Tatiyana Anatolievna Dilovarova

The short peptide AlaGluAspLeu (AEDL) stimulates shoot and root development in Nicotiana tabacum. Growing tobacco in the presence of AEDL was found to induce autophagy and programmed cell death, as demonstrated using immunodetection of the autophagy marker ATG8 and cytochrome c in the cytoplasm, as well as the detection of DNA breaks using the TUNEL assay. A detailed study of the ultrastructure of Nicotiana tabacum root cells grown in the presence of AEDL using transmission electron microscopy revealed fundamental structural differences from control cells. Control cells contained only lytic vacuoles, while in the presence of AEDL, tobacco root meristem cells contained predominantly protein-storing vacuoles and amyloplasts with numerous starch granules in the stroma. Characteristic types of phagophores were identified, forming numerous small autophagosomes with cytoplasmic regions, multivesicular bodies, or concentric membranes, possibly with cytoskeletal elements. Expression of autophagy protein genes revealed a decrease in TOR expression, which promoted autophagy activation and prevented ATG13 phosphorylation. ATG8 gene expression significantly increased in the presence of the AEDL peptide. Schematic diagrams of autophagy processes in root cells of control plants and those grown in the presence of AEDL are presented. Based on these data, it was concluded that stimulation of tobacco plant development in the presence of the AEDL peptide at a concentration of 10⁻⁷ M occurs due to the activation of metabolic processes and autophagy. Moreover, the synthesis of metabolites exceeds the required amount of nutrients, which accumulate in vacuoles and leucoplasts.

The overall survival of a plant depends on the development, growth, and functioning of the roots. Root development and growth are not only genetically programmed but are constantly influenced by environmental factors, with the roots adapting to such changes. The peptide AEDL (alanine–glutamine acid–asparagine acid–leucine) at a concentration of 10⁻⁷ M had an elongating effect on the root cells of Nicotiana tabacum seedlings. The action of this peptide at such a low concentration is similar to that of peptide phytohormones. In the presence of 150 mM NaCl, a strong distortion in the development and architecture of the tobacco roots was observed. However, the combined presence of AEDL and NaCl resulted in normal root development. In the presence of AEDL, reactive oxygen species (ROS) were detected in the elongation and root hair zones of the roots. The ROS marker fluorescence intensity in plant cells grown with AEDL was much lower than that of plant cells grown without the peptide. Thus, AEDL protected the root tissue from damage by oxidative stress caused by the toxic effects of NaCl. Localization and accumulation of AEDL at the root were tissue-specific. Fluorescence microscopy showed that FITC-AEDL predominantly localized in the zones of elongation and root hairs, with insignificant localization in the meristem zone. AEDL induced a change in the structural organization of chromatin. Structural changes in chromatin caused significant changes in the expression of numerous genes associated with the development and differentiation of the root system. In the roots of tobacco seedlings grown in the presence of AEDL, the expression of WOX family genes decreased, and differentiation of stem cells increased, which led to root elongation. However, in the presence of NaCl, elongation of the tobacco root occurred via a different mechanism involving genes of the expansin family that weaken the cell wall in the elongation zone. Root elongation of plants is of fundamental importance in biology and is especially relevant to crop production as it can affect crop yields.

In this work, we studied in vitro propagation of three cultivars of Mentha × piperita L. Murashige and Skoog medium (MS) supplemented with 0.5 mg·L⁻¹ BAP was the most optimal medium for micropropagation of the cultivars studied. The ability of peppermint plants field-acclimated after in vitro micropropagation to produce essential oils (EOs) was investigated. EO was obtained by hydrodistillation from dried leaves and flowering shoots from control (field grown) plants and plants acclimated in field conditions after in vitro propagation. The samples were collected at the first and second year of vegetation, and their chemical composition was investigated using gas chromatography-mass spectrometry (GC-MS). Differences were observed in the yield, as well as in the quantitative and qualitative composition of the EOs extracted from the control plants and field-acclimated plants after in vitro propagation. Menthol was the main component of the EO in control plants, while pulegone and menthone were dominant in the EO pattern in field-acclimated in vitro regenerants in the first year of the growing season. However, in the second year of vegetation, the content of the main EO components in field-acclimated peppermint plants was approximately the same as in control plants. The antioxidant activity of EOs extracted from field-acclimated after in vitro micropropagation plants was found to be the same as in control field-grown M. × piperita plants.

Various abiotic stresses cause the appearance of reactive oxygen species (ROS) in plant cells, which seriously damage the cellular structures. The engineering of transgenic plants with higher production of ROS-scavenging enzyme in plant cells could protect the integrity of such a fine intracellular structure as the cytoskeleton and each cellular compartment. We analyzed the morphological changes in root tip cells caused by the application of iso-osmotic NaCl and Na₂SO₄ solutions to tomato plants harboring an introduced superoxide dismutase gene. To study the roots of tomato plants cultivar Belyi Naliv (WT) and FeSOD-transgenic line, we examined the distribution of ROS and enzyme-linked immunosorbent detection of α-tubulin. In addition, longitudinal sections of the root apexes were compared. Transmission electronic microscopy of atypical cytoskeleton structures was also performed. The differences in the microtubules cortical network between WT and transgenic plants without salt stress were detected. The differences were found in the cortical network of microtubules between WT and transgenic plants in the absence of salt stress. While an ordered microtubule network was revealed in the root cells of WT tomato, no such degree of ordering was detected in transgenic line cells. The signs of microtubule disorganization in root cells of WT plants were manifested under the NaCl treatment. On the contrary, the cytoskeleton structural organization in the transgenic line cells was more ordered. Similar changes, including the cortical microtubules disorganization, possibly associated with the formation of atypical tubulin polymers as a response to salt stress caused by Na₂SO₄ treatment, were also observed. Changes in cell size, due to both vacuolization and impaired cell expansion in columella zone and cap initials, were responsible for the root tip tissue modification.

The toxicity of high concentrations of sodium chloride creates significant difficulties in realizing the productivity potential of wheat. The development of effective test systems for the identification and selection of resistant genotypes is an urgent task given the global increase in soil salinity in agricultural land. To identify the characteristics of the plant’s reaction to the toxic effect of sodium chloride, wheat genotypes with different resistance to ionic toxicity (the Orenburgskaya 10 and Orenburgskaya 22 varieties) were used. In model experiments, we used fluorescence, light-optical and electron microscopy to characterize the structural and functional features of the cells of the roots of wheat seedlings, and cytological markers suitable for creating a test system for the early diagnosis of the sensitivity of wheat genotypes to sodium chloride were established. The response of the plants to the effects of sodium chloride was assessed by changes in biometric data, respiration rate, peculiarities in the accumulation of reactive oxygen species (ROS) and mitochondrial staining, and the quantitative assessment of coleoptile cell viability as putative sensitivity markers. In the sodium chloride-sensitive genotype (Orenburgskaya 10), toxic effects resulted in oxidative damage in the root cells, while in the resistant genotype (Orenburgskaya 22), oxidative damage to the cells was minimal. A high level of expression of mitochondrial superoxide dismutase (MnSOD) was found in the roots of the Orenburgskaya 22 variety. The identification and functional analysis of cytological and molecular markers provide the basis for further studies of the resistance of wheat to sodium chloride stress.

Determining salt tolerance potential in wheat is one of the most important problems in breeding practice for areas with primary and secondary salinity. Presence of large areas of saline soils results in inhibition of growth, development and stability in obtaining high yields of agricultural plants. Therefore, there is a need for a comprehensive studying and improving of diagnostic methods during early growth stages. Different genotypes of wheat Triticum aestivum Host. and Triticum durum Desf. were used to identify salt tolerance markers. Both morphometric and some biochemical indicators of wheat varieties were used as salt tolerance markers. At this stage, it was shown that a comprehensive description of wheat varieties is needed to assess resistance of wheat varieties to chloride salinity.

Phytohormones, which are secreted peptides, play an important role in intercellular inter-actions, participating in the regulation of development and in numerous physiological processes and in responses to the influence of environmental factors. Short peptides in a concentration of 10-7-10-8 M regulate the growth and development of the callus culture of tobacco (Nicotiana tabacum L.) in culture in vitro. AlaGluAspGly, AlaAspGluLeu, GlyGly and GlyAsp on the 28th day of cultivation showed an increase in the biomass of tobacco by a factor of 1.5 to 2.5, an increase in the number of regenerants per explant by 10-30%, and also the area of the leaf plate of regenerants by 2-2.5 times. Exogenous peptides influence the expression of genes encoding the proteins of growth factor regulators. It was found that the expression values calculated by the PCR-PB method depend on the nature of the peptide. In addition, structurally different peptides differentially affect the different genes (growth regulating factor) of GRF. The greatest increase in the expression level of GRF family genes is observed in the presence of AlaAspGluLeu - GRF1, GRF3, GRF4 3.5-4 times, in the presence of AlaGluAspGly - GRF2 more than 4.5 times, GlyGly - GRF4 more than 3 times, GlyAsp - GRF3, GRF4 in 3-4 times. It is assumed that in the cell culture, short peptides can act as a regulator of the growth of new generation plants that can find application in biotechnology and practical plant growing.