Salt-Induced Early Changes in Photosynthesis Activity Caused by Root-to-Shoot Signaling in Potato.

Salinity is one of the most dangerous types of stress in agriculture. Acting on the root, salinity causes changes in physiological processes in the shoot, especially photosynthesis, which is crucial for plant productivity. In our study, we used potato plants, the most important crop, to investigate the role of salt-induced signals in changes in photosynthesis activity. We found a salt-induced polyphasic decrease in photosynthesis activity, and the earliest phase started several minutes after salt addition. We found that salt addition triggered rapid hydraulic and calcium waves from root to shoot, which occurred earlier than the first phase of the photosynthesis response. The inhibition of calcium signals by lanthanum decreased with the formation of rapid changes in photosynthesis. In addition to this, a comparison of the characteristic times of signal propagation and the formation of a response revealed the role of calcium waves in the modulation of rapid changes in photosynthesis. Calcium waves are activated by the ionic component of salinity. The salt-induced decrease in transpiration corresponds in time to the second phase of the photosynthetic response, and it can be the cause of this change. The accumulation of sodium in the leaves occurs a few hours after salt addition, and it can be the cause of the long-term suppression of photosynthesis. Thus, salinity modulates photosynthetic activity in plants in different ways: both through the activation of rapid distant signals and by reducing the water input and sodium accumulation.

The Morphological Parameters and Cytosolic pH of Cells of Root Zones in Tobacco Plants (Nicotiana tabacum L.): Nonlinear Effects of NaCl Concentrations.

Salinity impacts important processes in plants, reducing their yield. The effect of salinity on the cytosolic pH (pHcyt) has been little studied. In this research, we employed transgenic tobacco plants expressing the pH sensor Pt-GFP to investigate the alterations in pHcyt in cells across various root zones. Furthermore, we examined a wide spectrum of NaCl concentrations (ranging from 0 to 150 mM) and assessed morphological parameters and plant development. Our findings revealed a pattern of cytosolic acidification in cells across all root zones at lower NaCl concentrations (50, 100 mM). Interestingly, at 150 mM NaCl, pHcyt levels either increased or returned to normal, indicating a nonlinear effect of salinity on pHcyt. Most studied parameters related to development and morphology exhibited an inhibitory influence in response to NaCl. Notably, a nonlinear relationship was observed in the cell length within the elongation and differentiation zones. While cell elongation occurred at 50 and 100 mM NaCl, it was not evident at 150 mM NaCl. This suggests a complex interplay between stimulating and inhibitory effects of salinity, contributing to the nonlinear relationship observed between pHcyt, cell length, and NaCl concentration.

Comparison of the Efficiency of Hyperspectral and Pulse Amplitude Modulation Imaging Methods in Pre-Symptomatic Virus Detection in Tobacco Plants.

Early detection of pathogens can significantly reduce yield losses and improve the quality of agricultural products. This study compares the efficiency of hyperspectral (HS) imaging and pulse amplitude modulation (PAM) fluorometry to detect pathogens in plants. Reflectance spectra, normalized indices, and fluorescence parameters were studied in healthy and infected areas of leaves. Potato virus X with GFP fluorescent protein was used to assess the spread of infection throughout the plant. The study found that infection increased the reflectance of leaves in certain wavelength ranges. Analysis of the normalized reflectance indices (NRIs) revealed indices that were sensitive and insensitive to infection. NRI700/850 was optimal for virus detection; significant differences were detected on the 4th day after the virus arrived in the leaf. Maximum (Fv/Fm) and effective quantum yields of photosystem II (ΦPSII) and non-photochemical fluorescence quenching (NPQ) were almost unchanged at the early stage of infection. ΦPSII and NPQ in the transition state (a short time after actinic light was switched on) showed high sensitivity to infection. The higher sensitivity of PAM compared to HS imaging may be due to the possibility of assessing the physiological changes earlier than changes in leaf structure.

Several Characteristics of Oidiodendron maius G.L. Barron Important for Heather Plants' Controlled Mycorrhization.

Oidiodendron maius G.L. Barron is a recognized fungal species capable of forming ericoid mycorrhiza with various positive effects on host plants; therefore, newly found and previously uncharacterized O. maius strains may be valuable for heather plants' controlled mycorrhization. Characteristics of the O. maius F3860 strain were studied, i.e., mycelium growth on various nutrient media and the ability to secrete auxins and enzymes. O. maius F3860 grew rapidly on malt extract agar and potato dextrose agar. It was also able to grow on nutrient media suitable for heather plant cultivation. The presence of the flavonoids rutin and quercetin increased the mycelium growth rate compared to the control, starting from the 8th to the 13th days of cultivation. The ability to secrete auxins was confirmed with bioassay and thin-layer chromatography, and their content, as well as phytase activity, was estimated spectrophotometrically. Both in nutrient media with tryptophan and without it, O. maius F3860 secreted about 6 µg IAA/mL growth medium. O. maius F3860 possessed extracellular phytase, protease, and phenol oxidase activities. The investigation indicates O. maius F3860's promise for heather seedling inoculation as an approach to increase their fitness.

Effect of low-dose ionizing radiation on spatiotemporal parameters of functional responses induced by electrical signals in tobacco plants.

Plants growing under an increased radiation background may be exposed to additional stressors. Plant acclimatization is formed with the participation of stress signals that cause systemic responses-a change in the activity of physiological processes. In this work, we studied the mechanisms of the effect of ionizing radiation (IR) on the systemic functional responses induced by electrical signals. Chronic ß-irradiation (31.3 µGy/h) have a positive effect on the morphometric parameters and photosynthetic activity of tobacco plants (Nicotiana tabacum L.) at rest. An additional stressor causes an electrical signal, which, when propagated, causes a temporary change in chlorophyll fluorescence parameters, reflecting a decrease in photosynthesis activity. Irradiation did not significantly affect the electrical signals. At the same time, more pronounced photosynthesis responses are observed in irradiated plants: both the amplitude and the leaf area covered by the reaction increase. The formation of such responses is associated with changes in pH and stomatal conductance, the role of which was analyzed under IR. Using tobacco plants expressing the fluorescent pH-sensitive protein Pt-GFP, it was shown that IR enhances signal-induced cytoplasmic acidification. It was noted that irradiation also disrupts the correlation between the amplitudes of the electrical signal, pH shifts, changes in chlorophyll fluorescence parameters. Also stronger inhibition of stomatal conductance by the signal was shown in irradiated plants. It was concluded that the effect of IR on the systemic response induced by the electrical signal is mainly due to its effect on the stage of signal transformation into the response.

Cell-Type-Specific Length and Cytosolic pH Response of Superficial Cells of Arabidopsis Root to Chronic Salinity.

Soil salinity negatively affects the growth, development and yield of plants. Acidification of the cytosol in cells of glycophytes was reported under salinity, while various types of plant cells can have a specific reaction under the same conditions. Transgenic Arabidopsis plants expressing the pH sensor Pt-GFP in the cytosol were used in this work for determination of morphometric changes and cytosolic pH changes in the superficial cells of Arabidopsis roots under chronic salinity in vitro. We did not find changes in the length of the root cap cells, while there was a decrease in the length of the differentiation zone under 50, 75 mM NaCl and the size of the epidermal cells of the differentiation zone under 75 mM NaCl. The most significant changes of cytosolic pH to chronic salinity was noted in columella (decrease by 1 pH unit at 75 mM NaCl) and epidermal cells of the differentiation zone (decrease by 0.6 and 0.4 pH units at 50 and 75 mM NaCl, respectively). In developed lateral root cap cells, acidification of cytosol by 0.4 units occurred only under 75 mM NaCl in the medium. In poorly differentiated lateral cells of the root cap, there were no changes in pH under chronic salinity.

The Role of Phialocephala fortinii in Improving Plants' Phosphorus Nutrition: New Puzzle Pieces.

Plants' mineral nutrition in acidic soils can be facilitated by phosphate solubilizing fungi inhabiting the root systems of these plants. We attempt to find dark septate endophyte (DSE) isolates in the roots of wild-heather plants, which are capable of improving plants' phosphorus nutrition levels. Bright-field and confocal laser scanning microscopy were used for the visualization of endophytes. A model system of co-cultivation with Vaccinium macrocarpon Ait. was used to study a fungal isolate's ability to supply plants with phosphorus. Fungal phytase activity and phosphorus content in plants were estimated spectrophotometrically. In V. vitis-idaea L. roots, we obtained a Phialocephala fortinii Wang, Wilcox DSE2 isolate with acid phytase activity (maximum 6.91 ± 0.17 U on 21st day of cultivation on potato-dextrose broth medium) and the ability to accumulate polyphosphates in hyphae cells. The ability of the isolate to increase both phosphorus accumulation and biomass in V. macrocarpon is also shown. The data obtained for the same isolate, as puzzle pieces put together, indicate the possible mediation of P. fortinii DSE2 isolate in the process of phosphorus intake from inorganic soil reserves to plants.

Real-Time Fluorescence Visualization and Quantitation of Cell Growth and Death in Response to Treatment in 3D Collagen-Based Tumor Model.

The use of 3D in vitro tumor models has become a common trend in cancer biology studies as well as drug screening and preclinical testing of drug candidates. The transition from 2D to 3D matrix-based cell cultures requires modification of methods for assessing tumor growth. We propose the method for assessing the growth of tumor cells in a collagen hydrogel using macro-scale registration and quantification of the gel epi-fluorescence. The technique does not require gel destruction, can be used for real-time observation of fast (in seconds) cellular responses and demonstrates high agreement with cell counting approaches or measuring total DNA content. The potency of the method was proven in experiments aimed at testing cytotoxic activity of chemotherapeutic drug (cisplatin) and recombinant targeted toxin (DARPin-LoPE) against two different tumor cell lines genetically labelled with fluorescent proteins. Moreover, using fluorescent proteins with sensor properties allows registration of dynamic changes in cells' metabolism, which was shown for the case of sensor of caspase 3 activity.

Far-Red Fluorescent Murine Glioma Model for Accurate Assessment of Brain Tumor Progression.

Glioma is the most common brain tumor, for which no significant improvement in life expectancy and quality of life is yet possible. The creation of stable fluorescent glioma cell lines is a promising tool for in-depth studies of the molecular mechanisms of glioma initialization and pathogenesis, as well as for the development of new anti-cancer strategies. Herein, a new fluorescent glioma GL261-kat cell line stably expressing a far-red fluorescent protein (TurboFP635; Katushka) was generated and characterized, and then validated in a mouse orthotopic glioma model. By using epi-fluorescence imaging, we detect the fluorescent glioma GL261-kat cells in mice starting from day 14 after the inoculation of glioma cells, and the fluorescence signal intensity increases as the glioma progresses. Tumor growth is confirmed by magnetic resonance imaging and histology. A gradual development of neurological deficit and behavioral alterations in mice is observed during glioma progression. In conclusion, our results demonstrate the significance and feasibility of using the novel glioma GL261-kat cell line as a model of glioma biology, which can be used to study the initialization of glioma and monitor its growth by lifetime non-invasive tracking of glioma cells, with the prospect of monitoring the response to anti-cancer therapy.

Salt-Induced Changes in Cytosolic pH and Photosynthesis in Tobacco and Potato Leaves.

Salinity is one of the most common factors limiting the productivity of crops. The damaging effect of salt stress on many vital plant processes is mediated, on the one hand, by the osmotic stress caused by large concentrations of Na+ and Cl- outside the root and, on the other hand, by the toxic effect of these ions loaded in the cell. In our work, the influence of salinity on the changes in photosynthesis, transpiration, water content and cytosolic pH in the leaves of two important crops of the Solanaceae family-tobacco and potato-was investigated. Salinity caused a decrease in photosynthesis activity, which manifested as a decrease in the quantum yield of photosystem II and an increase in non-photochemical quenching. Along with photosynthesis limitation, there was a slight reduction in the relative water content in the leaves and a decrease in transpiration, determined by the crop water stress index. Furthermore, a decrease in cytosolic pH was detected in tobacco and potato plants transformed by the gene of pH-sensitive protein Pt-GFP. The potential mechanisms of the salinity influence on the activity of photosynthesis were analyzed with the comparison of the parameters' dynamics, as well as the salt content in the leaves.

Pre-Symptomatic Detection of Viral Infection in Tobacco Leaves Using PAM Fluorometry.

Chlorophyll fluorescence imaging was used to study potato virus X (PVX) infection of Nicotiana benthamiana. Infection-induced changes in chlorophyll fluorescence parameters (quantum yield of photosystem II photochemistry (ΦPSII) and non-photochemical fluorescence quenching (NPQ)) in the non-inoculated leaf were recorded and compared with the spatial distribution of the virus detected by the fluorescence of GFP associated with the virus. We determined infection-related changes at different points of the light-induced chlorophyll fluorescence kinetics and at different days after inoculation. A slight change in the light-adapted steady-state values of ΦPSII and NPQ was observed in the infected area of the non-inoculated leaf. In contrast to the steady-state parameters, the dynamics of ΦPSII and NPQ caused by the dark-light transition in healthy and infected areas differed significantly starting from the second day after the detection of the virus in a non-inoculated leaf. The coefficients of correlation between chlorophyll fluorescence parameters and virus localization were 0.67 for ΦPSII and 0.76 for NPQ. In general, the results demonstrate the possibility of reliable pre-symptomatic detection of the spread of a viral infection using chlorophyll fluorescence imaging.

The localization of the photosensitizer determines the dynamics of the secondary production of hydrogen peroxide in cell cytoplasm and mitochondria.

Photodynamic therapy (PDT) is based on the production of the cytotoxic reactive oxygen species (ROS) by light irradiation of a photosensitizer dye in the presence of molecular oxygen. Along with photochemical ROS production, it becomes evident that PDT induces massive secondary production of ROS which is registered long after the irradiation is completed. We created cell lines of human epidermoid carcinoma with the cytoplasmic and mitochondrial localization of protein sensor HyPer sensitive to hydrogen peroxide to compare its concentration in two cellular compartments. The lag-period between irradiation and accumulation of hydrogen peroxide in cells was registered; its duration was dose-dependent and increased up to 80 min when lowering the exposition dose from 50 to 15 J/cm2. We have shown that localization of the photosensitizer determines the spatiotemporal pattern of the cell response to PDT: secondary hydrogen peroxide accumulation in cell cytoplasm induced by photodynamic treatment with lysosome-localized phtalocyianine Photosens occurs several minutes prior to that in mitochondria; on the contrary, membranotropic arylcyanoporphyrazine dye leads to massive mitochondrial hydrogen peroxide production followed by its cytoplasmic accumulation. We hypothesize that photosensitizers with various physicochemical properties and intracellular localization can trigger different patterns not only of primary but also secondary ROS production leading to different cell fate outcomes.

Mechanisms of specific systemic response in wheat plants under different locally acting heat stimuli.

Mechanisms of the specific systemic response of plant to different adverse factors are poorly understood. We studied the mechanisms acting in wheat (Triticum aestivum L.) under the action of local burn and gradual heating. Both stimuli induce a variation potential (VP) propagation and a biphasic (fast and long-term phases) photosynthetic response in non-stimulated zones of plant with stimulus-specific parameters of the latter: the fast phase or long-term phase predominance in responses induced by burn or heating, respectively. The burn-induced VP and photosynthetic response attenuate with distance, while the heating-induced VP and photosynthetic response were of more stable amplitude in distant part of the stimulated plant. VP propagation in both cases induced apoplast alkalization with dynamics well corresponding to such of VP and of the fast phase of photosynthetic response. Gradual heating induced a significant rise in jasmonate production along with a decrease in stomatal conductance with characteristic times well corresponding to the long-term phase of the photosynthetic response. We suppose that the VP-induced pH shift is responsible for in the induction of the fast phase, while jasmonate production for the long-term phase of the photosynthetic response. The revealed differences in the systemic response to stressors studied, apparently, reflect two distinct plant adaptation strategies to fast and slow-growing stimuli. The immediate response in the tissue nearest to the damage zone is the most important under a fast-growing stimulus. The fundamentally different situation is under a slowly-growing stimulus which provokes long-term changes in the plant that ensure the preparation of the whole organism for impending environmental changes.

Comparative Analysis of Cell-Cell Contact Abundance in Ovarian Carcinoma Cells Cultured in Two- and Three-Dimensional In Vitro Models.

Tumor resistance to therapy is associated with the 3D organization and peculiarities of the tumor microenvironment, of which intercellular adhesion is a key participant. In this work, the abundance of contact proteins was compared in SKOV-3 and SKOV-3.ip human ovarian adenocarcinoma cell lines, cultivated in monolayers, tumor spheroids and collagen hydrogels. Three-dimensional models were characterized by extremely low expression of basic molecules of adherens junctions E-cadherin and demonstrated a simultaneous decrease in desmosomal protein desmoglein-2, gap junction protein connexin-43 and tight junction proteins occludin and ZO-1. The reduction in the level of contact proteins was most pronounced in collagen hydrogel, accompanied by significantly increased resistance to treatment with doxorubicin and targeted anticancer toxin DARPin-LoPE. Thus, we suggest that 3D models of ovarian cancer, especially matrix-based models, tend to recapitulate tumor microenvironment and treatment responsiveness to a greater extent than monolayer culture, so they can be used as a highly relevant platform for drug efficiency evaluation.

Spatial and Temporal Dynamics of Electrical and Photosynthetic Activity and the Content of Phytohormones Induced by Local Stimulation of Pea Plants.

A local leaf burning causes variation potential (VP) propagation, a decrease in photosynthesis activity, and changes in the content of phytohormones in unstimulated leaves in pea plants. The VP-induced photosynthesis response develops in two phases: fast inactivation and long-term inactivation. Along with a decrease in photosynthetic activity, there is a transpiration suppression in unstimulated pea leaves, which corresponds to the long-term phase of photosynthesis response. Phytohormone level analysis showed an increase in the concentration of jasmonic acid (JA) preceding a transpiration suppression and a long-term phase of the photosynthesis response. Analysis of the spatial and temporal dynamics of electrical signals, phytohormone levels, photosynthesis, and transpiration activity showed the most pronounced changes in the more distant leaf from the area of local stimulation. The established features are related to the architecture of the vascular bundles in the pea stem.

Photobiological properties of phthalocyanine photosensitizers Photosens, Holosens and Phthalosens: A comparative in vitro analysis.

Photobiological properties of phthalocyanine photosensitizers, namely, clinically approved Photosens and new compounds Holosens and Phthalosens were analyzed on transitional cell carcinoma of the urinary bladder (T24) and human hepatic adenocarcinoma (SK-HEP-1). Photosens is a sulfated aluminum phthalocyanine with the number of sulfo groups 3.4, which is characterized by a high degree of hydrophilicity, slow cellular uptake, localization in lysosomes and the lowest photodynamic activity. Holosens is an octacholine zinc phthalocyanine, a cationic compound with significant charge. Holosens more efficiently enters the cells; it is localized in Golgi apparatus in addition to lysosomes and exhibits a significant inhibitory effect on cell viability upon irradiation. The highest photodynamic activity was demostrated by Phthalosens. Phthalosens is a metal-free analog of Photosens with a number of sulfo groups 2.5, which determines its amphiphilicity. Phthalosens is characterized by the highest rate of cellular uptake through the outer cell membrane, localization in cell membrane as well as in lysosomes and Golgi apparatus, and the highest activity upon irradiation among the photosensitizers studied. In general, changes in the physicochemical properties of Holosens and Phthalosens ensured an increase in their efficiency in vitro compared to Photosens. The features of accumulation, intracellular distribution and their interrelation with photodynamic activity, revealed in this work, indicate the prospects of Phthalosens and Holosens for clinical practice.

Monitoring of hydrogen peroxide production under photodynamic treatment using protein sensor HyPer.

An interest to H2O2 accumulation under photodynamic treatment can be explained by its participation in intracellular signal cascades. It is important not only to detect H2O2 generation, but also to trace the dynamics of its intracellular content. In the present study the dynamics of cellular H2O2 content under photodynamic treatment was analyzed using genetically encoded reversible H2O2-sensitive sensor HyPer. Real-time detecting of H2O2 production after photodynamic treatment was performed using the protein sensor and individual features of action of different photosensitizers were revealed. Photodynamic treatment with a number of chlorin and phthalocyanine photosensitizers was found to induce secondary production of H2O2 in the cells. Three types of dynamic responses were registered: monotonous increase of H2O2 level during the entire observation time in the presence of Fotoditazin and Holosens; transient short-term accumulation in the presence of Radachlorin and Phthalosens; and relatively low-level stable increase in the presence of Photosens. The listed photosensitizers differ significantly in intracellular localization and physicochemical properties, which can determine the differences in the response of H2O2 after the photodynamic treatment. In general, it has been shown that the rapid transient H2O2 response is typical for hydrophobic compounds localized in membrane cell structures, whereas in the presence of more hydrophilic dyes a prolonged monotonous H2O2 accumulation occurs.

Effective delivery of porphyrazine photosensitizers to cancer cells by polymer brush nanocontainers.

Efficient drug delivery can be assigned to tasks that attract the most acute attention of researchers in the field of anticancer drug design. We have reported the first case of using amphiphilic polymer brushes as nanocontainers for photosensitizer delivery to cancer cells. Regular graft-copolymers of hydrophobic polyimides with hydrophilic polymethacrylic acid side chains were loaded with photosensitive dye tetra(4-fluorophenyl)tetracyanoporphyrazine (Pz) providing a sufficiently stable homogeneous fraction of fluorescent Pz-loaded nanoparticles with a size of 100-150 nm. Pz-loaded polymer brushes were substantially more efficient for Pz delivery into cells compared with other types of particles examined, Pz-polyethyleneglycol and Pz-methylcellulose. In vivo, an efficient Pz delivery to tumor can also be expected since the Pz-PB particle size is in the optimal range for passive targeting. Pz-PB showed pronounced photodynamic activity, while, that is important, in the absence of irradiation the PB carrier itself was significantly less toxic than the dye itself. Summing up, water-soluble polymer brushes with polyimide backbones and polymethacrylic acid side chains can be regarded as a novel type of nanocontainers providing efficient intracellular drug delivery for photodynamic therapy of cancers.

Passive and active targeting of quantum dots for whole-body fluorescence imaging of breast cancer xenografts.

Far-red and near-infrared fluorescent quantum dots (QDs) have become advancing contrast agents for efficient whole-body tumor imaging. In this study, we investigated the possibility of the vital fluorescence imaging of tumor using two contrast agents on the basis of QDs: bioinert QDs coated with polyethyleneglycol and QDs bound with anti-HER2/neu scFv antibodies. HER2/neu-positive breast cancer tumor xenografts in nude mice were used as a model. It was shown that both bioinert and tumor-targeted QD probes can be successfully applied for visualization of the tumor using in vivo imaging method, but fluorescent signal of QD-4D5scFv in tumors was considerably stronger than that of QD-PEG.