Application of confocal microscopy and flow cytometry to identify physiological responses of Prorocentrum micans to the herbicide glyphosate.

The physiological response of the dinoflagellate P. micans to the effect of the herbicide glyphosate at a concentration of 25-200 µg L-1 was evaluated. It has been shown that P. micans is able to grow due to the consumption of dissolved organic phosphorus formed as a result of the mineralization of glyphosate by bacteria. The addition of glyphosate to the medium inhibits the photosynthetic activity of cells; there is a pronounced inhibition of the relative electron transfer rate along the electron transport chain and the maximum quantum efficiency of the use of light energy. Morphological and ultrastructural changes in P. micans cells were evaluated at sublethal (150 µg L-1) and lethal (200 µg L-1) glyphosate concentrations. It has been shown that at a herbicide concentration of 150 µg L-1, the first signs of apoptosis appear in most P. micans cells: a decrease in lateral light scattering, cytoplasmic retraction, partial destruction of cytoplasmic organelles, a change in the morphology of nuclei, mitochondria, a change in the potential of mitochondrial membranes, and a decrease in the autofluorescence of chlorophyll in cells. At a glyphosate concentration of 200 µg L-1, P. micans showed signs of a late stage of apoptosis: violation of the integrity of intracellular organelles and chromatin organization, fragmentation of nuclei, condensation of cytoplasm, disorganization of chloroplasts in the cells, and the release of cell contents beyond the cell membrane. The effectiveness of using flow cytometry and laser scanning confocal microscopy methods for identifying signs and stages of cell apoptosis when exposed to glyphosate is discussed.

Class III Peroxidases in the Peach (Prunus persica): Genome-Wide Identification and Functional Analysis.

Class III peroxidases are plant-specific and play a key role in the response to biotic and abiotic stresses, as well as in plant growth and development. In this study, we investigated 60 POD genes from Prunus persica based on genomic and transcriptomic data available in NCBI and analysed the expression of individual genes with qPCR. Peroxidase genes were clustered into five subgroups using the phylogenetic analysis. Their exon-intron structure and conserved motifs were analysed. Analysis of the transcriptomic data showed that the expression of PpPOD genes varied significantly in different tissues, at different developmental stages and under different stress treatments. All genes were divided into low- and high-expressed genes, and the most highly expressed genes were identified for individual tissues (PpPOD12 and PpPOD42 in flower buds and PpPOD73, PpPOD12, PpPOD42, and PpPOD31 in fruits). The relationship between cold tolerance and the level of peroxidase expression was revealed. These studies were carried out for the first time in the peach and confirmed that chilling tolerance may be related to the specificity of antioxidant complex gene expression.

The comprehensive effect of copper oxide nanoparticles on the physiology of the diatom microalga Thalassiosira weissflogii.

The high rate of production and use of copper oxide nanoparticles (CuO NPs) results in its accumulation in the environment. However, the effect of large quantities of CuO NPs on aquatic ecosystems is not fully known. In aquatic ecosystems, phytoplankton is the primary producer of organic matter and the basis of all the trophic interactions; accordingly, the potential effect of CuO NPs on the microalgae community is of great concern. This study established the main patterns of changes in morphological, structural, functional, fluorescent and cytometric parameters in the marine diatom Thalassiosira weissflogii after adding CuO NPs to the medium at concentrations of 250-2500µgL-1 . As shown, the investigated pollutant has cytotoxic, genotoxic and mechanical effect on the microalga covering almost all the aspects of cell functioning. A two-fold decrease in the culture abundance relative to the control is observed at the toxicant content of 550µgL-1 in the medium. At CuO NPs content above 750µgL-1 , a pronounced inhibition of the alga growth is recorded, as well as a decrease in the efficiency of its photosynthetic apparatus, a disturbance of membrane integrity, an increase in cell volume, a rise in abundance of dead/inactive cells in the culture, enlargement and deformation of nuclei, an increase in reactive oxygen species production, and depolarisation of the mitochondrial membrane. Our results show that high CuO NPs concentrations in water can cause serious disruptions in phytoplankton functioning and in equilibrium of aquatic ecosystems in general.

Aldolase of Mytilus galloprovincialis, Lamarck, 1819: Gene structure, tissue specificity of expression level and activity.

In the present study, the structure of the fructose-1,6-bisphosphataldolase (FBA) gene in Mytilus galloprovincialis (Lamarck, 1819) was analyzed and its tissue specificity of expression level and activity was determined. A 1092 base pairs (bps) complete coding sequence of the FBA gene was assembled from M. galloprovincialis transcriptome. Only one gene encoding FBA (MgFBA) was identified in the M. galloprovincialis genome. The length of MgFBA was 363 amino acids with a molecular mass of 39.7 kDa. According to the amino acid residues, the detected MgFBA gene is a type I aldolase. The FBA gene in M. galloprovincialis had 7 exons; the maximum intron length was about 2.5 kbps. Intraspecific nucleotide diversity (15 mutations) between MgFBAs from the Mediterranean mussels and the Black Sea mussels (present study) was detected. All mutations were synonymous. Tissue specificity in FBA expression level and activity was established. No direct correlation between these functions was found. The highest level of FBA gene expression is found in muscle tissue. According to the phylogenetic analyses, FBA gene in invertebrates could be considered the ancestral gene of muscle type aldolase, which may explain the character of tissue-specific expression.

Responses of Prorocentrum cordatum (Ostenfeld) Dodge, 1975 (Dinoflagellata) to copper nanoparticles and copper ions effect.

In the present study, changes were determined in morphological, structural-functional, and fluorescent parameters of Prorocentrum cordatum with the addition of CuO nanoparticles (NPs) and copper ions (CuSO4). A stimulating effect of low Cu2+ concentrations (30 µg L-1) on algal growth characteristics was observed. Higher Cu2+ concentration of 60-600 µg L-1 and CuO NPs concentration of 100-520 µg L-1 inhibited algal growth. Ionic copper is more toxic to P. cordatum than NPs. After 72 h of algae cultivation in the medium supplemented with CuSO4 and CuO NPs, EC50 values (calculated based on cell abundance) were of 60 and 300 µg L-1 (in terms of copper ions), respectively. Reduction in algal growth rate is due to disruption in cell cycle, changes in nuclear morphology, chromatin dispersion, and DNA damage. The studied pollutants slightly affected the efficiency of P. cordatum photosynthetic apparatus. Addition of the pollutants resulted in an increased production of reactive oxygen species (ROS). At a concentration of Cu2+ of 120 µg L-1 and a concentration of CuO NPs 0-300 µg L-1 of CuO NPs increase in ROS production is short-term with a decrease at later stages of the experiment. This is probably due to the activation of antioxidant mechanisms in cells and an increase in the concentration of carotenoids (peridinin) in cells. The high values of ROS production persisted throughout the experiment at sublethal copper concentrations (400-600 µg L-1 of CuSO4 and 520 µg L-1 of CuO NPs). Sublethal concentrations of pollutants caused restructuring of cell membranes in P. cordatum. Shedding of cell membranes (ecdysis) and formation of immobile stages (temporary or resting cysts) were recorded. The pronounced mechanical impact of NPs on the cell surface was observed such as-deformation and damage of a cell wall, its "wrinkling" and shrinkage, and adsorption of NP aggregates.