelia and basidiocarps of studied Trametes species was analyzed by 1H NMR spectroscopy. Studied extracts showed low antifungal potential in comparison with ketoconazole. Basidiocarp extracts were more effective ABTS+ scavengers and Fe2+ reducers than mycelium ones but less effective in comparison with L-ascorbic acid. Results showed that mycelium extracts had stronger cytotoxic effects against three cancer cell lines than basidiocarp ones, and that cervix adenocarcinoma cells were the most sensitive to the extracts and commercial cytostatics. T. versicolor mycelium extract was the most effective inhibitor of acetylcholinesterase activity but double weaker than galantamine, and T. gibbosa mycelium extract was significantly better inhibitor of tyrosinase activity than kojic acid for 40.9%. Chemical analysis indicated strong synergistic action of triterpenes, sugars and polyphenols in applied assays. The results suggest that tested Trametes species have significant medicinal potentials which could be attributed to antioxidative and cytotoxic activity. Additionally both, basidiocarps and mycelia extracts can strongly inhibit activity of acetylcholinesterase and tyrosinase.

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

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