Superoxide Dismutase Premodulates Oxidative Stress in Plastids for Protection of Tobacco Plants from Cold Damage Ultrastructure Damage.

ROS-dependent induction of oxidative damage can be used as a trigger initiating genetically determined non-specific protection in plant cells and tissues. Plants are potentially able to withstand various specific (toxic, osmotic) factors of abiotic effects, but do not have sufficient or specific sensitivity to form an adequate effective response. In this work, we demonstrate one of the possible approaches for successful cold acclimation through the formation of effective protection of photosynthetic structures due to the insertion of the heterologous FeSOD gene into the tobacco genome under the control of the constitutive promoter and equipped with a signal sequence targeting the protein to plastid. The increased enzymatic activity of superoxide dismutase in the plastid compartment of transgenic tobacco plants enables them to tolerate the oxidative factor of environmental stresses scavenging ROS. On the other hand, the cost of such resistance is quite high and, when grown under normal conditions, disturbs the arrangement of the intrachloroplastic subdomains leading to the modification of stromal thylakoids, probably significantly affecting the photosynthesis processes that regulate the efficiency of photosystem II. This is partially compensated for by the fact that, at the same time, under normal conditions, the production of peroxide induces the activation of ROS detoxification enzymes. However, a violation of a number of processes, such as the metabolism of accumulation, and utilization and transportation of sugars and starch, is significantly altered, which leads to a shift in metabolic chains. The expected step for further improvement of the applied technology could be both the use of inducible promoters in the expression cassette, and the addition of other genes encoding for hydrogen peroxide-scavenging enzymes in the genetic construct that are downstream in the metabolic chain.

The Morphological Parameters and Cytosolic pH of Cells of Root Zones in Tobacco Plants (Nicotiana tabacum L.): Nonlinear Effects of NaCl Concentrations.

Salinity impacts important processes in plants, reducing their yield. The effect of salinity on the cytosolic pH (pHcyt) has been little studied. In this research, we employed transgenic tobacco plants expressing the pH sensor Pt-GFP to investigate the alterations in pHcyt in cells across various root zones. Furthermore, we examined a wide spectrum of NaCl concentrations (ranging from 0 to 150 mM) and assessed morphological parameters and plant development. Our findings revealed a pattern of cytosolic acidification in cells across all root zones at lower NaCl concentrations (50, 100 mM). Interestingly, at 150 mM NaCl, pHcyt levels either increased or returned to normal, indicating a nonlinear effect of salinity on pHcyt. Most studied parameters related to development and morphology exhibited an inhibitory influence in response to NaCl. Notably, a nonlinear relationship was observed in the cell length within the elongation and differentiation zones. While cell elongation occurred at 50 and 100 mM NaCl, it was not evident at 150 mM NaCl. This suggests a complex interplay between stimulating and inhibitory effects of salinity, contributing to the nonlinear relationship observed between pHcyt, cell length, and NaCl concentration.

Heterologous codA Gene Expression Leads to Mitigation of Salt Stress Effects and Modulates Developmental Processes.

Transgenic tobacco plants overexpressing the choline oxidase gene from A. globiformis showed an increase in resistance at the level of primary and secondary biosynthesis of metabolites, removing the damage characteristic of salinity and stabilizing the condition of plants. We used 200 mM NaCl, which inhibits the growth of tobacco plants at all stages of development. Leaves of transgenic and wild-type (WT) plants Nicotiána tabácum were used for biochemical, cytological and molecular biological analysis. However, for transgenic lines cultivated under normal conditions (without salinity), we noted juvenile characteristics, delay in flowering, and slowing down of development, including the photosynthetic apparatus. This caused changes in the amount of chlorophyll, a delay in the plastid grana development with the preservation of prolamellar bodies. It also caused changes in the amount of sugars and indirectly downstream processes. A significant change in the activity of antioxidant enzymes and a change in metabolism is probably compensated by the regulation of a number of genes, the expression level of which was also changed. Thus, the tolerance of transgenic tobacco plants to salinity, which manifested itself as a result of the constitutive expression of codA, demonstrates an advantage over WT plants, but in the absence of salinity, transgenic plants did not have such advantages due to juvenilization.

A Simple and Effective Bioassay Method Suitable to Comparative In Vitro Study of Tomato Salt Tolerance at Early Development Stages.

In vitro evaluation of tomato seeds and seedlings for salt tolerance has undoubted advantages (high productivity, as well as stability and reproducibility of the obtained experimental data due to the maintenance of constant controlled conditions) in comparison with open-field system and pot experiments. However, even high-quality seeds greatly differ in the uniformity of germination capacity and germination energy. Heterogeneous germination in the habit and developmental stage of plant material significantly distorts the obtaining of relevant experimental data suitable for correct interpretation. In our study, we propose a simple and effective bioassay method suitable to comparative in vitro study of tomato salt tolerance using shoot apex of seedlings at the early first-true-leaf stage. Shoot apexes cultured the on the root induction medium (RIM) supplemented with 0.2 mg/L indole-3-butyric acid (IBA) and NaCl at different concentrations (0-250 mM NaCl) revealed significant differences between two tomato genotypes (line YaLF and cv. Rekordsmen) at the organismal (measurements of CO2 gas exchange), organ (rhizogenesis frequency; number and length of de novo regenerated roots; root fresh (RFW) and dry (RDW) weights; shoot fresh (SFW) and dry (SDW) weights), tissue (the average cross-sectional area of epidermal and mesophylls cotyledonary cells) and cellular (ultrastructure of chloroplast and nuclear compartments) development levels. In addition, a quantitative comparison of proline and photosynthetic pigments contents under 75 and 150 mm NaCl treatments showed a different response between two tomato genotypes. The proposed methodological approach can be used for other plants with a high response to auxin-induced rhizogenesis in vitro, as well as for the comparative in vitro assessment of other abiotic stresses.

Expression of Cytochrome c3 from Desulfovibrio vulgaris in Plant Leaves Enhances Uranium Uptake and Tolerance of Tobacco.

Cytochrome c3 (uranyl reductase) from Desulfovibrio vulgaris can reduce uranium in bacterial cells and in cell-free systems. This gene was introduced in tobacco under control of the RbcS promoter, and the resulting transgenic plants accumulated uranium when grown on a uranyl ion containing medium. The uptaken uranium was detected by EM in chloroplasts. In the presence of uranyl ions in sublethal concentration, the transgenic plants grew phenotypically normal while the control plants' development was impaired. The data on uranium oxidation state in the transgenic plants and the possible uses of uranium hyperaccumulation by plants for environmental cleanup are discussed.

Morpho-Physiological Testing of NaCl Sensitivity of Tobacco Plants Overexpressing Choline Oxidase Gene.

In this study the transgenic lines (TLs) of tobacco (Nicotianatabacum L.), which overexpress the heterologous gene encoding the bacterial enzyme choline oxidase were evaluated. The goal of our work is to study the effect of choline oxidase gene expression on the sensitivity of plant tissues to the action of NaCl. The regenerative capacity, rhizogenesis, the amount of photosynthetic pigments and osmotically active compounds (proline and glycine betaine) were assessed by in vitro cell culture methods using biochemical and morphological parameters. Transgenic lines with confirmed expression were characterized by high regeneration capacity from callus in the presence of 200 mmol NaCl, partial retention of viability at 400 mmol NaCl. These data correlated with the implicit response of regenerants and whole plants to the harmful effects of salinity. They turned out to be less sensitive to the presence of 200 mmol NaCl in the cultivation medium, in contrast to the WT plants.