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Glutathione is the most important part of a plant’s antioxidant defense system. Two enzymes are involved in cellular glutathione biosynthesis: glutamylcysteine ligase and glutathione synthetase, of which the latter catalyzes the transfer of glycine to the glutamylcysteine dipeptide. Transgenic plants with an elevated expression level of the glutathione synthetase genes are known to be tolerant to heavy metals. However, our knowledge on their tolerance to other types of the abiotic stress is insufficient. The goal of this study is to produce transgenic tobacco plants with a constitutive expression of the glutathione synthetase gene BnGSH from rapeseed and to estimate their growth parameters under the normal conditions, as well as under the effects of salt, drought, and cold stresses. We generate 17 lines of transgenic plants with the rapeseed BnGSH gene under the control of the 35S promoter by agrobacterium-mediated transformation. The transgene presence is confirmed by the PCR and histochemical analysis of the activity of the GUS reporter gene. Twelve lines with the highest expression level of the BnGSH gene are chosen based on the results of RT-PCR. The following morphological parameters are measured: stem height, leaf area, flower length, fresh and dry weights of shoots, and root length. Some transgenic plants demonstrated increased productivity both under normal conditions and under the effect of a high salinity stress. However, no change in the tolerance to drought and cold was observed in the transgenic plants.

Summary: Glutathione is the most important part of plant antioxidant defense system. Biosynthesis of glutathione in the cells is performed by two enzymes: glutamylcysteine ligase and glutathione synthetase, the latter catalyzing the attachment of glycine to a dipeptide glutamylcysteine. In literature there is information on the improvement of heavy metal-tolerance of transgenic plants due to the increase in the expression level of glutathione synthetase genes. However there is not enough data on the tolerance of these plants to other types of abiotic stress. Therefore the aim of our research was to make transgenic tobacco plants with constitutive expression of rapeseed glutathione synthetase gene BnGSH and to estimate their growth parameters in normal conditions and under salt, drough and cold stress. Using agrobacterial transformation method, we generated 17 lines of transgenic plants containing rapeseed BnGSH gene under control of 35S promoter. The presence of transgenes was confirmed by PCR method and histochemical analysis of the activity of GUS reporter gene. 12 lines with the highest expression of BnGSH gene were chosen on the basis of the results of RT-PCR. We performed morphological analysis, including measurements of stem hight, leaf area, flower length, fresh and dry weight of shoots and root length. Some transgenic plants demonstrated increased productivity in normal conditions as well as under NaCl stress. However, no change in drought and cold tolerance was observed in transgenic plants.

Despite the intensive study of xyloglucan endotransglucosylases/hydrolases, their multifaceted role in plant growth regulation in changing environmental conditions is not yet clarified. The functional role of the large number of genes encoding this group of enzymes is also still unclear. NtEXGT gene encodes one of xyloglucan endotransglucosylases/hydrolases (XTHs) of Nicotiana tabacum L. The highest level of NtEXGT gene expression was detected in young flowers and leaves near the shoot apex. Expression of the NtEXGT gene in leaves was induced by cytokinins, auxins, brassinosteroids and gibberellins. NtEXGT gene was also up-regulated by salinity, drought, cold, cadmium and 10 μM abscisic acid treatments and down-regulated in response to 0 °C and 100 μM abscisic acid. Pretreatment of leaves with fluridone contributed to smaller increase in the level of NtEXGT transcripts in response to drought stress. These data suggest that NtEXGT gene is ABA-regulated and probably implicated in ABA-dependent signaling in response to stress factors. 35S::NtEXGT plants of tobacco showed higher rate of root growth under salt-stress conditions, greater frost and heat tolerance as compared with the wild type tobacco plants.

Screening of nodule bacteria (rhizobia) forming symbiotic relationships with legumes has been performed in order to isolate strains resistant to cadmium ions in a wide range of concentrations (6-132 mg/kg). The effect ofcadmium salts (6, 12, 24 mg/kg) on the legume-rhizobium symbiosis ofthe pea Pisum sativum L. with Rhizobium leguminosarum and of the fodder galega Galega orientalis Lam. with Rhizobium galegae has been studied under experimental laboratory conditions. No statistically significant differences have been revealed in the growth and biomass of plants with regard to the control in the range of concentrations given above. However, it was found that cadmium inhibited nodulation in P. sativum and stimulated it in G. orientalis.

Transgenic tobacco plants expressing the fragments of the ARGOS and NtEXPA4 genes in antisense orientation have been created. Eleven lines of transgenic plants were investigated and five of them were characterized by a decrease in the sizes of the leaves and flowers as compared to control. Stem sizes decreased when only the NtEXPA4 gene fragment was used. The organ size of the experimental plants decreased because of a reduction in the level of both cell division and cell expansion. Two lines of transgenic tobacco plants expressing the part of the ARGOS gene in antisense orientation were characterized by a reduction in the level of the NtEXPA1 and NtEXPA4 gene expression.

The effect of cadmium acetate (200 and 400 μM Cd(CH3COO)2) on the transcriptional promoter activity of rice (Oryza sativa L.) phytochelatin synthase gene 900 bp in size was studied. The reporter gene of glucuronidase was placed under the control of this promoter in transgenic tobacco (Nicotiana tabacum L.) plants. For determining boundaries of this gene promoter responsive to cadmium, deletion constructs were created and their transcriptional activity was measured under the influence of different cadmium concentrations. Deletion of 189 bp from the 5′-terminus of this gene promoter resulted in some transcription activation, whereas deletion of successive 95 bp from the 5′-terminus resulted in cadmium-independent activation of the promoter.

The AINTEGUMENTA gene encodes a transcription factor involved in the regulation of growth of both reproductive and vegetative plant organs. Two conserved regions of this gene were amplified and cloned in antisense orientation. The produced genetic constructs were used to obtain transgenic tobacco plants with the AINTEGUMENTA gene expression downregulated. Over half of the transgenic plants were characterized by smaller leaves, stems, and flowers with their shapes and symmetry not changed. The transgenic plants ceased to grow earlier, flowered later, and had fewer flowers than controls. The main reason for smaller organs was shown to be the lower cell number in an organ with cell sizes unchanged. The genetic constructs can be used to obtain transgenic plants of various species with smaller organs.

In the process of obtaining transgenic plants suitable for phytoremediation of heavy metal-contaminated soils, a synthetic pseudophytochelatin gene coding for a phytochelatin analog Met(GluCys)6Gly was de novo designed and cloned. Contrary to natural enzymatically synthesized phytochelatins, this peptide can be made by a template synthesis. A construct carrying the gene in question under the control of the viral constitutive 35S promoter was made on the basis of a binary vector pCAMBIA 1305.1. This construct was used to express the pseudophytochelatin gene in the model transgenic tobacco plants (Nicotiana tabacum L.), and the above plants acquired additional resistance to various cadmium concentrations.

New approaches to the detection of impaired nucleotides based on the allele specific ligation of a "C probe" followed by rolling circle amplification have been developed. The detection of amplification products was realized by using enzymatic and deoxyribozyme digestion of fluorescently-labeled DNA-RNA-DNA chimeric oligonucleotide structures in cycling probe technology (CPT) in real-time mode.

Screening of nodule bacteria (rhizobia) forming symbiotic relationships with legumes has been performed in order to isolate strains resistant to cadmium ions in a wide range of concentrations (6-132 mg/kg). The effect ofcadmium salts (6, 12, 24 mg/kg) on the legume-rhizobium symbiosis ofthe pea Pisum sativum L. with Rhizobium leguminosarum and of the fodder galega Galega orientalis Lam. with Rhizobium galegae has been studied under experimental laboratory conditions. No statistically significant differences have been revealed in the growth and biomass of plants with regard to the control in the range of concentrations given above. However, it was found that cadmium inhibited nodulation in P. sativum and stimulated it in G. orientalis.

Transgenic tobacco plants expressing the fragments of the ARGOS and NtEXPA4 genes in antisense orientation have been created. Eleven lines of transgenic plants were investigated and five of them were characterized by a decrease in the sizes of the leaves and flowers as compared to control. Stalk sizes decreased when only the NtEXPA4 gene fragment was used. The organ size of the experimental plants decreased because of a reduction in the level of both cell division and cell expansion. Two lines of transgenic tobacco plants expressing the part of the ARGOS gene in antisense orientation were characterized by a reduction in the level of the NtEXPA1 and NtEXPA4 gene expression.