Modifications in Ultrastructural Characteristics and Redox Status of Plants under Environmental Stress: A Review.

The rate of global environmental change is unprecedented, with climate change causing an increase in the oscillation and intensification of various abiotic stress factors that have negative impacts on crop production. This issue has become an alarming global concern, especially for countries already facing the threat of food insecurity. Abiotic stressors, such as drought, salinity, extreme temperatures, and metal (nanoparticle) toxicities, are recognized as major constraints in agriculture, and are closely associated with the crop yield penalty and losses in food supply. In order to combat abiotic stress, it is important to understand how plant organs adapt to changing conditions, as this can help produce more stress-resistant or stress-tolerant plants. The investigation of plant tissue ultrastructure and subcellular components can provide valuable insights into plant responses to abiotic stress-related stimuli. In particular, the columella cells (statocytes) of the root cap exhibit a unique architecture that is easily recognizable under a transmission electron microscope, making them a useful experimental model for ultrastructural observations. In combination with the assessment of plant oxidative/antioxidative status, both approaches can shed more light on the cellular and molecular mechanisms involved in plant adaptation to environmental cues. This review summarizes life-threatening factors of the changing environment that lead to stress-related damage to plants, with an emphasis on their subcellular components. Additionally, selected plant responses to such conditions in the context of their ability to adapt and survive in a challenging environment are also described.

Protective effect of Aronia melanocarpa juice against acrylamide-induced cellular toxicity.

Acrylamide (AA) a widely used industrial chemical is also formed during food processing by the Maillard reaction, which makes its exposure to humans almost unavoidable. In this study, we used Schizosaccharomyces pombe as a model organism to investigate AA toxicity (10 or 20 mM concentration) in eukaryotes. In S. pombe, AA delays cell growth causes oxidative stress by enhancement of ROS production and triggers excitement of the antioxidant defence system resulting in the division arrest. Aronia fruit contains a variety of health-promoting substances with considerable antioxidant potential. Therefore, Aronia juice supplementation was tested to evaluate its protective effect against AA-derived perturbations of the organism. Cell treatment with several Aronia juice concentrations ranging from 0 to 2% revealed the best protective effect of 1 or 2% Aronia juice solutions. Both chosen Aronia juice concentrations alleviated AA toxicity through the improvement of the antioxidant cell capacity and metabolic activity by their strong ROS scavenging property. Efficiency of Aronia juice cell protection is dose dependent as the 2% solution led to significantly higher cellular defence compared with 1%. Due to the high similarity of biological processes of S. pombe with higher eukaryotes, the protective effect of Aronia juice against AA toxicity might also apply to higher organisms.

Curative Potential of Substances with Bioactive Properties to Alleviate Cd Toxicity: A Review.

Rapid urbanization and industrialization have led to alarming cadmium (Cd) pollution. Cd is a toxic heavy metal without any known physiological function in the organism, leading to severe health threat to the population. Cd has a long half-life (10-30 years) and thus it represents serious concern as it to a great extent accumulates in organs or organelles where it often causes irreversible damage. Moreover, Cd contamination might further lead to certain carcinogenic and non-carcinogenic health risks. Therefore, its negative effect on population health has to be minimalized. As Cd is able to enter the body through the air, water, soil, and food chain one possible way to defend and eliminate Cd toxicities is via dietary supplements that aim to eliminate the adverse effects of Cd to the organism. Naturally occurring bioactive compounds in food or medicinal plants with beneficial, mostly antioxidant, anti-inflammatory, anti-aging, or anti-tumorigenesis impact on the organism, have been described to mitigate the negative effect of various contaminants and pollutants, including Cd. This study summarizes the curative effect of recently studied bioactive substances and mineral elements capable to alleviate the negative impact of Cd on various model systems, supposing that not only the Cd-derived health threat can be reduced, but also prevention and control of Cd toxicity and elimination of Cd contamination can be achieved in the future.

Acrylamide-Derived Ionome, Metabolic, and Cell Cycle Alterations Are Alleviated by Ascorbic Acid in the Fission Yeast.

Acrylamide (AA), is a chemical with multiple industrial applications, however, it can be found in foods that are rich in carbohydrates. Due to its genotoxic and cytotoxic effects, AA has been classified as a potential carcinogen. With the use of spectrophotometry, ICP-OES, fluorescence spectroscopy, and microscopy cell growth, metabolic activity, apoptosis, ROS production, MDA formation, CAT and SOD activity, ionome balance, and chromosome segregation were determined in Schizosaccharomyces pombe. AA caused growth and metabolic activity retardation, enhanced ROS and MDA production, and modulated antioxidant enzyme activity. This led to damage to the cell homeostasis due to ionome balance disruption. Moreover, AA-induced oxidative stress caused alterations in the cell cycle regulation resulting in chromosome segregation errors, as 4.07% of cells displayed sister chromatid non-disjunction during mitosis. Ascorbic acid (AsA, Vitamin C), a strong natural antioxidant, was used to alleviate the negative impact of AA. Cell pre-treatment with AsA significantly improved AA impaired growth, and antioxidant capacity, and supported ionome balance maintenance mainly due to the promotion of calcium uptake. Chromosome missegregation was reduced to 1.79% (44% improvement) by AsA pre-incubation. Results of our multiapproach analyses suggest that AA-induced oxidative stress is the major cause of alteration to cell homeostasis and cell cycle regulation.

Nickel induced cell impairments are negatively regulated by the Tor1 kinase in Schizosaccharomyces pombe.

In our study we investigated the effect of different nickel (NiSO4·6H2O) (Ni) concentrations on cell division, cellular morphology and ionome homeostasis of the eukaryotic model organism Schizosaccharomyces pombe. Target of rapamycin (TOR) protein kinase is one of the key regulators of cell growth under different environmental stresses. We analyzed the effect of Ni on cell strains lacking the Tor1 signaling pathway utilizing light-absorbance spectroscopy, visualization, microscopy and inductively coupled plasma optical emission spectroscopy. Interestingly, our findings revealed that Ni mediated cell growth alterations are noticeably lower in Tor1 deficient cells. Greater size of Tor1 depleted cells reached similar quantitative parameters to wild type cells upon incubation with 400 µM Ni. Differences of ion levels among the two tested yeast strains were detected even before Ni addition. Addition of high concentration (1 mM) of the heavy metal, representing acute contamination, caused considerable changes in the ionome of both strains. Strikingly, Tor1 deficient cells displayed largely reduced Ni content after treatment compared to wild type controls (644.1 ± 49 vs. 2096.8 ± 75 µg/g), suggesting its significant role in Ni trafficking. Together our results predict yet undefined role for the Tor1 signaling in metal uptake and/or metabolism.

Ascorbic acid mitigates cadmium-induced stress, and contributes to ionome stabilization in fission yeast.

Cadmium is a highly toxic environmental pollutant which through enhancement of reactive oxygen species (ROS) production triggers oxidative stress to the cell. Cell growth, a fundamental feature of all living organisms is closely connected to the cell shape and homeostasis. As these processes largely depend on cell fitness status and environmental conditions we have analyzed, the impact of different cadmium concentrations and the effect of ascorbic acid (ascorbate, AsA) supplementation on cell growth parameters, cell morphology, and ionome balance maintenance in Schizosaccharomyces pombe. We show that cadmium causes membrane lipid peroxidation resulting in cell shape alterations leading to growth impairment and through mineral elements disequilibrium affects ionome homeostasis in a dose- and time-dependent manner. AsA recognized as one of the most prominent antioxidants, when overdosed, displays considerable pro-oxidant activity, though precise dosing of its supplementation is desired. We present here that AsA under efficacious concentration largely improves cell condition affected by cadmium. Although, we clearly demonstrate the beneficial feature of AsA, further studies are required to fully understand its protective nature on cell homeostasis maintenance under conditions of the broken environment.

Cadmium-Induced Cell Homeostasis Impairment is Suppressed by the Tor1 Deficiency in Fission Yeast.

Cadmium has no known physiological function in the body; however, its adverse effects are associated with cancer and many types of organ system damage. Although much has been shown about Cd toxicity, the underlying mechanisms of its responses to the organism remain unclear. In this study, the role of Tor1, a catalytic subunit of the target of rapamycin complex 2 (TORC2), in Cd-mediated effects on cell proliferation, the antioxidant system, morphology, and ionome balance was investigated in the eukaryotic model organism Schizosaccharomyces pombe. Surprisingly, spectrophotometric and biochemical analyses revealed that the growth rate conditions and antioxidant defense mechanisms are considerably better in cells lacking the Tor1 signaling. The malondialdehyde (MDA) content of Tor1-deficient cells upon Cd treatment represents approximately half of the wild-type content. The microscopic determination of the cell morphological parameters indicates the role for Tor1 in cell shape maintenance. The ion content, determined by inductively coupled plasma optical emission spectroscopy (ICP-OES), showed that the Cd uptake potency was markedly lower in Tor1-depleted compared to wild-type cells. Conclusively, we show that the cadmium-mediated cell impairments in the fission yeast significantly depend on the Tor1 signaling. Additionally, the data presented here suggest the yet-undefined role of Tor1 in the transport of ions.

Impact of cadmium and nickel on ion homeostasis in the yeast Schizosaccharomyces pombe.

Toxicity of heavy metals to living organisms is a worldwide research topic. Although, much has been discovered about cadmium and nickel impact on biological systems, a lot still remains unclear. We used inductively coupled plasma - optical emission spectroscopy to address the question of the effect of two different heavy metals nickel, and cadmium on intracellular ion balance. Increase or decrease of the content of several essential cations including Ca2+, Na+, K+, Mg2+, Cu2+, Fe3+ in the yeast Schizosaccharomyces pombe was determined. Our results revealed that the cell exposure to high nickel and cadmium concentrations led to significant elevation of Ca2+, Na+, Mg2+, Cu2+, Fe3+ levels in the yeast cell, while the content of K+ decreased. Correlation analyses showing in the presence of nickel and cadmium strong positive correlation among each tested element (Ca2+, Na+, Cu2+, Mg2+ and Fe3+) except for K+, demonstrate the significant impact of heavy metal treatment to ion homeostasis of the cell. Our data indicate that acute nickel and cadmium contamination leads to substantial ionome misbalance in yeast.

Sexual Reproduction: Preventing Re-fertilization in Fission Yeast.

During sexual reproduction, two haploid cells fuse to produce a diploid cell called a zygote. A new study describes how fission yeast prevents a zygote from being formed by the fusion of more than two cells.

Regulation of hematogenous tumor metastasis by acid sphingomyelinase.

Metastatic dissemination of cancer cells is the ultimate hallmark of malignancy and accounts for approximately 90% of human cancer deaths. We investigated the role of acid sphingomyelinase (Asm) in the hematogenous metastasis of melanoma cells. Intravenous injection of B16F10 melanoma cells into wild-type mice resulted in multiple lung metastases, while Asm-deficient mice (Smpd1(-/-) mice) were protected from pulmonary tumor spread. Transplanting wild-type platelets into Asm-deficient mice reinstated tumor metastasis. Likewise, Asm-deficient mice were protected from hematogenous MT/ret melanoma metastasis to the spleen in a mouse model of spontaneous tumor metastasis. Human and mouse melanoma cells triggered activation and release of platelet secretory Asm, in turn leading to ceramide formation, clustering, and activation of α5ß1 integrins on melanoma cells finally leading to adhesion of the tumor cells. Clustering of integrins by applying purified Asm or C16 ceramide to B16F10 melanoma cells before intravenous injection restored trapping of tumor cells in the lung in Asm-deficient mice. This effect was revertable by arginine-glycine-aspartic acid peptides, which are known inhibitors of integrins, and by antibodies neutralizing ß1 integrins. These findings indicate that melanoma cells employ platelet-derived Asm for adhesion and metastasis.

Prp4 kinase is required for proper segregation of chromosomes during meiosis in Schizosaccharomyces pombe.

Chromosome segregation during meiosis is a complex process, which leads to production of four haploid gametes from two precursor cells. Reversible phosphorylation of proteins plays a crucial role in this process. The Schizosaccharomyces pombe Prp4 is an essential serine/threonine protein kinase, which belongs to the Clk/Sty family. To study the role of Prp4 in meiosis, we analysed chromosome segregation in a strain carrying conditional analog-sensitive allele of Prp4 protein kinase (prp4-as2). Our data show, that Prp4 protein kinase plays important role in chromosome segregation during meiosis, as revealed by enhanced missegregation of chromosomes in prp4-as2 mutant cells.

New evidence that SAC can tolerate misaligned chromosomes in mouse oocytes.

Comment on: Sebestova J, et al. Cell Cycle 2012; 11:3011-8.

Casein kinase 1 is required for efficient removal of Rec8 during meiosis I.

Segregation of chromosomes during meiosis depends on separase cleavage of Rec8, the meiosis-specific alpha-kleisin subunit of cohesin. We mapped Rec8 phosphorylation sites by mass spectrometry and show that Rec8 phosphorylation is required for proper chromosome disjunction during meiosis. We further show that the fission yeast casein kinase 1 (CK1) delta/epsilon isoforms Hhp1 and Hhp2 are required for full levels of Rec8 phosphorylation and for efficient removal of Rec8 at the onset of anaphase I. Our data are consistent with the model that Hhp1/Hhp2-dependent phosphorylation of Rec8 is required for separase-mediated cleavage of Rec8 during meiosis I.

Inhibition of platelet GPIb alpha and promotion of melanoma metastasis.

Platelet glycoprotein Ibalpha (GPIb alpha) is part of the receptor complex GPIb-V-IX, which has a critical role in hemostasis, especially through interactions with the subendothelial von Willebrand factor. As there is accumulating evidence for a contribution of platelet receptors to hematogenous tumor metastasis, GPIb alpha is an interesting molecule to study in this context. We have investigated the effect of GPIb alpha inhibition by monovalent Fab fragments on experimental pulmonary metastasis in a syngeneic mouse model using C57BL/6 mice and B16F10 melanoma cells. The early fate of green fluorescent protein (GFP)-transfected melanoma cells under GPIb alpha blockade was also assessed, as was the effect of GPIb alpha inhibition on pulmonary metastasis in mice lacking P-selectin. Surprisingly and, to our knowledge previously unreported, GPIb alpha inhibition led to a significant increase in pulmonary metastasis, and assessment of the early fate of circulating GFP-labeled B16F10 showed improved survival and pulmonary arrest of tumor cells shortly after GPIb alpha inhibition, indicating that inhibition of a platelet protein can, in some cases, promote metastasis of a malignant tumor. In contrast, GPIb alpha blockade in P-selectin-deficient mice had no enhancing effect on metastasis, suggesting the involvement of GPIb alpha in the initial, P-selectin-dependent steps of metastasis. These findings suggest that GPIb alpha contributes to the control of tumor metastasis, in addition to its role in hemostasis.

Rac1 is essential for phospholipase C-gamma2 activation in platelets.

Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+) mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCbeta isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCgamma2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP(3)) and intracellular calcium mobilization suggesting inappropriate activation of PLCgamma2 despite normal tyrosine phosphorylation of the enzyme. Rac1 ( -/- ) platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA(2) analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1 ( -/- ) mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCgamma2 activation in platelets and that this is critical for thrombus formation in vivo.

Kindlin-3 is essential for integrin activation and platelet aggregation.

Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel. The shift is mediated through binding of the cytoskeletal protein Talin to the beta subunit cytoplasmic tail. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of beta-integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.

Role of murine integrin alpha2beta1 in thrombus stabilization and embolization: contribution of thromboxane A2.

Platelets stably interact with collagen via glycoprotein (GP)VI and alpha2beta1integrin. With alpha2-null mice, we investigated the role of alpha2beta1 in thrombus formation and stability in vivo and in vitro. Using a FeCl(3)-induced thrombosis model, in arteries from alpha2-null mice smaller thrombi were formed with more embolization compared to vessels from wild-type mice. Aspirin treatment of wild-type mice causes similar effects, while the thromboxane A(2) analogue U46619 was borderline effective in suppressing the embolisation in alpha2-null mice. In vitro, perfusion of alpha2-null blood over collagen resulted in formation of thrombi that were smaller and looser in appearance, regardless of the presence or absence of coagulation. Aspirin treatment or blockage of thromboxane receptors provoked embolus formation in wildtype blood, while U46619 normalized thrombus formation in blood from alpha2-null mice. We conclude that integrin alpha2beta1 plays a role in stabilizing murine thrombi, likely by enhancing GPVI activation and thromboxane A(2) release. The increased embolization in alpha2-null mice may argue against the use of alpha2beta1 integrin inhibitors for antithrombotic therapy.

Diminished thrombus formation and alleviation of myocardial infarction and reperfusion injury through antibody- or small-molecule-mediated inhibition of selectin-dependent platelet functions.

BACKGROUND AND OBJECTIVES: P-selectin ctin has been implicated in important platelet functions. However, neither its role in thrombus formation and cardiovascular disorders nor its suitability as a therapeutic target structure is entirely clear. DESIGN AND METHODS: Platelet aggregation was assessed in complementary in vitro settings by measurements of static aggregation, standardized aggregometry and dynamic flow chamber assays. Degradation of aggregates was also analyzed under flow conditions using video microscopy. In vivo, platelet rolling in cutaneous venules was assessed by intravital microscopy in wild-type mice treated with selectin-blocking compounds as well as in P-selectin-deficient mice. FeCl3-induced arterial thrombosis was studied by intravital microscopy in untreated mice or mice treated with an inhibitor of selectin functions. Finally, inhibition of selectin functions was studied in an ischemia/reperfusion injury model in rats. RESULTS: Antibody- or small-molecule-mediated inhibition of P-selectin functions significantly diminished platelet aggregation (p<0.03) and platelet-neutrophil adhesion in vitro (p<0.01) as well as platelet aggregate sizes under flow (p<0.03). Established aggregates were degraded, either via detachment of single platelets following addition of efomycine M, or via detachment of multicellular clumps when P-selectin-directed Fab-fragments were used. In vivo, selectin inhibition resulted in a greater than 50% reduction of platelet rolling in cutaneous venules (p<0.01), producing rolling fractions similar to those observed in P-selectin-deficient mice (p<0.05). Moreover, inhibition of selectin functions significantly decreased the thrombus size in FeCl3-induced arterial thrombosis in mice (p<0.05). In an ischemia/reperfusion injury model in rats, small-molecule-mediated selectin inhibition significantly reduced myocardial infarct size from 18.9% to 9.42% (p<0.001) and reperfusion injury (p<0.001). INTERPRETATION AND CONCLUSIONS: Inhibition of P-selectin functions reduces platelet aggregation and can alleviate platelet-related disorders in disease-relevant preclinical settings.

Targeting platelets in acute experimental stroke: impact of glycoprotein Ib, VI, and IIb/IIIa blockade on infarct size, functional outcome, and intracranial bleeding.

BACKGROUND: Ischemic stroke is a frequent and serious disease with limited treatment options. Platelets can adhere to hypoxic cerebral endothelial cells by binding of their glycoprotein (GP) Ib receptor to von Willebrand factor. Exposure of subendothelial matrix proteins further facilitates firm attachment of platelets to the vessel wall by binding of collagen to their GPVI receptor. In the present study, we addressed the pathogenic role of GPIb, GPVI, and the aggregation receptor GPIIb/IIIa in experimental stroke in mice. METHODS AND RESULTS: Complete blockade of GPIb alpha was achieved by intravenous injection of 100 microg Fab fragments of the monoclonal antibody p0p/B to mice undergoing 1 hour of transient middle cerebral artery occlusion. At 24 hours after transient middle cerebral artery occlusion, cerebral infarct volumes were assessed by 2,3,5-triphenyltetrazolium chloride staining. In mice treated with anti-GPIb alpha Fab 1 hour before middle cerebral artery occlusion, ischemic lesions were reduced to approximately 40% compared with controls (28.5+/-12.7 versus 73.9+/-17.4 mm3, respectively; P<0.001). Application of anti-GPIb alpha Fab 1 hour after middle cerebral artery occlusion likewise reduced brain infarct volumes (24.5+/-7.7 mm3; P<0.001) and improved the neurological status. Similarly, depletion of GPVI significantly diminished the infarct volume but to a lesser extent (49.4+/-19.1 mm3; P<0.05). Importantly, the disruption of early steps of platelet activation was not accompanied by an increase in bleeding complications as revealed by serial magnetic resonance imaging. In contrast, blockade of the final common pathway of platelet aggregation with anti-GPIIb/IIIa F(ab)2 fragments had no positive effect on stroke size and functional outcome but increased the incidence of intracerebral hemorrhage and mortality after transient middle cerebral artery occlusion in a dose-dependent manner. CONCLUSIONS: Our data indicate that the selective blockade of key signaling pathways of platelet adhesion and aggregation has a different impact on stroke outcome and bleeding complications. Inhibition of early steps of platelet adhesion to the ischemic endothelium and the subendothelial matrix may offer a novel and safe treatment strategy in acute stroke.