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 Production of Helianthus Haploids: A Review of Its Current Status and Future Prospects.

The genus Helianthus comprises 52 species and 19 subspecies, with the cultivated sunflower (Helianthus annuus L.) representing one of the most important oilseed crops in the world, which is also of value for fodder and technical purposes. Currently, the leading direction in sunflower breeding is to produce highly effective heterosis F1 hybrids with increased resistance to biotic and abiotic stresses. The production of inbred parental lines via repeated self-pollination takes 4-8 years, and the creation of a commercial hybrid can take as long as 10 years. However, the use of doubled haploid technology allows for the obtainment of inbred lines in one generation, shortening the time needed for hybrid production. Moreover, it allows for the introgression of the valuable genes present in the wild Helianthus species into cultivated sunflowers. Additionally, this technology makes it possible to manipulate the ploidy level, thereby restoring fertility in interspecific hybridization. This review systematizes and analyzes the knowledge available thus far about the production of haploid and dihaploid Helianthus plants using male (isolated anther and microspore cultures) and female (unpollinated ovaries and ovules culture) gametophytes, as well as by induced parthenogenesis using γ-irradiated pollen and interspecific hybridization. The genetic, physiological, and physical factors influencing the efficiency of haploid plant production are considered. A special section focuses on the approaches used to double a haploid chromosome set and the direct and indirect methods for determining the ploidy level. The current analyzed data on the successful application of haploid sunflower plants in breeding are summarized.

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.

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.

Effects of Different ß-Lactam Antibiotics on Indirect Tomato (Solanum lycopersicum L.) Shoot Organogenesis and Agrobacterium tumefaciens Growth Inhibition In Vitro.

A ß-lactams that act by inhibiting the bacterial cell wall biosynthesis are one of the most common classes of antibiotics applied to suppress the growth of latent bacterial infection associated with the plant tissue culture, as well as in the Agrobacterium-mediated transformation techniques. Plant sensitivity to antibiotics usually is species-, genotype-, or even tissue-specific and mainly depends on concentrations, growth conditions, and culture system. In the presented article, we estimated a comparative effect of four ß-lactam antibiotics (Claforan®, timentin, amoxicillin, and Amoxiclav®) at different concentrations in an agar-solidified Murashige and Skoog (MS) culture medium supplemented with 5 mg L-1 6-benzylaminopurine (6-BA) and 0.1 mg L-1 indole-3-acetic acid (IAA) on in vitro callus induction and shoot organogenesis from hypocotyl and cotyledon explants of two tomato cultivars (Rekordsmen, Moryana). The role of clavulanic acid in combination with amoxicillin (Amoxiclav®) in the shoot organogenesis frequency and number of shoots per explant has been demonstrated. Additionally, the growth inhibition of Agrobacterium tumefaciens AGL0 strain according to agar disk-diffusion assay was studied. As a result, both stimulatory (timentin, amoxicillin, and Amoxiclav®) and inhibitory (Claforan®) effects of ß-lactam antibiotics on in vitro morphogenetic responses of tomato were noted. It was found that clavulanic acid, which is part of the commercial antibiotic Amoxiclav®, significantly increased the shoot regeneration frequency from cotyledon and hypocotyl explants of Rekordsmen tomato cultivar. Possible reasons for the stimulating effect of clavulanic acid on the induction of shoot organogenesis are discussed. According to agar disk-diffusion assay, the maximum diameter of growth inhibition zones (43.9 mm) was identified using 200 mg L-1 timentin. The in vitro antibacterial activity of tested ß-lactam antibiotics was arranged in the following order: timentin > Claforan® > amoxicillin ≥ Amoxiclav®. Thus, to suppress the growth of internal and latent bacterial infection of tomato plant tissue culture, as well as for transformation of Moryana and Rekordsmen cultivars by A. tumefaciens strain AGL0, we recommend adding of 100-200 mg L-1 timentin or 400-800 mg L-1 Amoxiclav® to the shoot induction medium.

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.

A key enzyme of animal steroidogenesis can function in plants enhancing their immunity and accelerating the processes of growth and development.

BACKGROUND: The initial stage of the biosynthesis of steroid hormones in animals occurs in the mitochondria of steroidogenic tissues, where cytochrome P450SCC (CYP11A1) encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone - the general precursor of all the steroid hormones, starting with progesterone. This stage is missing in plants where mitochondrial cytochromes P450 (the mito CYP clan) have not been found. Generating transgenic plants with a mitochondrial type P450 from animals would offer an interesting option to verify whether plant mitochondria could serve as another site of P450 monooxygenase reaction for the steroid hormones biosynthesis. RESULTS: For a more detailed comparison of steroidogenic systems of Plantae and Animalia, we have created and studied transgenic tobacco and tomato plants efficiently expressing mammalian CYP11A1 cDNA. The detailed phenotypic characterization of plants obtained has shown that through four generations studied, the transgenic tobacco plants have reduced a period of vegetative development (early flowering and maturation of bolls), enlarged biomass and increased productivity (quantity and quality of seeds) as compared to the only empty-vector containing or wild type plants. Moreover, the CYP11A1 transgenic plants show resistance to such fungal pathogen as Botrytis cinerea. Similar valuable phenotypes (the accelerated course of ontogenesis and/or stress resistance) are also visible in two clearly distinct transgenic tomato lines expressing CYP11A1 cDNA: one line (No. 4) has an accelerated rate of vegetative development, while the other (No. 7) has enhanced immunity to abiotic and biotic stresses. The progesterone level in transgenic tobacco and tomato leaves is 3-5 times higher than in the control plants of the wild type. CONCLUSIONS: For the first time, we could show the compatibility in vivo of even the most specific components of the systems of biosynthesis of steroid hormones in Plantae and Animalia. The hypothesis is proposed and substantiated that the formation of the above-noted special phenotypes of transgenic plants expressing mammalian CYP11A1 cDNA is due to the increased biosynthesis of progesterone that can be considered as a very ancient bioregulator of plant cells and the first real hormone common to plants and animals.