The principal goal of this paper was to generate flax (Linum usitatissimum L.) plants with increased antioxidant properties. To accomplish this a vector containing a multigene construct was prepared, and transgenic plants overexpressing essential flavonoid biosynthesis pathway enzymes were generated and analyzed. The simultaneous expression of genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) resulted in a significant increase of flax antioxidant capacity. To investigate the determinants of higher antioxidant properties of transgenic plants, the phenolic acids and lignans compound contents were measured. In both green part and seed extracts from transgenic plants, the phenolic acids level was increased when compared to the control. The calculated correlation coefficient between phenolic acids content and antioxidant capacity (0.82 and 0.70 for green part and flaxseed, respectively) perfectly reflects their strong relationship. The increase in yield of transgenic plants and their higher resistance to Fusarium culmorum and Fusarium oxysporum when compared to the control plants was a characteristic feature. It was assessed a very high correlation (correlation coefficient = 0.9) between phenolic acids level in flaxseed extract and resistance to F. culmorum. The flowering date of transgenic plants was approximately 3 weeks earlier than that of the control plants. Interestingly, a significant increase in monounsaturated fatty acids and a slight increase in lignans content accompanied the increase in antioxidant properties of flaxseeds.

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SUMMARYThe green peach aphid (GPA) (Myzus persicae Sü lzer) is an important sap-sucking pest of a large variety of plants, including Arabidopsis thaliana. Arabidopsis utilizes a combination of defenses that deter insects from settling on the plant, limit insect feeding and curtail insect reproduction. We demonstrate that the previously uncharacterized Arabidopsis MPL1 (MYZUS PERSICAE-INDUCED LIPASE1) gene has an important role in defense against the GPA. MPL1 expression was rapidly induced to high level in GPA-infested plants. Furthermore, the GPA population was larger on the mpl1 mutant than the wild-type (WT) plant. In contrast, constitutive over-expression of MPL1 from the Cauliflower mosaic virus 35S gene promoter curtailed the size of the GPA population. Insect settling and feeding behavior were unaffected on the mpl1 mutant. However, compared with the phloem-sap enriched petiole exudate from the WT plant, mpl1 petiole exudate was deficient in an activity that restricts insect reproduction on a synthetic diet. Furthermore, MPL1 was required for the heightened accumulation of an antibiotic activity in petiole exudate of the Arabidopsis ssi2 mutant, which exhibits enhanced resistance to GPA. These results indicate that MPL1 has an essential function in antibiosis against GPA. The MPL1 protein exhibits homology to lipases and recombinant MPL1 has lipase activity, thus suggesting that a MPL1-dependent lipid, or a product thereof, has an important role in antibiosis against GPA.

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.

The principal goal of this paper was to investigate the significance of anthocyanin 5-O-glucosyltransferase (5-UGT) for potato tuber metabolism. The ectopic expression of a 5-UGT cDNA in the tuber improved the plant's defense against pathogen infection. The resistance of transgenic lines against Erwinia carotovora subsp. carotovora was about 2-fold higher than for nontransformed plants. In most cases the pathogen resistance was accompanied by a significant increase in tuber yield. To investigate the molecular basis of transgenic potato resistance, metabolic profiling of the plant was performed. In tuber extracts, the anthocyanin 3,5-O-substituted level was significantly increased when compared to that of the control plant. Of six anthocyanin compounds identified, the highest quantity for pelargonidin 3-rutinoside-5-glucoside acylated with p-coumaric acid and peonidin 3-rutinoside-5-glucoside acylated with p-coumaric acid was detected. A significant increase in starch and a decrease in sucrose level in transgenic tubers have been detected. The level of all other metabolites (amino acids, organic acids, polyamines, and fatty acids) was quite the same as in nontransformants. The plant resistance to bacterial infection correlates with anthocyanin content and sucrose level. The properties of recombinant glucosyltransferase were analyzed in in vitro experiments. The enzyme kinetics and its biochemical properties were similar to those from other sources.

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Nitric oxide (NO) is a small molecule involved in the regulation of many physiological processes. It plays a crucial role in the regulation of nervous system, immune and inflammatory responses, and blood flow. NO is synthesized by nitric oxide synthase (NOS) during two-step oxidation of l-arginine to l-citrulline. Intermediates and derivatives of NO metabolism, such as l-arginine, l-citrulline, asymmetrical dimethylarginine (ADMA), symmetrical dimethylarginine (SDMA), and dimethylamine (DMA), are investigated as potential biomarkers. In this article, we present a novel analytical method that allowed for simultaneous analysis of l-arginine, ADMA, SDMA, l-citrulline, and DMA, in a single-step extraction and derivatization using benzoyl chloride. In brief, aliquots of serum were mixed with internal standard solution mixture (50 µM D6-DMA, 20 µM D7-ADMA, and 100 µM D7-arginine) and 0.025 M borate buffer, pH 9.2 (10:1:5). The derivatization process was performed at 25 °C for 5 min using 10% benzoyl chloride. A reverse phase column was used for chromatographic separation. Quantitation was performed using following ions (m/z): 279.1457, 286.1749, 307.1717, 314.2076, 280.1297, 150.0919, and 156.1113 for l-arginine, D7-arginine, ADMA, SDMA, D7-ADMA, l-citrulline, DMA, and D6-DMA, respectively. The method was validated, and its assay linearity, accuracy and precision, recovery, and limits of detection (1.7 µM l-arginine, 0.03 µM ADMA, 0.02 µM SDMA, 0.36 µM l-citrulline, 0.06 µM DMA) and quantification (3.2 µM l-arginine, 0.08 µM ADMA, 0.05 µM SDMA, 1.08 µM l-citrulline, 0.19 µM DMA) were determined. The method is sensitive, reliable, repeatable, and reproducible. It can be applied in the routine clinical/diagnostic laboratory.Graphical abstract