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The influence of 50 and 100 µM Ni on the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT) was studied in the wheat roots. Root fresh weight, tissue Ni, nitrate, ammonium, glutamate and protein concentrations were also determined. Exposure to Ni resulted in a marked reduction in fresh weight of the roots accompanied by a rapid accumulation of Ni in these organs. Both nitrate and ammonium contents in the root tissue were considerably enhanced by Ni stress. While protein content was not significantly influenced by Ni application, glutamate concentration was slightly reduced on the first day after treatment with the higher Ni dose. Treatment of the wheat seedlings with 100 µM Ni led to a decrease in NR activity; however, it did not alter the activation state of this enzyme. Decline in NiR activity observed after application of 100 µM Ni was more pronounced than that in NR. The activities of GS and NADH-GOGAT also showed substantial decreases in response to Ni stress with the latter being more susceptible to this metal. Starting from the fourth day, both aminating and deaminating GDH activities in the roots of the seedlings supplemented with Ni were lower in comparison to the control. While the activity of AspAT remained unaltered after Ni application that of AlaAT showed a considerable enhancement. The results indicate that exposure of the wheat seedlings to Ni resulted in a general depression of nitrogen assimilation in the roots. Increase in the glutamate-producing activity of AlaAT may suggest its involvement in supplying the wheat roots with this amino acid under Ni stress.
Effects of two Ni concentrations (50 and 100 µM) on growth, Ni accumulation as well as the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were studied in shoots of wheat seedlings. Exposure to Ni caused rapid accumulation of this metal in the shoots accompanied by a substantial decrease in the length and fresh weight of these organs. Both aminating (NADH-GDH) and deaminating (NAD-GDH) glutamate dehydrogenase activities were significantly influenced by Ni stress, while GS activity did not change in response to Ni application. The activity of NADH-GDH showed an increase at the end of the experiment and 7 days after Ni treatment it was 68% and 76% higher than in the control, at 50 and 100 µM Ni, respectively. NAD-GDH activity after 1 and 4 days of exposure to a higher concentration of Ni was reduced by 24% and 37%, respectively. However, on the 7th day the activity of this enzyme was enhanced by 150% and 72% over the control level, at 50 and 100 µM Ni, respectively. The obtained results suggest that GDH can play an important role in response of wheat seedlings to Ni toxicity.
We have studied the influence of 50 sulfate/selenate ratios in the range of 2; 1.5; 1; 0.5; 0 mM/0.125; 0.25; 0.375; 0.5; 0.75; 1; 1.5; 2; 2.5; 3 mM on biomass growth, glucosinolate biosynthesis and selenium accumulation in T. majus and A. caucasica transformed roots producing Phe-derived glucotropaeolin and Met-derived glucoiberverin, respectively. Gradual decrease in glucosinolate production, more significant in glucoiberverin, was correlated both with the decrease in sulfate and increase in selenate concentrations and with increasing Se accumulation in the biomass of the roots. Both T. majus and A. caucasica transformed roots accumulated high amounts of Se in biomass, up to 2177 ± 81.7 µg·g⁻¹ DM and 3110 ± 97.4 µg·g⁻¹ DM, respectively, in the media with sulfate 0.5; 0 mM/selenate 2.5 mM ratios. In transformed roots cultured at sulfate 1.5; 1; 0.5; 0 mM/selenate 0.75; 1; 1.5; 2; 2.5; 3 mM ratios accumulation of Se into glucosinolate fractions occurred, it reached the maxima at sulfate 0.5 mM/selenate 2.5 mM ratio. In T. majus roots Se accumulation in glucosinolate fraction was by about 50% lower than in A. caucasica ones. These results indicated that selenium enriched hairy roots of T. majus and A. caucasica could be fully controlled sources of both glucosinolates and selenium as supplements for cancer chemoprevention.
We compared the biochemical profiles of Physalis ixocarpa hairy roots transformed with Agrobacterium rhizogenes ATCC and A4 strains with non-transformed root cultures. The studied clones of A4- and ATCC-induced hairy roots differed significantly; the latter showed greater growth potential and greater ability to produce secondary metabolites (tropane alkaloids) and to biotransform hydroquinone to arbutin. We compared glucose content, alanine and aspartate aminotransferase activity, and L-phenylalanine ammonia-lyase activity. We analyzed markers of prooxidant/antioxidant homeostasis: catalase, ascorbate peroxidase, oxidase, glutathione peroxidase and transferase activity, and the levels of ascorbate, glutathione, tocopherol and lipid peroxidation. We found that transformation induced strain-specific regulation, including regulation based on redox signals, determining the rate of allocation of carbon and nitrogen resources to secondary metabolism pathways. Our results provide evidence that A. rhizogenes strain-specific modification of primary metabolites contributed to regulation of secondary metabolism and could determine the ability of P. ixocarpa hairy root clones to produce tropane alkaloids and to convert exogenously applied hydroquinone to pharmaceutically valuable arbutin. Of the studied parameters, glucose content, L-phenylalanine ammonia-lyase activity and alanine aminotransferases activity may be indicators of the secondary metabolite-producing potential of different P. ixocarpa hairy root clones.
P. ixocarpa hairy root cultures were obtained after the transformation with A. rhizogenes strain ATCC 15834. The ability of P. ixocarpa hairy roots to biotransform HQ to arbutin was examined. The roots were treated 3 times with the same HQ concentration on 3 consecutive days or every 3 days. Despite these differences the highest arbutin yield and the highest biotransformation ratio were similar in both variants, 13.1 and 14.4 mg·25 cm⁻³ of the cultures and 67.6% and 70.6%, respectively. However, in the case of shorter intervals between treatments the highest levels of these parameters were achieved earlier. Multiple treatment of lower HQ concentration reduced its harmful effects on root biomass growth.
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