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 Accumulation of Volatile Compounds and the Change in the Morphology of the Leaf Wax Cover Accompanied the "Anti-Aging" Effect in Anethum graveolens L. Plants Sprayed with 6-Benzylaminopurine.

Essential oils (EOs) are of commercial importance for medicine, food, cosmetics, the perfume industry, and agriculture. In plants, EOs, like the wax cover, serve as protection against abiotic stresses, such as high temperatures and water deficiency. The use of spraying with exogenous hormones of aromatic plants affects the accumulation and composition of volatile compounds, as well as tolerance to abiotic stress. As a result of cytokinin treatment with 6-BAP (6-benzylaminopurine) (200 mg L-l) of Anetum graveolens L. "Uzory" and "Rusich" varieties, several responses to its action were revealed: a change in the division of leaf blades, inhibition of flowering, an increase in the content of EO and its main components α-phellandrene and p-cymene in leaves, and limonene in umbels and fruits. It was revealed that the increased accumulation of EO in dill leaves was longer with sufficient moisture. In contrast, under conditions of heat and water deficiency, the effect of 6-BAP treatment on accumulations of the EO in leaves was short-lived and did not appear on umbels and fruits. The study of the cytokinin effect on a fine structure of a wax cover on the adaxial side of leaves by scanning electron microscopy revealed a change in its elements (from amorphous layers with scales to thin tubules), which probably increased the sensitivity of leaves to water deficiency and, consequently, led to a decrease in the biosynthetic activity of leaf tissue. Thus, 6-BAP had an impact on the adaptive properties of dill plants, prolonging the "youth" of vegetative organs and the ability to EO biosynthesis under conditions of sufficient moisture.

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.

Unexpected Effects of Sulfate and Sodium Chloride Application on Yield Qualitative Characteristics and Symmetry Indicators of Hard and Soft Wheat Kernels.

The heterogeneity of grain quality can lead to limited predictability of qualitative and quantitative characteristics of the wheat yield, especially with an increase in the importance of drought and salinity caused by climate change. This study was undertaken with the aim of creating basic tools for phenotyping and assessing the sensitivity of genotypes to salt effects at the level of some wheat kernel attributes. The study considers 36 variants of the experiment, including four wheat cultivars-Zolotaya, Ulyanovskaya 105, Orenburgskaya 10, Orenburgskaya 23; three treatment variants-control (without salt) and two salts exposure (NaCl at a concentration of 1.1 g L-1 and Na2SO4 at a concentration of 0.4 g L-1); as well as three options for the arrangement of kernels in a simple spikelet-left, middle, and right. It has been established that the salt exposure had a positive effect on the percentage of kernel fulfilling in the cultivars Zolotaya, Ulyanovskaya 105, and Orenburgskaya 23 compared to control. The kernels of the Orenburgskaya 10 variety matured better in the experiment with Na2SO4 exposure, while the control variant and NaCl gave the same effect. When exposed to NaCl, significantly greater values of weight, transverse section area, and transverse section perimeter of the kernel were noted in the cv Zolotaya and Ulyanovskaya 105. Cv Orenburgskaya 10 responded positively to the use of Na2SO4. This salt caused an increase in the area, length, and width of the kernel. The fluctuating asymmetry of the left, middle, and right kernels in the spikelet was calculated. In the cv Orenburgskaya 23 the salts affected only the kernel perimeter among parameters examined. The indicators of the general (fluctuating) asymmetry were lower in the experiments with the use of salts, i.e., kernels were more symmetrical than in the control variant, both for the cultivar as a whole and when compared taking into account the kernel location in spikelet. However, this result was unexpected, since salt stress inhibited a number of morphological parameters: the number and average length of embryonic, adventitious, and nodal roots, flag leaf area, plant height, dry biomass accumulation, and plant productivity indicators. The study showed that low concentrations of salts can positively affect the fulfilling of kernels (the absence of a cavity inside the kernel) and the symmetry of the left and right sides of the kernel.

Bread Wheat in Space Flight: Is There a Difference in Kernel Quality?

Planning long-term space flights necessarily includes issues of providing food for the crew. One of the areas of research is the development of technologies for independent production of food by the crew. Extensive research on lettuce has confirmed that the "space production" of lettuce is not inferior to that on Earth, even in the absence of gravity, but the same deep understanding of the quality of grain crops has not yet been achieved. Therefore, the goal of our work is to establish whether the conditions for growing wheat in outer space without gravity affect the weight and basic parameters of the grain, and whether this leads to increased asymmetry of the kernel and distortion of the starch composition. The objects of the study were wheat (Triticum aestivum L.) kernels of the Super Dwarf cultivar. Of which, 100 kernels matured in outer space conditions in the Lada growth chamber on the International Space Station (ISS), and 85 kernels of the control wheat grown in a similar growth chamber under terrestrial conditions. It has been established that kernels from ISS have significant differences to a smaller extent in weight, area, length, and width of the kernel. However, the kernels under both conditions were predominantly large (the average weight of a kernel in space is 0.0362 g, and in terrestrial conditions-0.0376 g). The hypothesis that the level of fluctuating asymmetry will increase in outer space was not confirmed; significant differences between the options were not proven. In general, the kernels are fairly even (coefficients of variation for the main parameters of the kernel are within 6-12%) and with a low or very low level of asymmetry. The length of starch granules of type A in filled and puny kernels is significantly greater in kernels from ISS than in the control, and in terms of the width of starch granules B and roundness indices, both experimental variants are the same. It can be assumed that the baking qualities of earthly kernels will be slightly higher, since the ratio of type B starch granules to type A is 5-8% higher than on the ISS. Also, the width of the aleurone layer cells in mature kernels was significantly inferior to the result obtained on Earth. The work proposes a new method for establishing the asymmetry of kernels without a traumatic effect (in early works, it was supposed to study asymmetry in transverse sections of the kernels). Perhaps this will make it possible to further develop a computer scanning program that will determine the level of asymmetry of the wheat fruit.

Formation of Unique Placental Seed Capsules in the Maturation Process of the Tomato Fruit.

The morphological and anatomical study of the seed formation features in a juicy tomato fruit was carried out. The ovules, which form on the placenta, have been shown to be gradually enveloped by the protrusions of placental tissue that arises simultaneously with them. As a result of this process, each seed is enclosed in an individual capsule. These seed capsules have been shown in vivo to be airtight and air-filled. Tomato seeds, as has been shown in this study, develop inside these capsules until the full maturity of the fruit and do not come into contact with the detached and moldered cells of the placenta protrusions, which convert into a gel (pulp). Using scanning electron microscopy, it was possible to reveal the details of a ribbon-like "pubescence" formation of the tomato seed, as well as to understand the mechanism of cracking of the outer layer cells in the seed coat, associated with the detection of calcium oxalate crystals in these cells. The unique outer layer of the tomato seed coat seems to play the role of a scaffold that maintains a constant volume of the protective capsule.

Effects of Hydrogen Peroxide on In Vitro Cultures of Tea (Camellia sinensis L.) Grown in the Dark and in the Light: Morphology, Content of Malondialdehyde, and Accumulation of Various Polyphenols.

Tea plants (Camellia sinensis L.) are phenol-accumulating crops that are widely used for public health. The healing effect of tea leaf products is due to the biosynthesis of such phenolic compounds (PCs) as flavans, which have P-vitamin capillary-strengthening activity. Due to their limited habitat and the value of their specialized metabolites of a phenolic nature, a promising approach is to establish in vitro cultures from them that retain the ability to form PCs, which is characteristic of ex vivo tea plants. The aim of this study was to investigate the effect of exogenic H2O2 (0.01 mM; 0.1 mM; 1 mM) on the growth, morphology, degree of stress response, and accumulation of various phenolic compounds in tea plant callus cultures of different ages (24 or 36 days) grown under different cultivation conditions (darkness or light). According to the results obtained, the H2O2 effect on tea callus cultures of different ages did not cause changes in their morphophysiological characteristics, both after 2 h of exposure (rapid response of callus culture, RRCC) and after 48 h (delayed response of callus culture, DRCC). The determination of the malondialdehyde (MDA) content, which serves as an indicator of changes in the level of lipid peroxidation (LPO) and the presence of stress responses in plant cells, indicated either its maintenance at the control level, a decrease, or an increase. All these effects depended on the growth conditions of the tea callus cultures (darkness or light), their age, the duration of exposure (rapid or delayed response), and the H2O2 concentration. Similar trends were noted for the total content of PCs as well as the amount of flavans, proanthocyanidins (soluble and insoluble forms), and lignin. The plant cell responses reflected changes in its adaptation programs, when specialized metabolites act as a target for the action of H2O2, thereby contributing to an increase in their resistance.

Morphological and Structural Details of Tomato Seed Coat Formation: A Different Functional Role of the Inner and Outer Epidermises in Unitegmic Ovule.

In order to understand how and what structures of the tomato ovule with a single integument form the seed coat of a mature seed, a detailed study of the main development stages of the tomato ovule integument was carried out using the methods of light and electron microscopy. The integument itself it was shown to transform in the course of development into the coat (skin) of a mature seed, but the outer and inner epidermises of the integument and some layers of the integument parenchyma are mainly involved in this process. The outer epidermis cells are highly modified in later stages; their walls are thickened and lignified, creating a unique relatively hard outer coat. The fate of the inner epidermis of integument is completely different. It is separated from the other parenchyma cells of integument and is transformed into an independent new secretory tissue, an endothelium, which fences off the forming embryo and endosperm from the death zone. Due to the secretory activity of the endothelium, the dying inner parenchyma cells of the integument are lysed. Soon after the cuticle covers the endosperm, the lysis of dead integument cells stops and their flattened remnants form dense layers, which then enter the final composition of the coat of mature tomato seed. The endothelium itself returns to the location of the integument inner epidermis.

Morphological and Ultrastructural Features of Formation of the Skin of Wheat (Triticum aestivum L.) Kernel.

The integumentary tissues of plant seeds protect the embryo (new sporophyte) forming in them from unfavorable external conditions; therefore, comprehensive knowledge about the structural and functional specificity of seed covers in various plants may be of both theoretical and practical interest. As a result of our study, additional data were obtained on the morphological and ultrastructural features of the formation of a multilayer skin of wheat (Triticum aestivum L.) kernel (caryopsis). The ultrastructure research analysis showed that differentiation of the pericarp and inner integument of the ovule leads to the formation of functionally different layers of the skin of mature wheat grain. Thus, the differentiation of exocarp and endocarp cells is accompanied by a significant thickening of the cell walls, which reliably protect the ovule from adverse external conditions. The cells of the two-layer inner integument of the ovule differentiate into cuticular and phenolic layers, which are critical for protecting daughter tissues from various pathogens. The epidermis of the nucellus turns into a layer of mucilage, which apparently helps to maintain the water balance of the seed. Morphological and ultrastructural data showed that the formation of the kernel's skin occurs in coordination with the development of the embryo and endosperm up to the full maturity of the kernel. This is evidenced by the structure of the cytoplasm and nucleus, characteristic of metabolically active protoplasts of cells, which is observed in most integumentary layers at the late stages of maturation. This activity can also be confirmed by a significant increase in the thickness of the cell walls in the cells of two layers of the exocarp and in cross cells in comparison with the earlier stages. Based on these results, we came to the conclusion that the cells of a majority in the covering tissues of the wheat kernel during its ontogenesis are transformed into specialized layers of the skin by terminal differentiation.

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.

Possible Role of Crystal-Bearing Cells in Tomato Fertility and Formation of Seedless Fruits.

Crystal-bearing cells or idioblasts, which deposit calcium oxalate, are located in various tissues and organs of many plant species. The functional significance of their formation is currently unclear. Idioblasts in the leaf parenchyma and the development of crystal-bearing cells in the anther tissues of transgenic tomato plants (Solanum lycopersicon L.), expressing the heterologous FeSOD gene and which showed a decrease in fertility, were studied by transmission and scanning electron microscopy. The amount of calcium oxalate crystals was found to increase significantly in the transgenic plants compared to the wild type (WT) ones in idioblasts and crystal-bearing cells of the upper part of the anther. At the same time, changes in the size and shape of the crystals and their location in anther organs were noted. It seems that the interruption in the break of the anther stomium in transgenic plants was associated with the formation and cell death regulation of a specialized group of crystal-bearing cells. This disturbance caused an increase in the pool of these cells and their localization in the upper part of the anther, where rupture is initiated. Perturbations were also noted in the lower part of the anther in transgenic plants, where the amount of calcium oxalate crystals in crystal-bearing cells was reduced that was accompanied by disturbances in the morphology of pollen grains. Thus, the induction of the formation of crystal-bearing cells and calcium oxalate crystals can have multidirectional effects, contributing to the regulation of oxalate metabolism in the generative and vegetative organs and preventing fertility when the ROS balance changes, in particular, during oxidative stresses accompanying most abiotic and biotic environmental factors.

Salt Stress-Induced Structural Changes Are Mitigated in Transgenic Tomato Plants Over-Expressing Superoxide Dismutase.

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.

Morphological Features of the Anther Development in Tomato Plants with Non-Specific Male Sterility.

The study was devoted to morphological and cytoembryological analysis of disorders in the anther and pollen development of transgenic tomato plants with a normal and abnormal phenotype, which is characterized by the impaired development of generative organs. Various abnormalities in the structural organization of anthers and microspores were revealed. Such abnormalities in microspores lead to the blocking of asymmetric cell division and, accordingly, the male gametophyte formation. Some of the non-degenerated microspores accumulate a large number of storage inclusions, forming sterile mononuclear pseudo-pollen, which is similar in size and appearance to fertile pollen grain (looks like pollen grain). It was discussed that the growth of tapetal cells in abnormal anthers by increasing the size and ploidy level of nuclei contributes to this process. It has been shown that in transgenic plants with a normal phenotype, individual disturbances are also observed in the development of both male and female gametophytes. The reason for the developmental arrest of some ovules was the death of endosperm at different stages of the globular embryo. At the same time, noticeable hypertrophy of endothelial cells performing a secretory function was observed. In the ovules of transgenic plants with abnormalities, the endothelium forms a pseudo-embryo instead of the embryo sac, stimulating the development of parthenocarpic fruits. The data obtained in this study can be useful for a better understanding of the genetic and molecular mechanisms of cytoplasmic male sterility and parthenocarpic fruit development in tomatoes.

Wheat Space Odyssey: "From Seed to Seed". Kernel Morphology.

The long-term autonomous existence of man in extraterrestrial conditions is associated with the need to cultivate plants-the only affordable and effective means for both providing oxygen and CO2 utilization, and providing one of the most habitual and energetically valuable products: plant food. In this study, we analyzed the results of the space odyssey of wheat and compared the morphological features of parental grains harvested from soil grown wheat plants, the grains obtained from plants grown in a specialized device for plant cultivation-the "Lada" space greenhouses during space flight in the ISS, and the grains obtained from plants in the same device on Earth. The seeds obtained under various conditions were studied using scanning electron microscopy. We studied the mutual location of the surface layers of the kernel cover tissues, the structural features of the tube and cross cells of the fruit coat (pericarp), and the birsh hairs of the kernels. It was found that the grains obtained under wheat plants cultivation on board of the ISS in near space had some specific differences from the parental, original grains, and the grains obtained from plants grown in the "Lada" greenhouse in ground conditions. These changes were manifested in a shortening of the birsh hairs, and a change in the size and relative arrangement of the cells of the kernel coat. We suggest that such changes are a manifestation of the sensitivity of the cytoskeleton reorganization systems and water exchange to the influence of particular physical conditions of space flight (microgravity, increased doses of radiation, etc.). Thus, the revealed changes did not hinder the wheat grains production "from seed to seed", which allows the cultivation of this crop in stable life support systems in near earth orbit.