Различные стрессовые факторы приводят к повышению уровня активных форм кислорода (АФК) и усилению повреждения различных тканей растений. Избыточная продукция АФК при окислительном стрессе является частью многих стрессовых ситуаций, включая гипоксию и действие перекиси водорода. Целью данной работы было изучение влияния стрессовых факторов, таких как гипоксия и действие перекиси водорода, на образование и локализацию АФК у двух генотипов пшеницы Triticum aestivum и Triticum durum и защитного действия кверцетина от АФК.

The aim of the study was to study the state and reaction of cytoskeletal elements, microtubules and actin filaments, in root cells of Samsun tobacco plants and its transgenic line expressing the FeSOD1 gene from Arabidopsis thaliana with the pea rbcS leader sequence for the localization of the gene product in chloroplasts encoding Fe-dependent superoxide dismutase, constitutively inducing intracellular oxidative stress, by increasing the H2O2 pool for a long-term effect of moderate concentrations of NaCl and Na2SO4. The main hypothesis was to identify the positive protective effect of controlled constant oxidative stress on the stability of the most sensitive system that provides growth by division and growth by extension (the tubulin cytoskeleton) and effective intracellular transport and structural stability (the actin filament system). Localization of the microtubule cytoskeleton and actin filaments using antibodies to tubulin clone DM1α and actin clone 10-B3 by transmission electron microscopy and immunocytologically, detected by treatment with the second antibodies conjugated with Alexa‑488, made it possible to establish signs of reorganization and disassembly of the actin filament network under the action of NaCl and Na2SO4 as in control and in transgenic plants. At the same time, in transgenic plants, differences can be noted even without exposure, which indicates the effectiveness of this method for stimulating a protective response. These data suggest that the state of the system of the tubulin cytoskeleton and actin filaments may be an indicator of the resistance of FeSOD1 transgenic plants to salinity. A relationship has also been established between the reorganization of the cytoskeleton and vacuolization, especially with Na2SO4.

A comparative analysis of tomato roots regenerated in vitro on media supplemented with different NaCl concentrations (0–250 mM) has been carried out. A morphogenetic study performed at the organ level has determined rhizogenesis-inhibiting NaCl concentrations, while the morphometric analysis of regenerated roots in juvenile seedlings has registered such characteristics as their number, length, and fresh/dry weight. A cytological study has revealed some tissue disorders, such as changes in the vacuolization of root cap cells and root cortex cells (RCC) under salinity conditions. At some NaCl concentrations, significant changes in the cells of these tissues have been observed in relation to such parameters as the root cap length (50–100 and 250 mM NaCl), the number of root cap layers (50, 75, 150–250 mМ NaCl), the area of central cylinder cells (CCCs, 75–150 and 250 mM NaCl), the RCC area (100–200 mM NaCl), and the nucleolus to nucleus ratio (25, 50, and 200 mM NaCl). Using cytophotometry, we have shown there to be an increase in the number of interphase cells of the root meristem in the G2 phase with a simultaneous decrease of this parameter in the G1 phase. Immunofluorescent labeling has revealed various disorganizations in the alpha-tubulin cytoskeleton of interphase root meristem cells. Using transmission electron microscopy, we have revealed structural changes in plastids of root cap cells, RCC, and CCC, as well as changes in the organization of a nuclear compartment in RCC occurred in the presence of 150 mM NaCl. The studied characteristics can be used for a comparative evaluation of tomato genotypes under salinity at different levels of their organization. The proposed approach can be also used for crops that have no difficulties with induced rhizogenesis in vitro.

It is revealed that dipeptides glycylglycine (GlyGly) and glycylaspartic acid (GlyAsp), as well as the amino acid glycine (Gly), appreciably stimulated the growth and development of tobacco ( Nicotiana tabacum L.) regenerants and seedlings if they were present in a medium at 10 –7 М. All three compounds GlyGly, GlyAsp, and Gly influenced the cell differentiation and morphogenic processes in the calli. The compounds modulated expression of the KNOX and GRF family genes. The profiles of induction or repression of gene expression by one and the same peptide were found to differ in tobacco regenerants and seedlings. It is concluded that GlyGly, GlyAsp, and Gly may be considered as regulators of plant growth and development, the mode of action of which is signaling and mainly epigenetic.

The dipeptides glycylglycine (GlyGly) and glycylaspartic acid (GlyAsp) and amino acid glycine (Gly) in a concentration of 10 –7 M in a medium essentially stimulate the growth and development of tobacco ( Nicotiana tabacum ) calli. GlyGly, GlyAsp, and Gly influence the cell differentiation and tissue formation processes. They stimulate formation and growth of leaves and roots. After incubation of tobacco seedling roots in the presence of 10 –5 M fluorescent FITC-labeled peptides or glycine, marked fluorescence was observed in cells of the root cap and epidermis. The fluorescence was detected in the cell walls, cytoplasm, and nuclei. Thus, the peptides used can penetrate into the plant cell and be located in the nucleus and other cell compartments. Therefore, they may potentially interact with different structures and components of the cytoplasm and nucleus including various proteins, RNAs, and DNA. The penetration and accumulation of peptides in cells are tissue specific. In the tobacco callus, peptides modulate expression of the KNOX and GRF family genes that are responsible for cell differentiation and code for transcription factors. Thus, the dipeptides GlyGly and GlyAsp and amino acid Gly have marked physiological activity and can be related to efficient plant growth regulators.

The transformation of the structural organization of interphase nuclei of a plant cell, depending on the type of tissue, ploidy, and the action of abiotic factors of the medium, has been studied by light and transmission electron microscopy. It is shown that the location and the quantitative relationship between condensed and decondensed chromatin, the presence and localization of nuclear bodies in the plant nucleus, and the presence of invaginations of the nuclear membrane and inclusions depend on the tissue type and cell age as well as the intensity, time, and type of exposure to abiotic factors. Examples of different degrees of chromatin condensation in one plant organism are given. It has been established that the degree of chromatin condensation and decondensation, the state of other domains and nonspecific inclusions of the cell nucleus can be artificially modeled for research purposes or subsequent modification. The issue of application of such technologies for the creation and selection of stable forms of agricultural plants taking into account the controlled modification of their genome is discussed. A possible mechanism for incorporation of nonspecific inclusions in a nuclear compartment is proposed.

Using the methods of light microscopy and DNA cytophotometry, structural organization of the calli of the barley hybrid line Duet x Bios grown in the absence and in the presence of aluminum ions (20 and 40 mg/L) was studied. In the morphogenic calli grown in the standard conditions without aluminum, three main cell types were identified: meristematic, differentiated, and dying. Meristematic cells form a morphogenic zone. A characteristic feature of differentiated cells is a progressive accumulation of vacuoles with storage substances. Structurally separated zones of meristematic cells were not found the calli surviving in the presence of aluminum. Besides, the number of differentiated cells with storage substances is dramatically reduced and the number of dying cells is increased in such calli. According to the cytophotometry data, morphological changes in the aluminum-tolerant calli are accompanied by partial suppression of proliferation, accumulation of cells in postsynthetic phase of the cell cycle, and polyploidization, presumably caused by the DNA endoreduplication. It is noteworthy that the aluminum-tolerant calli exhibit a high regenerative capacity: the proportion of morphogenic calli surviving in the presence of 40 mg/L aluminum is about 5 times greater than that in the calli grown in the control medium. To identify cellular targets of the aluminum effect in differentiated tissues, the roots of rhizogenic calli were studied. For this purpose, the calli derived from aluminum-tolerant plants (cultivar Kupetz), from aluminum-sensitive line 999-93, and from aluminum-tolerant line 917-1 obtained by the method of cell selection were used. It was found that aluminum induces the death of differentiated cells of epidermis and vascular system ingrowing roots of calli derived from non-tolerant lines, whereas undifferentiated cells of meristem remain undamaged. In contrast, rhizogenic calli derived from tolerant forms produce normal roots. This observation has an important prognostic value because it makes it possible to select aluminum-tolerant regenerants at the early stages of cell selection. The data suggest that most likely cause of the emergence of resistant lines is somaclonal variation induced by high concentrations of the selecting agent. Under stressful conditions such variability may result in the in vitro system from the activation of epigenetic factors, such as, for example, methylation of DNA or modification of the chromatin proteins.

C использованием методов световой микроскопии и цитофотометрии ДНК проведен сравнительный анализ структурной организации каллусов гибридной линии ячменя Дуэт ? Биос, выращенных в стандартных условиях культивирования и на средах, содержащих ионы алюминия (20 и 40 мг/л). В морфогенных каллусах, растущих на среде без алюминия, идентифицировано три основных типа клеток: меристематические, дифференцированные и гибнущие. Меристематические клетки формируют морфогенные зоны, характерным признаком дифференцированных клеток является прогрессирующее накопление вакуолей с запасными веществами. В каллусах, выживающих в присутствии алюминия, отсутствуют структурно обособленные зоны меристематических клеток, резко снижается количество дифференцированных клеток с запасными веществами и возрастает количество гибнущих клеток. По данным цитофотометрии изменение морфологического статуса устойчивых каллусов сопровождается частичным подавлением пролиферации, накоплением клеток в постсинтетической фазе клеточного цикла и полиплоидизацией, по-видимому, вызванной эндоредупликацией ДНК. Принципиально важно, что устойчивые к токсическому действию алюминия каллусы обладают высокой способностью к регенерации: доля морфогенетически активных каллусов, выживших в среде с алюминием (40 мг/л) примерно в 5 раз превосходит долю морфогенных каллусов в контроле. Для выявления клеточных мишеней токсического действия алюминия в сформированных тканях изучены корешки ризогенных каллусов. С этой целью использовали каллусы, полученные от растений, толерантных к токсическому действию алюминия (сорт Купец), чувствительной линия 999-93 и полученной методом клеточной селекции линии, устойчивой к действию алюминия линия 917-1. Оказалось, что в растущих корнях каллусов, полученных от неустойчивых линий, алюминий индуцирует гибель дифференцированных клеток эпидермиса и проводящей системы, тогда как недифференцированные клетки меристемы остаются интактными. В противоположность этому ризогенные каллусы, полученные от устойчивых форм, дают нормальные корешки. Это наблюдение имеет важное прогностическое значение, так как позволяет отбирать устойчивые к алюминию регенеранты на ранних этапах клеточной селекции. Полученные данные позволяют высказать предположение, что появление устойчивых линий обусловлено, по-видимому, сомаклональной изменчивостью, индуцированной высокими концентрациями селективного агента. Такого рода изменчивость возникает в системе in vitro в условиях стресса, в результате активации таких эпигенетических факторов, как, например, метилирование ДНК или ацетилирование белков хроматина.

In determination of salt resistance of wild plants, cytophotometry has been used for evaluation of distribution of nuclei of the root meristem cells. Salt resistant and salt sensitive plants of two Thellungiella species whose seeds were collected at the places of their vegetation, as well as of two Aegilops L. species from the collection of the All-Russia Research Institute of Plant Industry (VIR), have been analyzed in the experiments. Salt resistant cells of Thel. botschantzevii and Ae. taushii (k-677) were accumulated in the G1 period of the interphase, which indicates high adaptation of these plants to high concentrations of salts. It has been demonstrated that cytophotometry can be used for determination of salt resistance of wild plants growing in the climatic zones appropriate for their natural propagation.

In this study, the morphological and cytoembryological analyses of the tomato plants transformed with the genes encoding chitin-binding proteins (ac and RS-intron-Shir) from Amaranthus caudatus L. and A. retroflexus L., respectively, as well as the gene amp2 encoding hevein-like antimicrobial peptides from Stellaria media L., have been performed. The transgenic lines were adapted to soil and grown in the greenhouse. The analysis of putative transgenic tomato plants revealed several lines that did not differ phenotypically from the wild type plants and three lines with disruption in differentiation of the inflorescence shoot and the flower, as well as the fruit formation (modified plants of each line were transformed with a single gene as noted before). Abnormalities in the development of the generative organs were maintained for at least six vegetative generations. These transgenic plants were shown to be defective in the mail gametophyte formation, fertilization, and, consequently, led to parthenocarpic fruits. The detailed analysis of growing ovules in the abnormal transgenic plants showed that the replacement tissue was formed and proliferated instead of unfertilized embryo sac. The structure of the replacement tissue differed from both embryonic and endosperm tissue of the normal ovule. The formation of the replacement tissue occurred due to continuing proliferation of the endothelial cells that lost their ability for differentiation. The final step in the development of the replacement tissue was its death, which resulted in the cell lysis. The expression of the genes used was confirmed by RTPCR in all three lines with abnormal phenotype, as well as in several lines that did not phenotypically differ from the untransformed control. This suggests that abnormalities in the organs of the generative sphere in the transgenic plants do not depend on the expression of the foreign genes that were introduced in the tomato genome. Here, we argue that agrobacterial transformation affects, directly or indirectly, expression of genes encoding for transcription factors that can activate a gene cascade responsible for the normal plant development.

В работе проведен морфологический и цитоэмбриологический анализ растений томата, трансформированных генами, кодирующими хитинсвязывающие белки из Amaranthus caudatus L. (ac) и A. retroflexus L. (RS-intron-Shir), а также гевеиноподобные антимикробные пептиды из Stellaria media L. (amp2). Полученные трансгенные линии были адаптированы к почвенным условиям и выращены в защищенном грунте. Анализ трансгенных растений поколения Т0 выявил линии, фенотипически не отличающиеся от растений дикого типа, и три линии (по одной линии с каждым из вышеперечисленных генов), которые имели существенные нарушения в дифференцировке цветоносных побегов, строении цветков и плодов. Продемонстрировано сохранение нарушений в развитии генеративных органов в 6 вегетативных поколениях. Показано, что у трансгенных растений с вегетативно наследуемыми аномалиями наблюдаются нарушения в формировании мужского гаметофита, отсутствие нормального оплодотворения и, как следствие, развитие партенокарпических плодов. Детальный анализ растущих семяпочек аномальных трансгенных линий показал, что на месте неоплодотворенного зародышевого мешка формируется и разрастается замещающая ткань, по структуре отличающаяся как от зародышевой, так и от эндоспермальной ткани нормальной семяпочки. Формирование замещающей ткани происходит в результате продолжающейся пролиферации клеток эндотелия, утративших способность к нормальной дифференцировке. Конечным этапом развития замещающей ткани является ее гибель, сопровождающаяся лизисом клеток. Методом ОТ-ПЦР экспрессия целевых генов была подтверждена у всех трех линий с аномальным фенотипом, а также у ряда линий, фенотипически не отличающихся от нетрансформированного контроля. Это означает, что нарушения органов генеративной сферы у трансформированных растений не зависят от экспрессии привнесенных в геном томата гетерологичных генов. Обсуждается, что причиной возникновения нарушений у трансгенных растений является прямое или опосредованное влияние агробактериальной трансформации на изменение экспрессии генов, кодирующих транскрипционные факторы и контролирующих включение каскада генов, необходимых для нормального развития растений.