Uncoupling proteins and their role in the regulation of brain and heart tolerance to impact of ischemia and reperfusion

Experimental data indicate that moderate uncoupling oxidative phosphorylation induces reduction in production of reactive oxygen species (ROS) and promotes an increase in survival of neurons and cardiomyocytes under hypoxia and re-oxygenation conditions. Uncoupling proteins (UCP) are expressed by cardiomyocytes and neurons. These proteins are involved in the thermogenesis, inhibit ROS generation by mitochondria, reduce deltaphi, elevate respiration rate of these organelles. It was established that UCP contributed to the elevation of cardiomyocyte and neuron tolerance of an impact of hypoxia and re-oxygenation. They also promote cell resistance to oxidative stress. Experimental data indicate the important role of the UCP in the neuroprotective and cardioprotective effects of ischemic preconditioning. At the same time, real contribution of the UCP in preconditioning is still to be verified.     

Low dose (-)deprenyl is cytoprotective: it maintains mitochondrial membrane potential and eliminates oxygen radicals

Hypoxia leads to a collapse in mitochondrial transmembrane potential (Deltapsi(M)), a fall in the ATP/ADP ratio, and finally cell death. Since (-)deprenyl directly modulates Deltapsi(M) and production of reactive oxygen species (ROS) by altering the respiratory function of mitochondria, we were interested in the dose-response relations of these effects. The changes in JC-1 red/green signal ratios {mitochondrial transmembrane potential}, and the changes in the cerium staining (intracellular ROS) in hypoxic and normoxic PC12 cell cultures were measured following 1 h of Argon hypoxia and 24 h of re-oxygenation in the absence and in the presence of various concentrations of (-)deprenyl. Deltapsi(M) shifted to lower values following hypoxia/re-oxygenation and all cells had decreased and uniform Deltapsi(M) levels. The amount of ROS increased. Following 24 h of treatment with various concentrations of (-)deprenyl during the re-oxygenation period, survival increased, the Deltapsi(M) shift caused by oxygen deprivation was reversed and the peroxy radical levels decreased except for at 10(-3) M.     

G proteins as regulators in ethylene-mediated hypoxia signaling

Waterlogging or flooding are frequently or constitutively encountered by many plant species. The resulting reduction in endogenous O₂ concentration poses a severe threat. Numerous adaptations at the anatomical, morphological and metabolic level help plants to either escape low oxygen conditions or to endure them. Formation of aerenchyma or rapid shoot elongation are escape responses, as is the formation of adventitious roots. The metabolic shift from aerobic respiration to anaerobic fermentation contributes to a basal energy supply at low oxygen conditions. Ethylene plays a central role in hypoxic stress signaling, and G proteins have been recognized as crucial signal transducers in various hypoxic signaling pathways. The programmed death of parenchyma cells that results in hypoxia-induced aerenchyma formation is an ethylene response. In maize, aerenchyma are induced in the absence of ethylene when G proteins are constitutively activated. Similarly, ethylene induced death of epidermal cells that cover adventitious roots at the stem node of rice is strictly dependent on heterotrimeric G protein activity. Knock down of the unique Gα gene RGA1 in rice prevents epidermal cell death. Finally, in Arabidopsis, induction of alcohol dehydrogenase with resulting increased plant survival relies on the balanced activities of a small Rop G protein and its deactivating protein RopGAP4. Identifying the general mechanisms of G protein signaling in hypoxia adaptation of plants is one of the tasks ahead.Key words: submergence, hypoxia, ethylene, G protein, reactive oxygen species, H₂O₂     

How plants sense low oxygen

The recent identification of the oxygen-sensing mechanism in plants is a breakthrough in plant physiology. The presence of a conserved N-terminal motif on some ethylene responsive factors (ERFs), targets the protein for post-translational modifications finally leading to degradation under normoxia and thus providing a mechanism for sensing the presence of oxygen. The stabilization of the N-terminus under low oxygen activates these ERFs, which regulate low oxygen core genes that enable plants to tolerate abiotic stress such as flooding. Additional mechanisms that signal low-oxygen probably also exist, and the production of reactive oxygen species (ROS) has been observed under low oxygen, suggesting that ROS might be part of the network involved in plant acclimation. Here, we review the most recent findings related to oxygen sensing.     

Plant DELLAs restrain growth and promote survival of adversity by reducing the levels of reactive oxygen species

Plant growth is adaptively modulated in response to environmental change. The phytohormone gibberellin (GA) promotes growth by stimulating destruction of the nuclear growth-repressing DELLA proteins [1-7], thus providing a mechanism for environmentally responsive growth regulation [8, 9]. Furthermore, DELLAs promote survival of adverse environments [8]. However, the relationship between these survival and growth-regulatory mechanisms was previously unknown. Here, we show that both mechanisms are dependent upon control of the accumulation of reactive oxygen species (ROS). ROS are small molecules generated during development and in response to stress that play diverse roles as eukaryotic intracellular second messengers [10]. We show that Arabidopsis DELLAs cause ROS levels to remain low after either biotic or abiotic stress, thus delaying cell death and promoting tolerance. In essence, stress-induced DELLA accumulation elevates the expression of genes encoding ROS-detoxification enzymes, thus reducing ROS levels. In accord with recent demonstrations that ROS control root cell expansion [11, 12], we also show that DELLAs regulate root-hair growth via a ROS-dependent mechanism. We therefore propose that environmental variability regulates DELLA activity [8] and that DELLAs in turn couple the downstream regulation of plant growth and stress tolerance through modulation of ROS levels.     

Antioxidative Responses and Morpho-anatomical Alterations for Coping with Flood-Induced Hypoxic Stress in Grass Pea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.) in Comparison with Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

Flooding stress constrains crop growth and yield because most agricultural species are flood-sensitive. However, many of the plant species that live in permanently or temporarily flooded habitats have evolved specific traits to cope with these harsh conditions. Grass pea (Lathyrus sativus L.) is a legume that tolerates stresses such as drought, diseases, and pests; however, it is unclear whether grass pea has a tolerance mechanism for flooding stress. To understand if grass pea tolerates hypoxia and how it deals with hypoxic stress, the effects of hypoxia on root tip death, physiological, and morpho-anatomical alterations in grass pea and pea (Pisum sativum), which is sensitive to hypoxia, were compared. The results showed that activities of antioxidant enzymes, namely superoxide dismutase, catalase, ascorbate peroxidase, and glutathione content in grass pea were greater than in pea during hypoxia, which protected the root tip from oxidative damage and reduced ion leakage, which helped maintain membrane integrity. Furthermore, aerenchyma and lateral root development accompanied by ethylene production, moderate ROS accumulation-mediated cell death, and Ca²⁺ spatial-temporal heterogeneity developed well in grass pea compared to pea, which may not only facilitate internal gas diffusion but also promote removal of toxic by-products under hypoxic conditions. These results demonstrate that grass pea is more tolerant to hypoxic stress induced by flooding than garden pea seedlings. This discovery not only provides significant information for understanding the hypoxia-tolerant mechanisms in plants, but also promotes the usability of grass pea in flood-prone areas.     

Formation of reactive species and induction of antioxidant defence systems in polar and temperate marine invertebrates and fish

High oxygen solubility at cold-water temperature is frequently considered to be responsible for an apparently elevated level of antioxidant protection in marine ectotherms from polar environments. However, tissue oxidative stress is in most cases a function of elevated or variable pO2, rather than of an elevated tissue oxygen concentration. This review summarizes current knowledge on pro- and antioxidant processes in marine invertebrates and fish, and relates reactive oxygen species (ROS) formation in polar ectotherms to homeoviscous adaptations of membrane and storage lipids, as well as to tissue hypoxia and re-oxygenation during physiological stress.     

Hypoxic reactive oxygen species regulate the integrated stress response and cell survival

Under hypoxic conditions, cells suppress energy-intensive mRNA translation by modulating the mammalian target of rapamycin (mTOR) and pancreatic eIF2alpha kinase (PERK) pathways. Much is known about hypoxic inhibition of mTOR activity; however, the cellular processes activating PERK remain unclear. Since hypoxia is known to increase intracellular reactive oxygen species (ROS), we hypothesized that hypoxic ROS regulate mTOR and PERK to control mRNA translation and cell survival. Our data indicate that although exogenous ROS inhibit mTOR, eIF2alpha, and eEF2, mTOR and eEF2 were largely refractory to ROS generated under moderate hypoxia (0.5% O(2)). In direct contrast, the PERK/eIF2alpha/ATF4 integrated stress response (ISR) was activated by hypoxic ROS and contributed to global protein synthesis inhibition and adaptive ATF4-mediated gene expression. The ISR as well as exogenous growth factors were critical for cell viability during extended hypoxia, since ISR inhibition decreased the viability of cells deprived of O(2) and growth factors. Collectively, our data support an important role for ROS in hypoxic cell survival. Under conditions of moderate hypoxia, ROS induce the ISR, thereby promoting energy and redox homeostasis and enhancing cellular survival.     

Effect of cadmium and temperature on the lipoxygenase activity in barley root tip

An analysis of different cell fractions isolated from barley roots revealed that lipoxygenase (LOX) activity occurred both extra- and intracellulary. Cadmium (Cd)-induced LOX activity was observed in the fraction containing cell walls, plasma membrane and the cytoplasm. High temperature-induced root growth inhibition and elevated LOX activity did not induce lipid peroxidation. In contrast, Cd inhibited root growth and caused both enhanced lipid peroxidation and elevated LOX activity at each of the temperatures analyzed. Spatial distribution studies revealed that the patterns of apoplastic LOX activity were different from those of cytoplasmic activity. Cd-induced intracellular LOX activity increased equally along the barley root tip, while Cd-induced apoplastic LOX activity was associated mainly with the differentiation zone of the barley root tip. Our results suggest the involvement of Cd-induced LOX activity in the premature differentiation of the barley root tip during Cd stress. We hypothesize that the role of LOX in plant metabolic processes in the root may depend on the level of reactive oxygen species in the roots: at physiological concentrations of ROS, LOX may be involved in the processes of root growth, while at the elevated harmful concentrations of ROS induced by different stress conditions, it may be involved in root growth inhibition through ectopic differentiation.     

Nitric oxide as a critical factor for perception of UV-B irradiation by microtubules in Arabidopsis

Influence of ultraviolet-B (UV-B) as an abiotic stress factor on plant microtubules (MTs) and involvement of nitric oxide (NO) as a secondary messenger mediating plant cell response to environmental stimuli were investigated in this study. Taking into account that endogenous NO content in plant cells has been shown to be increased under a broad range of abiotic stress factors, the effects of UV-B irradiation and also the combined action of UV-B and NO donor sodium nitroprusside (SNP) or NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) on the MTs organization in different root cells of Arabidopsis thaliana were tested. Subsequently, realization of the MT-mediated processes such as root growth and development was studied under these conditions. Arabidopsis thaliana seedlings expressing the chimeric gene gfp-map4 were exposed to the enhanced UV-B with or without SNP or c-PTIO pretreatment. The UV-B irradiation alone led to a dose-dependent root growth inhibition and to morphological alterations of the primary root manifested in their swelling and excessive root hair formation. Moreover, dose-dependent randomization and depolymerization of MTs in both epidermal and cortical cells under the enhanced UV-B were found. However, SNP pretreatment of the UV-B irradiated A. thaliana seedlings recovered the UV-B inhibited root growth as compared to c-PTIO pretreatment. It has been shown that in 24 h after UV-B irradiation the organization of MTs in root epidermal cells of SNP-pretreated A. thaliana seedlings was partially recovered, whereas in c-PTIO-pretreated ones the organization of MTs has not been distinctly improved. Therefore, we suppose that the enhanced NO levels in plant cells can protect MTs organization as well as MT-related processes of root growth and development against disrupting effects of UV-B.     

Physiological integration enhanced the tolerance of Cynodon dactylon to flooding

Many flooding‐tolerant species are clonal plants; however, the effects of physiological integration on plant responses to flooding have received limited attention. We hypothesise that flooding can trigger changes in metabolism of carbohydrates and ROS (reactive oxygen species) in clonal plants, and that physiological integration can ameliorate the adverse effects of stress, subsequently restoring the growth of flooded ramets. In the present study, we conducted a factorial experiment combining flooding to apical ramets and stolon severing (preventing physiological integration) between apical and basal ramets of Cynodon dactylon, which is a stoloniferous perennial grass with considerable flooding tolerance. Flooding‐induced responses including decreased root biomass, accumulation of soluble sugar and starch, as well as increased activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in apical ramets. Physiological integration relieved growth inhibition, carbohydrate accumulation and induction of antioxidant enzyme activity in stressed ramets, as expected, without any observable cost in unstressed ramets. We speculate that relief of flooding stress in clonal plants may rely on oxidising power and electron acceptors transferred between ramets through physiological integration.     

Disrupted actin dynamics trigger an increment in the reactive oxygen species levels in the Arabidopsis root under salt stress

Changes in actin dynamics represent the primary response of the plant cell to extracellular signaling. Recent studies have now revealed that actin remodeling is involved in abiotic stress tolerance in plants. In our current study, the relationship between the changes in actin dynamics and the reactive oxygen species (ROS) level at the initial stages of salt stress was investigated in the elongation zone of the Arabidopsis root tip. We found that a 200 mM NaCl treatment disrupted the dynamics of the actin filaments within 10 min and increased the ROS levels in the elongation zone cells of the Arabidopsis root tip. We further found that the NADPH oxidase activity inhibitor, diphenyleneiodonium, treatment blocked this ROS increase under salt stress conditions. The roles of actin dynamics and the NADPH oxidases in ROS generation were further analyzed using the actin-specific agents, latrunculin B (Lat-B) and jasplakinolide (Jasp), and mutants of Arabidopsis NADPH oxidase AtrbohC. Lat-B and Jasp promote actin depolymerization and polymerization, respectively, and both were found to enhance the ROS levels following NaCl treatment. However, this response was abolished in the atrbohC mutants. Our present results thus demonstrate that actin dynamics are involved in regulating the ROS level in Arabidopsis root under salt stress conditions. KEY MESSAGE: Salt stress disrupts the dynamics of the actin filaments in Arabidopsis in the short term which are involved in regulating the ROS levels that arise under salt stress conditions via the actions of the AtrbohC.     

Roles of phosphatidylinositol 3-kinase in root hair growth

The root hair is a model system for understanding plant cell tip growth. As phosphatidylinositol 3-phosphate [PtdIns(3)P] has been shown in other plant cell types to regulate factors that affect root hair growth, including reactive oxygen species (ROS) levels, cytoskeleton, and endosomal movement, we hypothesized that PtdIns(3)P is also important for root hair elongation. The enzyme that generates PtdIns(3)P, phosphatidylinositol 3-kinase (PI3K), was expressed in root hair cells of transgenic plants containing the PI3K promoter:beta-glucuronidase reporter construct. To obtain genetic evidence for the role of PtdIns(3)P in root hair elongation, we attempted to isolate Arabidopsis (Arabidopsis thaliana) mutant plants that did not express the gene VPS34 encoding the PI3K enzyme. However, the homozygous mutant was lethal due to gametophytic defects, and heterozygous plants were not discernibly different from wild-type plants. Alternatively, we made transgenic plants expressing the PtdIns(3)P-binding FYVE domain in the root hair cell to block signal transduction downstream of PtdIns(3)P. These transgenic plants had shorter root hairs and a reduced hair growth rate compared with wild-type plants. In addition, LY294002, a PI3K-specific inhibitor, inhibited root hair elongation but not initiation. In LY294002-treated root hair cells, endocytosis at the stage of final fusion of the late endosomes to the tonoplast was inhibited and ROS level decreased in a dose-dependent manner. Surprisingly, the LY294002 effects on ROS and root hair elongation were similar in rhd2 mutant plants, suggesting that RHD2 was not the major ROS generator in the PtdIns(3)P-mediated root hair elongation process. Collectively, these results suggest that PtdIns(3)P is required for maintenance of the processes essential for root hair cell elongation.     

Production of reactive oxygen species and antioxidant enzymes of pea and soybean plants under hypoxia and high CO<Subscript>2</Subscript> concentration in medium

Generation of reactive oxygen species (ROS) and activities of antioxidant enzymes (catalase, peroxidase, ascorbate peroxidase) in pea (Pisum sativum L.) and soybean (Glycine max L.) under hypoxia (3–24 h) and high CO₂ concentration in medium were studied. In sensitive to hypoxia pea seedlings, hypoxia enhanced markedly production of superoxide anion-radical, hydroperoxides, and especially hydrogen peroxide. In more tolerant soybean plants, these changes were less pronounced. During first hours of hypoxia, activity of lipoxygenase in plant cells increased. This allows a suggestion that this enzyme is involved in the processes of hydroperoxide accumulation in plant tissues under oxygen deficit. In pea and soybean plants, a correlation between tolerance to hypoxia, the rate of ROS generation, and antioxidant enzyme activities was established. During the first hours of hypoxia, the catalase activity in soybean plants increased stronger than in sensitive to hypoxia pea plants. At longer exposure to hypoxia (24 h), peroxidases started to play the higher role in cell defense against hypoxia, but only in soybean plants. The medium with the higher CO₂ content induced higher changes in the processes of ROS accumulation and activities of lipoxygenase and antioxidant enzymes. This permits us to refer CO₂, accumulated as a product of respiration in the cells, to low-molecular signal molecules switching on plant adaptation to hypoxic stress.