Involvement of ROS and calcium ions in developing heat resistance and inducing antioxidant system of wheat seedlings under melatonin's effects.

The stress-protective effect of melatonin (N-acetyl-5-methoxytryptamine) on plant cells is mediated by key signaling mediators, in particular calcium ions and reactive oxygen species (ROS). However, the links between changes in calcium and redox homeostasis and the formation of adaptive responses of cultivated cereals (including wheat) to the action of high temperatures have not yet been studied. In the present study, we investigated the possible involvement of ROS and calcium ions as signaling mediators in developing heat resistance in wheat (Triticum aestivum L.) seedlings and activating their antioxidant system. Treatment of 3-day-old etiolated seedlings with melatonin solutions at concentrations 0.01-10 µM increased their survival after exposure to 45 °C for 10 min. The most significant stress-protective effect was exerted by melatonin treatment at 1 µM concentration. Under the influence of melatonin, a transient enhancement of superoxide anion radical (O2•-) generation and an increase in hydrogen peroxide content were observed in roots, with a maximum at 1 h. Four hours after treatment with melatonin, the activity of catalase and guaiacol peroxidase increased in roots, while the activity of superoxide dismutase did not change significantly. After exposure to 45 °C, the activity of catalase and guaiacol peroxidase was higher in the roots of melatonin-treated wheat seedlings, and the indices of ROS generation, content of the lipid peroxidation product malonic dialdehyde, and cell membrane damage were lower than in control seedlings. Melatonin-induced changes in root ROS generation and antioxidant enzyme activities were eliminated by pretreatment with the hydrogen peroxide scavenger dimethylthiourea (DMTU), NADPH oxidase inhibitor imidazole, and calcium antagonists (the extracellular calcium chelator EGTA and phospholipase C inhibitor neomycin). Treatment with DMTU, imidazole, EGTA, and neomycin also abolished the melatonin-induced increase in survival of wheat seedlings after heat stress. The role of calcium ions and ROS, generated with the participation of NADPH oxidase, as signaling mediators in the melatonin-induced antioxidant system and heat stress resistance of wheat seedlings have been demonstrated.

Autophagy formation, microtubule disorientation, and alteration of ATG8 and tubulin gene expression under simulated microgravity in Arabidopsis thaliana.

Autophagy plays an important role in plant growth and development, pathogen invasion and modulates plant response and adaptation to various abiotic stress stimuli. The biogenesis and trafficking of autophagosomes involve microtubules (MTs) as important actors in the autophagic process. However, initiation of autophagy in plants under microgravity has not been previously studied. Here we demonstrate how simulated microgravity induces autophagy development involving microtubular reorganization during period of autophagosome formation. It was shown that induction of autophagy with maximal autophagosome formation in root cells of Arabidopsis thaliana is observed after 6 days of clinostating, along with MT disorganization, which leads to visible changes in root morphology. Gradual decrease of autophagosome number was indicated on 9th and 12th days of the experiment as well as no significant re-orientation of MTs were identified. Respectively, analysis of α- and ß-tubulins and ATG8 gene expression was carried out. In particular, the most pronounced increase of expression on both 6th and 9th days in response to simulated microgravity was detected for non-paralogous AtATG8b, AtATG8f, AtATG8i, and AtTUA2, AtTUA3 genes, as well as for the pair of ß-tubulin duplicates, namely AtTUB2 and AtTUB3. Overall, the main autophagic response was observed after 6 and 9 days of exposure to simulated microgravity, followed by adaptive response after 12 days. These findings provide a key basis for further studies of cellular mechanisms of autophagy and involvement of cytoskeletal structures in autophagy biogenesis under microgravity, which would enable development of new approaches, aimed on enhancing plant adaptation to microgravity.

Strain-specific features of Pleurotus ostreatus growth in vitro and some of its biological activities.

BACKGROUND: The production of Pleurotus ostreatus mycelium as a promising object for use in food and other industries is hampered by a lack of information about the strain-specificity of this fungus mycelium growth and its acquisition of various biological activities. Therefore, this research aimed to investigate mycelial growth of different P. ostreatus strains on varies solid and liquid media as well as to evaluate strains antagonistic, antibacterial, antiradical scavenging activities, and total phenolic content. RESULTS: Potato Dextrose Agar medium was suitable for all strains except P. ostreatus strain 2460. The best growth rate of P. ostreatus 2462 strain on solid culture media was 15.0 ± 0.8 mm/day, and mycelia best growth on liquid culture media-36.5 ± 0.2 g/l. P. ostreatus strains 551 and 1685 were more susceptible to positive effect of plant growth regulators Ivin, Methyur and Kamethur. Using of nutrient media based on combination of natural waste (amaranth flour cake and wheat germ, wheat bran, broken vermicelli and crumbs) has been increased the yield of P. ostreatus strains mycelium by 2.2-2.9 times compared to the control. All used P. ostreatus strains displayed strong antagonistic activity in co-cultivation with Aspergillus niger, Candida albicans, Issatchenkia orientalis, Fusarium poae, Microdochium nivale in dual-culture assay. P. ostreatus 2462 EtOAc mycelial extract good inhibited growth of Escherichia coli (17.0 ± 0.9 mm) while P. ostreatus 2460 suppressed Staphylococcus aureus growth (21.5 ± 0.5 mm) by agar well diffusion method. The highest radical scavenging effect displayed both mycelial extracts (EtOH and EtOAc) of P. ostreatus 1685 (61 and 56%) by DPPH assay as well as high phenolic content (7.17 and 6.73 mg GAE/g) by the Folin-Ciocalteu's method. The maximal total phenol content (7.52 mg GAE/g) demonstrated of P. ostreatus 2461 EtOH extract. CONCLUSIONS: It is found that the growth, antibacterial, antiradical scavenging activity as well as total phenolic content were dependent on studied P. ostreatus strains in contrast to antagonistic activity. The proposed culture mediums of natural waste could be an alternative to commercial mediums for the production mycelial biomass of P. ostreatus strains.

The effect of human PBMCs immobilization on their Аß42 aggregates-dependent proinflammatory state on a cellular model of Alzheimer's disease.

The leading pathological mechanisms of Alzheimer's disease are amyloidosis and inflammation. The presented work was aimed to study the effect of human peripheral blood mononuclear cells (hPBMcs) cells-matrix adhesion on their pro-inflammatory state in vitro. Although direct interaction of Аß42 to PBMC is not a cellular model of Alzheimer's disease, PBMCs may serve as test cells to detect Аß42-dependent molecular effects in monitoring disease progression. Peripheral blood mononuclear cells (PBMCs) are used to assess changes in cytokines released in response to diseases or Alzheimer's disease-specific cytotoxic molecules such as Aß42. The effect of recombinant amyloid ß-peptide rАß42 on the concentration of endogenous amyloid ß-peptide Aß40 and pro-inflammatory cytokines TNFα and IL-1ß in human peripheral blood mononuclear cells that were cultured in suspension and immobilized in alginate microcarriers for 24 h were investigated. The localization and accumulation of Aß40 and rAß42 peptides in cells, as well as quantitative determination of the concentration of Aß40 peptide, TNFα and IL-1ß cytokines, was performed by intravital fluorescence imaging. The results were qualitatively similar for both cell models. It was determined that the content of TNFα and Aß40 in the absence of rAß42 in the incubation medium did not change for 24 h after incubation, and the content of IL-1ß was lower compared to the cells that were not incubated. Incubation of cells in vitro with exogenous rAß42 led to an increase in the intracellular content of TNFα and Aß40, and no accumulation of IL-1ß in cells was observed. The accumulation of Aß40 in the cytoplasm was accompanied by the aggregation of rAß42 on the outer surface of the cell plasma membrane. It was shown that the basic levels of indicators and the intensity of the response of immobilized cells to an exogenous stimulus were significantly greater than those of cells in suspension. To explore whether non-neuronal cells effects in alginate microcarriers were cell-matrix adhesion mediated, we tested the effect of blocking ß1 integrins on proamyloidogenic and proinflammation cellular state. Immobilization within alginate hydrogels after incubation with the ß1 integrins blocking antibodies showed a remarkable inhibition of TNFα and Aß40 accumulation in rAß42-treated cells. It can be concluded that activation of signal transduction and synthesizing activity of a portion of mononuclear cells of human peripheral blood is possible (can significantly increase) in the presence of cell-matrix adhesion.

Chromosome-level assembly and analysis of Camelina neglecta: a novel diploid model for Camelina biotechnology research.

Camelina neglecta is a new diploid Brassicaceae species, which has great research value because of its close relationship with the hexaploid oilseed crop Camelina sativa. Here, we report a chromosome-level assembly of C. neglecta with a total length of 210 Mb. By adopting PacBio sequencing and Hi-C technology, the C. neglecta genome was assembled into 6 chromosomes with scaffold N50 of 29.62 Mb. C. neglecta has undergone the whole-genome triplication (γ) shared among eudicots and two whole-genome duplications (α and ß) shared by crucifers, but it has not undergone a specific whole-genome duplication event. By synteny analysis between C. neglecta and C. sativa, we successfully used the method of calculating Ks to distinguish the three subgenomes of C. sativa and determined that C. neglecta was closest to the first subgenome (SG1) of C. sativa. Further, transcriptomic analysis revealed the key genes associated with seed oil biosynthesis and its transcriptional regulation, including SAD, FAD2, FAD3, FAE1, ABI3, WRI1 and FUS3 displaying high expression levels in C. neglecta seeds. The high representability of C. neglecta as a model species for Camelina-based biotechnology research has been demonstrated for the first time. In particular, floral Agrobacterium tumefaciens infiltration-based transformation of C. neglecta, leading to overexpression of CvLPAT2, CpDGAT1 and CvFatB1 transgenes, was demonstrated for medium-chain fatty acid accumulation in C. neglecta seed oil. This study provides an important genomic resource and establishes C. neglecta as a new model for oilseed biotechnology research.

Ivermectin affects Arabidopsis thaliana microtubules through predicted binding site of ß-tubulin.

The ivermectin is a potent nematocide and insecticide, which has low toxicity for humans and domestic animals, but due to low biotransformation, it can be dangerous for non-target organisms. The recent determination of ivermectin absorption and accumulation in tissues of higher plants and multiple shreds of evidence of its negative impact on plant physiology provide a basis for the search for ivermectin's molecular targets and mechanisms of action in plant cells. In this research, for the first time, the ivermectin effect on microtubules of Arabidopsis thaliana cells was studied. It was revealed that ivermectin (250 µg mL-1) disrupts the microtubule network, induces the loss of microtubule orientation, leads to microtubule curvature and shrinkage, and their longitudinal and cross-linked bundling in various cells of A. thaliana primary roots. Further, the previously proposed binding of ivermectin to the ß1-tubulin taxane site was developed and confirmed using molecular dynamics simulations of ivermectin complexes with Haemonchus contortus and A. thaliana ß1-tubulins. It was predicted that similar to other microtubule stabilizing agents ivermectin binding causes M-loop stabilization in both H. contortus and A. thaliana ß-tubulin, which leads to the enhancement of lateral contacts between subunits of adjacent protofilaments preventing microtubule depolymerization.

Overcoming genetic paucity of Camelina sativa: possibilities for interspecific hybridization conditioned by the genus evolution pathway.

Camelina or false flax (Camelina sativa) is an emerging oilseed crop and a feedstock for biofuel production. This species is believed to originate from Western Asian and Eastern European regions, where the center of diversity of the Camelina genus is located. Cultivated Camelina species arose via a series of polyploidization events, serving as bottlenecks narrowing genetic diversity of the species. The genetic paucity of C. sativa is foreseen as the most crucial limitation for successful breeding and improvement of this crop. A potential solution to this challenge could be gene introgression from Camelina wild species or from resynthesized allohexaploid C. sativa. However, both approaches would require a complete comprehension of the evolutionary trajectories that led to the C. sativa origin. Although there are some studies discussing the origin and evolution of Camelina hexaploid species, final conclusions have not been made yet. Here, we propose the most complete integrated evolutionary model for the Camelina genus based on the most recently described findings, which enables efficient improvement of C. sativa via the interspecific hybridization with its wild relatives. We also discuss issues of interspecific and intergeneric hybridization, aimed on improving C. sativa and overcoming the genetic paucity of this crop. The proposed comprehensive evolutionary model of Camelina species indicates that a newly described species Camelina neglecta has a key role in origin of tetra- and hexaploids, all of which have two C. neglecta-based subgenomes. Understanding of species evolution within the Camelina genus provides insights into further research on C. sativa improvements via gene introgression from wild species, and a potential resynthesis of this emerging oilseed crop.

In silico induced effect of N-ε-lysine acetylation on microtubule stability and subsequent interaction of microtubule-associated proteins.

Plant systems have been considered valuable models for addressing fundamental questions of microtubule (MT) organization due to their considerable practical utility. Protein acetylation is a very common protein modification, and therate of acetylation can be modulated in cells in different biological states, and these changes can be detected at a molecular level. Here, we focused on K40, K112, and K394 residues as putative acetylation sites, which were shown to exist in both plants and mammals. Such residual effect of acetylation causes critical but unclear effect on MT stability. In turn, it was shown that acetylation indirectly affects the probability of interaction with different MAPs (Microtubule-associated proteins). In a multiscale study using an all-atom force field to reproduce several lattice-forming elements found on the surface the microtubule, we assembled a fragment of a plant microtubule composed of nine tubulins and used it as a model object along with the existing human complex. Triplets of tubulins assembled in a lattice cell were then simulated for both human and plant protein complexes, using a coarse-grained force field. We then analyzed the trajectories and identified some critical deformations of the MAP interaction surface. The initial coordinates were used to investigate the structural scenario in which autophagy-related protein 8 (ATG8) was able to interact with the MT fragment.

The role of nitric oxide and hydrogen sulfide in regulation of redox homeostasis at extreme temperatures in plants.

Nitric oxide and hydrogen sulfide, as important signaling molecules (gasotransmitters), are involved in many functions of plant organism, including adaptation to stress factors of various natures. As redox-active molecules, NO and H2S are involved in redox regulation of functional activity of many proteins. They are also involved in maintaining cell redox homeostasis due to their ability to interact directly and indirectly (functionally) with ROS, thiols, and other molecules. The review considers the involvement of nitric oxide and hydrogen sulfide in plant responses to low and high temperatures. Particular attention is paid to the role of gasotransmitters interaction with other signaling mediators (in particular, with Ca2+ ions and ROS) in the formation of adaptive responses to extreme temperatures. Pathways of stress-induced enhancement of NO and H2S synthesis in plants are considered. Mechanisms of the NO and H2S effect on the activity of some proteins of the signaling system, as well as on the state of antioxidant and osmoprotective systems during adaptation to stress temperatures, were analyzed. Possibilities of practical use of nitric oxide and hydrogen sulfide donors as inductors of plant adaptive responses are discussed.

Wheat Genes Associated with Different Types of Resistance against Stem Rust (Puccinia graminis Pers.).

Stem rust is one wheat's most dangerous fungal diseases. Yield losses caused by stem rust have been significant enough to cause famine in the past. Some races of stem rust are considered to be a threat to food security even nowadays. Resistance genes are considered to be the most rational environment-friendly and widely used way to control the spread of stem rust and prevent yield losses. More than 60 genes conferring resistance against stem rust have been discovered so far (so-called Sr genes). The majority of the Sr genes discovered have lost their effectiveness due to the emergence of new races of stem rust. There are some known resistance genes that have been used for over 50 years and are still effective against most known races of stem rust. The goal of this article is to outline the different types of resistance against stem rust as well as the effective and noneffective genes, conferring each type of resistance with a brief overview of their origin and usage.

Genome-wide identification and analysis of the cytokinin oxidase/dehydrogenase (ckx) gene family in finger millet (Eleusine coracana).

Cytokinin dehydrogenase/oxidase (CKX) enzymes play a key role in regulating cytokinin (CK) levels in plants by degrading the excess of this phytohormone. CKX genes have proven an attractive target for genetic engineering, as their silencing boosts cytokinin accumulation in various tissues, thereby contributing to a rapid increase in biomass and overall plant productivity. We previously reported a similar effect in finger millet (Eleusine coracana) somaclonal lines, caused by downregulation of EcCKX1 and EcCKX2. However, the CKX gene family has numerous representatives, especially in allopolyploid crop species, such as E. coracana. To date, the entire CKX gene family of E. coracana and its related species has not been characterized. We offer here, for the first time, a comprehensive genome-wide identification and analysis of a panel of CKX genes in finger millet. The functional genes identified in the E. coracana genome are compared with the previously-identified genes, EcCKX1 and EcCKX2. Exon-intron structural analysis and motif analysis of FAD- and CK-binding domains are performed. The phylogeny of the EcCKX genes suggests that CKX genes are divided into several distinct groups, corresponding to certain isotypes. Finally, the phenotypic effect of EcCKX1 and EcCKX2 in partially silencing the SE7 somaclonal line is investigated, showing that lines deficient in CKX-expression demonstrate increased grain yield and greater bushiness, enhanced biomass accumulation, and a shorter vegetation cycle.

Quantum Dot-Antibody Conjugates for Immunofluorescence Studies of Biomolecules and Subcellular Structures.

Quantum dots, or nanoscale semiconductors, are one of the most important materials for various research and development purposes. Due to their advantageous photoluminescence and electronic properties, namely, their unique photostability, high brightness, narrow emission spectra from visible to near-infrared wavelengths, convey them significant advantages over widely used fluorochromes, including organic dyes, fluorescent probes. Quantum dots are a unique instrument for a wide range of immunoassays with antibodies. The paper provides an overview of the developed and already applied methods of quantum dot surface modification, quantum dots conjugation to different antibodies (non-covalent, direct covalent linkage or with the use of special adapter molecules), as well as practical examples of recent quantum dot-antibody applications in the immunofluorescence microscopy for cell and cell structure imaging, fluorescent assays for biomolecules detection and in diagnostics of various diseases. The review presents advantages of quantum dot-antibody conjugation technology over the existing methods of immunofluorescence studies and a forward look into its potential prospects in biological and biomedical research.

Synthesis, Properties and Bioimaging Applications of Silver-Based Quantum Dots.

Ag-based quantum dots (QDs) are semiconductor nanomaterials with exclusive electrooptical properties ideally adaptable for various biotechnological, chemical, and medical applications. Silver-based semiconductor nanocrystals have developed rapidly over the past decades. They have become a promising luminescent functional material for in vivo and in vitro fluorescent studies due to their ability to emit at the near-infrared (NIR) wavelength. In this review, we discuss the basic features of Ag-based QDs, the current status of classic (chemical) and novel methods ("green" synthesis) used to produce these QDs. Additionally, the advantages of using such organisms as bacteria, actinomycetes, fungi, algae, and plants for silver-based QDs biosynthesis have been discussed. The application of silver-based QDs as fluorophores for bioimaging application due to their fluorescence intensity, high quantum yield, fluorescent stability, and resistance to photobleaching has also been reviewed.

Brassinosteroids application induces phosphatidic acid production and modify antioxidant enzymes activity in tobacco in calcium-dependent manner.

Brassinosteroids (BRs) are steroid hormones regulating various aspects of plant metabolism, including growth, development and stress responses. However, little is known about the mechanism of their impact on antioxidant systems and phospholipid turnover. Using tobacco plants overexpressing H+/Ca2+vacuolar Arabidopsis antiporter CAX1, we showed the role of Ca2+ ion balance in the reactive oxygen species production and rapid phosphatidic acid accumulation induced by exogenous BR. Combination of our experimental results with public transcriptomic and proteomic data revealed a particular role of Ca2+-dependent phospholipid metabolizing enzymes in BR signaling. Here we provide novel insights into the role of calcium balance and lipid-derived second messengers in plant responses to exogenous BRs and propose a complex model integrating BR-mediated metabolic changes with phospholipid turnover.

Cadmium, nickel, copper, and zinc influence on microfilament organization in Arabidopsis root cells.

The plant cytoskeleton orchestrates such fundamental processes in cells as division, growth and development, polymer cross-linking, membrane anchorage, etc. Here, we describe the influence of Cd2+ , Ni2+ , Zn2+ , and Cu2+ on root development and vital organization of actin filaments into different cells of Arabidopsis thaliana line expressing GFP-FABD2. CdSO4 , NiSO4 , CuSO4 , and ZnSO4 were used in concentrations of 5-20 µM in this study. It was found that Cd, Ni, and Cu cause dose-dependent primary root growth inhibition and alteration of the root morphology, whereas Zn slightly stimulates root growth and does not affect the morphology of Arabidopsis roots. This growth inhibition/stimulation correlated with the various sensitivities of microfilaments to Cd, Ni, Cu, and Zn action. It was established that Cd, Ni, and Cu affected predominantly the actin filaments of meristematic cells. Cells of transition and elongation zones demonstrated strong actin filament sensitivity to Cd and Cu. Microfilaments of elongating root cells were more sensitive to Ni and Cu. Although Cd, Ni, and Cu stimulated root hair growth after long-term treatment, actin filaments were destroyed after 1 h exposure with these metals. Zn did not disrupt native actin filament organization in root cells. Thus, our investigation shows that microfilaments act as sensitive cellular targets for Cd, Ni, and Cu. More data on effects on native actin filaments organization would contribute to a better understanding of plant tolerance mechanisms to the action of these metals.

Proposal of Potent Inhibitors for a Bacterial Cell Division Protein FtsZ: Molecular Simulations Based on Molecular Docking and ab Initio Molecular Orbital Calculations.

The inhibition of a bacterial cell division protein, filamentous temperature-sensitive Z (FtsZ), prevents the reproduction of Mycobacteria. To propose potent inhibitors of FtsZ, the binding properties of FtsZ with various derivatives of Zantrin ZZ3 were investigated at an electronic level, using molecular simulations. We here employed protein-ligand docking, classical molecular mechanics (MM) optimizations, and ab initio fragment molecular orbital (FMO) calculations. Based on the specific interactions between FtsZ and the derivatives, as determined by FMO calculations, we proposed novel ligands, which can strongly bind to FtsZ and inhibit its aggregations. The introduction of a hydroxyl group into ZZ3 was found to enhance its binding affinity to FtsZ.

Specific interactions between tau protein and curcumin derivatives: Molecular docking and ab initio molecular orbital simulations.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the world, and there is currently no potent medicine for the treatment of ADs. Curcumin, a primary chemical contained in the ancient Indian herb known as turmeric, has been extensively studied and shown to be effective in inhibiting the aggregations of amyloid-ß and tau proteins, both of which are observed in the brains of AD patients. In the present study, we focused on the tau protein and investigated its specific interactions with curcumin derivatives, using molecular simulations based on molecular docking, molecular mechanics and ab initio fragment molecular orbital calculations. Based on the results, we attempted to propose novel potent inhibitors against the tau protein aggregation. Our molecular simulations provide useful information for developing novel medicines for the treatment of ADs.

Flax tubulin and CesA superfamilies represent attractive and challenging targets for a variety of genome- and base-editing applications.

Flax is both a valuable resource and an interesting model crop. Despite a long history of flax genetic transformation only one transgenic linseed cultivar has been so far registered in Canada. Implementation and use of the genome-editing technologies that allow site-directed modification of endogenous genes without the introduction of foreign genes might improve this situation. Besides its potential for boosting crop yields, genome editing is now one of the best tools for carrying out reverse genetics and it is emerging as an especially versatile tool for studying basic biology. A complex interplay between the flax tubulin family (6 α-, 14 ß-, and 2 γ-tubulin genes), the building block of microtubules, and the CesA (15-16 genes), the subunit of the multimeric cellulose-synthesizing complex devoted to the oriented deposition of the cellulose microfibrils is fundamental for the biosynthesis of the cell wall. The role of the different members of each family in providing specificities to the assembled complexes in terms of structure, dynamics, activity, and interaction remains substantially obscure. Genome-editing strategies, recently shown to be successful in flax, can therefore be useful to unravel the issue of functional redundancy and provide evidence for specific interactions between different members of the tubulin and CesA gene families, in relation to different phase and mode of cell wall biosynthesis.

A journey through a plant cytoskeleton: Hot spots in signaling and functioning.

This survey paper contains a brief analysis of publications included in the special issue of the scientific journal Cell Biology International titled "Plant Cytoskeleton Structure, Dynamics and Functions". The manuscripts in this special issue reflect some new aspects of plant cytoskeleton organization, signaling and functioning, and results from different Ukrainian research groups, and focuses on bringing together scientists working across different instrumental scales.