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.

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.

Short Peptides Induce Development of Root Hair Nicotiana tabacum.

Root hairs absorb soil nutrients and water, and anchor the plant in the soil. Treatment of tobacco (Nicotiana tabacum) roots with glycine (Gly) amino acid, and glycilglycine (GlyGly) and glycilaspartic acid (GlyAsp) dipeptides (10-7 M concentration) significantly increased the development of root hairs. In the root, peptide accumulation was tissue-specific, with predominant localization to the root cap, meristem, elongation zone, and absorption zone. Peptides penetrated the epidermal and cortical cell and showed greater localization to the nucleus than to the cytoplasm. Compared with the control, tobacco plants grown in the presence of Gly, GlyGly, and GlyAsp exhibited the activation of WER, CPC, bHLH54, and bHLH66 genes and suppression of GTL1 and GL2 genes during root hair lengthening. Although Gly, GlyGly, and GlyAsp have a similar structure, the mechanism of regulation of root hair growth in each case were different, and these differences are most likely due to the fact that neutral Gly and GlyGly and negatively charged GlyAsp bind to different motives of functionally important proteins. Short peptides site-specifically interact with DNA, and histones. The molecular mechanisms underlying the effect of exogenous peptides on cellular processes remain unclear. Since these compounds acted at low concentrations, gene expression regulation by short peptides is most likely of epigenetic nature.

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.

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.

Chimeric Virus Made from crTMV RNA and the Coat Protein of Potato Leafroll Virus is Targeted to the Nucleolus and Can Infect Nicotiana benthamiana Mechanically.

A genetically engineered chimeric virus crTMV-CP-PLRV composed of the crucifer-infecting tobacco mosaic virus (crTMV) RNA and the potato leafroll virus (PLRV) coat protein (CP) was obtained by agroinfiltration of Nicotiana benthamiana with the binary vector pCambia-crTMV-CPPLRV. The significant levels of the chimeric virus enabled direct visualization of crTMV-CP-PLRV in the cell and to investigate the mechanism of the pathogenesis. Localization of the crTMV-CP-PLRV in plant cells was examined by immunoblot techniques, as well as light, and transmission electron microscopy. The chimera can transfer between vascular and nonvascular tissues. The chimeric virus inoculum is capable to infect N. benthamiana mechanically. The distinguishing feature of the chimeric virus, the RNA virus with the positive genome, was found to localize in the nucleolus. We also investigated the role of the N-terminal sequence of the PLRV P3 coat protein in the cellular localization of the virus. We believe that the gene of the PLRV CP can be substituted with genes from other challenging-to-study plant pathogens to produce other useful recombinant viruses.

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.

Distinct Differentiation Characteristics of Endothelium Determine Its Ability to Form Pseudo-Embryos in Tomato Ovules.

The endothelium is an additional cell layer, differentiating from the inner epidermis of the ovule integument. In tomato (Solanum lycopersicum L.), after fertilization, the endothelium separates from integument and becomes an independent tissue developing next to the growing embryo sac. In the absence of fertilization, the endothelium may proliferate and form pseudo-embryo. However, the course of the reorganization of endothelium into pseudo-embryo in tomato ovules is poorly understood. We aimed to investigate specific features of endothelium differentiation and the role of the endothelium in the development of fertilized and unfertilized tomato ovules. The ovules of tomato plants ("YaLF" line), produced by vegetative growth plants of transgenic tomato line expressing the ac gene, encoding chitin-binding protein from Amaranthus caudatus L., were investigated using light and transmission electron microscopy. We showed that in the fertilized ovule of normally developing fruit and in the unfertilized ovule of parthenocarpic fruit, separation of the endothelium from integument occurs via programmed death of cells of the integumental parenchyma, adjacent to the endothelium. Endothelial cells in normally developing ovules change their structural and functional specialization from meristematic to secretory and back to meristematic, and proliferate until seeds fully mature. The secretory activity of the endothelium is necessary for the lysis of dying cells of the integument and provides the space for the growth of the new sporophyte. However, in ovules of parthenocarpic fruits, pseudo-embryo cells do not change their structural and functional organization and remain meristematic, no zone of lysis is formed, and pseudo-embryo cells undergo programmed cell death. Our data shows the key role of the endothelium as a protective and secretory tissue, needed for the normal development of ovules.

Development of Wolffia arrhiza as a Producer for Recombinant Human Granulocyte Colony-Stimulating Factor.

To date, the expression of recombinant proteins in transgenic plants is becoming a powerful alternative to classical expression methods. Special efforts are directed to the development of contained cultivation systems based on cell culture or rhyzosecretion, which reliably prevents the heterologous DNA releasing into the environment. A promising object for the development of such systems is the tiny aquatic plant of Wolffia arrhiza, which can be used as a dipped culture in bioreactors. Herein we have expressed the human granulocyte colony-stimulating factor (hG-CSF) in nuclear-transformed Wolffia. The nucleotide sequence of hG-CSF was optimized for expression in Wolffia and cloned into the vector pCamGCSF downstream of double CaMV 35S promoter. Wolffia plants were successfully transformed and 34 independent transgenic lines with hG-CSF gene were obtained, PCR and Southern blot analysis confirmed the transgenic origin of these lines. Western blot analysis revealed accumulation of the target protein in 33 transgenic lines. Quantitative ELISA of protein extracts from these lines showed hG-CSF accumulation up to 35.5 mg/kg of Wolffia fresh weight (0.194% of total soluble protein). This relatively high yield holds promise for the development of Wolffia-based expression system in strictly controlled format to produce various recombinant proteins.

Abnormal development of floral meristem triggers defective morphogenesis of generative system in transgenic tomatoes.

Parthenocarpy and fruit malformations are common among independent transgenic tomato lines, expressing genes encoding different pathogenesis-related (PR) protein and antimicrobal peptides. Abnormal phenotype developed independently of the expression and type of target genes, but distinctive features during flower and fruit development were detected in each transgenic line. We analyzed the morphology, anatomy, and cytoembryology of abnormal flowers and fruits from these transgenic tomato lines and compared them with flowers and fruits of wild tomatoes, line YaLF used for transformation, and transgenic plants with normal phenotype. We confirmed that the main cause of abnormal flower and fruit development was the alterations of determinate growth of generative meristem. These alterations triggered different types of anomalous growth, affecting the number of growing ectopic shoots and formation of new flowers. Investigation of the ovule ontogenesis did not show anomalies in embryo sac development, but fertilization did not occur and embryo sac degenerated. Nevertheless, the ovule continued to differentiate due to proliferation of endothelium cells. The latter substituted embryo sac and formed pseudoembryonic tissue. This process imitated embryogenesis and stimulated ovary growth, leading to the development of parthenocarpic fruit. We demonstrated that failed fertilization occurred due to defective male gametophyte formation, which was manifested in blocked division of the nucleus in the microspore and arrest of vegetative and generative cell formation. Maturing pollen grains were overgrown microspores, not competent for fertilization but capable to induce proliferation of endothelium and development of parthenocarpic ovary. Thus, our study provided new data on the structural transformations of reproductive organs during development of parthenocarpic fruits in transgenic tomato.

Visualization of chromosome condensation in plants with large chromosomes.

BACKGROUND: Most data concerning chromosome organization have been acquired from studies of a small number of model organisms, the majority of which are mammals. In plants with large genomes, the chromosomes are significantly larger than the animal chromosomes that have been studied to date, and it is possible that chromosome condensation in such plants was modified during evolution. Here, we analyzed chromosome condensation and decondensation processes in order to find structural mechanisms that allowed for an increase in chromosome size. RESULTS: We found that anaphase and telophase chromosomes of plants with large chromosomes (average 2C DNA content exceeded 0.8 pg per chromosome) contained chromatin-free cavities in their axial regions in contrast to well-characterized animal chromosomes, which have high chromatin density in the axial regions. Similar to animal chromosomes, two intermediates of chromatin folding were visible inside condensing (during prophase) and decondensing (during telophase) chromosomes of Nigella damascena: approximately 150 nm chromonemata and approximately 300 nm fibers. The spatial folding of the latter fibers occurs in a fundamentally different way than in animal chromosomes, which leads to the formation of chromosomes with axial chromatin-free cavities. CONCLUSION: Different compaction topology, but not the number of compaction levels, allowed for the evolution of increased chromosome size in plants.

Protocol for efficient regulation of in vitro morphogenesis in einkorn (Triticum monococcum L.), a recalcitrant diploid wheat species.

Einkorn (Triticum monococcum L.) is A-genome diploid wheat that has a potential to become a useful model for understanding the biology and genomics in Triticeae. Unfortunately, the application of modern technologies such as genetic engineering, RNAi-based gene silencing and genome editing is not available for einkorn as there is no efficient in vitro tissue culture and plant regeneration system. In the present study an efficient and simple protocol for plant regeneration via direct or indirect somatic embryogenesis and organogenesis has been developed. Various auxins used as sole inductors in einkorn displayed low effect for morphogenesis (0-8%) and plant regeneration (1-2 shoots per explant). The addition of Daminozide, the inhibitor of biosynthesis of gibberellins, together with auxin significantly improved the formation of morphogenic structures, especially when Dicamba (51.4%) and Picloram (56.6%) were used for combination; furthermore, the simultaneous addition of cytokinin into induction medium significantly promoted in vitro performance. Among the tested cytokinins, the urea-type substances, such as TDZ and CPPU were more effective than the adenine type ones, BA and Zeatin, for the regulation of morphogenesis; especially, TDZ was more effective than CPPU for shoot formation (11.73 vs. 7.04 per regenerating callus). The highest morphogenic response of 90.2% with the production of more than 10 shoots per initial explant was observed when 3.0 mg/L Dicamba, 50.0 mg/L Daminozide and 0.25 mg/L TDZ were combined together. Along with the identification of appropriate induction medium, the optimal developmental stage for einkorn was found as partially transparent immature embryo in size of around 1.0 mm. Although in the present study the critical balance between plant growth regulators was established for einkorn only, we assume that further the proposed strategy could be successfully applied to other recalcitrant cereal species and genotypes.