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Currently, due to the increasing impact of anthropogenic factors and changes in solar activity, the temperature on Earth is rising, posing a threat to biodiversity. Lichens are among the most sensitive organisms to climate change. Elevated ambient temperatures can have a significant impact on lichens, resulting in more frequent and intense drying events that can impede metabolic activity. It has been suggested that the possession of a diverse sterol composition may contribute to the tolerance of lichens to adverse temperatures and other biotic and abiotic stresses. The major sterol found in lichens is ergosterol (ERG); however, the regulation of the ERG biosynthetic pathway, specifically the step of epoxidation of squalene to 2,3-oxidosqualene catalyzed by squalene epoxidase during stress, has not been extensively studied. In this study, we used lichen Lobaria pulmonaria as a model species that is well known to be sensitive to air pollution and habitat loss. Using in silico analysis, we identified cDNAs encoding squalene epoxidase from L. pulmonaria , designating them as LpSQE1 for the mycobiont and SrSQE1 for the photobiont Symbiochloris reticulata . Our results showed that compared with a control kept at room temperature (+20 °C), mild temperatures (+4 °C and +30 °C) did not affect the physiology of L. pulmonaria , assessed by changes in membrane integrity, respiration rates, and PSII activity. An extreme negative temperature (-20 °C) noticeably inhibited respiration but did not affect membrane stability. In contrast, treating lichen with a high positive temperature (+40 °C) significantly reduced all physiological parameters. Quantitative PCR analysis revealed that exposing thalli to -20 °C, +4 °C, +30 °C, and +40 °C stimulated the expression levels of LpSQE1 and SrSQE1 and led to a significant upregulation of Hsps . These data provide new information regarding the roles of sterols and Hsps in the response of lichens to climate change.

Melanin is a dark pigment from the group of phenolic or indole polymers with inherent biocompatibility and antioxidant capacity. In extremophilic lichen Lobaria pulmonaria, melanin is responsible for protective properties against hostile environments. Herein, the ability of melanin extracted from L. pulmonaria to counteract oxidative stress and related damages was studied in the mouse diaphragm, the main respiratory muscle. Initial in vitro experiments demonstrated ultraviolet (UV)-absorbing, antioxidant and metal chelating activities of melanin. This melanin can form nanoparticles and stabile colloidal system at concentration of 5 μg/ml. Pretreatment of the muscle with melanin (5 μg/ml) markedly reduced UV-induced increase in intracellular and extracellular reactive oxygen species (ROS) as well as antimycin A-mediated enhancement in mitochondrial ROS production accompanied by lipid peroxidation and membrane asymmetry loss. In addition, melanin attenuated suppression of neuromuscular transmission and alterations of contractile responses provoked by hydrogen peroxide. Thus, this study shed the light on the perspectives of the application of a lichen melanin as a protective component for treatment of skeletal muscle disorders, which are accompanied with an increased ROS production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lichens are symbiotic organisms that effectively survive in harsh environments, including arid regions. Maintaining viability with an almost complete loss of water and the rapid restoration of metabolism during rehydration distinguishes lichens from most eukaryotic organisms. The lichen Xanthoria parietina is known to have high stress tolerance, possessing diverse defense mechanisms, including the presence of the bright-orange pigment parietin. While several studies have demonstrated the photoprotective and antioxidant properties of this anthraquinone, the role of parietin in the tolerance of lichens to desiccation is not clear yet. Thalli, which are exposed to solar radiation and become bright orange, may require enhanced desiccation tolerance. Here, we showed differences in the anatomy of naturally pale and bright-orange thalli of X. parietina and visualized parietin crystals on the surface of the upper cortex. Parietin was extracted from bright-orange thalli by acetone rinsing and quantified using HPLC. Although acetone rinsing did not affect PSII activity, thalli without parietin had higher levels of lipid peroxidation and a lower membrane stability index in response to desiccation. Furthermore, highly pigmented thalli possess thicker cell walls and, according to thermogravimetric analysis, higher water-holding capacities than pale thalli. Thus, parietin may play a role in desiccation tolerance by stabilizing mycobiont membranes, providing an antioxidative defense, and changing the morphology of the upper cortex of X. parietina .

Lichens are unique extremophilic organisms due to their phenomenal resistance to adverse environmental factors, including ultraviolet (UV) irradiation. Melanization plays a special role in the protection of lichens from UV-B stress. In the present study, we analyzed the binding of melanins with the components of cell walls of the mycobiont of the upper cortex in the melanized lichen thalli Lobaria pulmonaria . Using scanning electron and atomic force microscopy, the morphological and nanomechanical characteristics of the melanized layer of mycobiont cells were visualized. Melanization of lichen thalli led to the smoothing of the surface relief and thickening of mycobiont cell walls, as well as the reduction in adhesion properties of the lichen thallus. Treatment of thalli with hydrolytic enzymes, especially chitinase and lichenase, enhanced the yield of melanin from melanized thalli and promoted the release of carbohydrates, while treatment with pectinase increased the release of carbohydrates and phenols. Our results suggest that melanin can firmly bind with hyphal cell wall carbohydrates, particularly chitin and 1,4-β-glucans, strengthening the melanized upper cortex of lichen thalli, and thereby it can contribute to lichen survival under UV stress.

Plant dehydration-responsive element binding (DREB) transcription factors (TFs) play important roles during stress tolerance by regulating the expression of numerous genes involved in stresses. DREB TFs have been extensively studied in a variety of angiosperms and bryophytes. To date, no information on the identification and characterization of DREB TFs in Dicranum scoparium has been reported. In this study, a new DBF1 gene from D. scoparium was identified by cloning and sequencing. Analysis of the conserved domain and physicochemical properties revealed that DsDBF1 protein has a classic AP2 domain encoding a 238 amino acid polypeptide with a molecular mass of 26 kDa and a pI of 5.98. Subcellular prediction suggested that DsDBF1 is a nuclear and cytoplasmic protein. Phylogenetic analysis showed that DsDBF1 belongs to group A-5 DREBs. Expression analysis by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) revealed that DsDBF1 was significantly upregulated in response to abiotic stresses such as desiccation/rehydration, exposure to paraquat, CdCl 2 , high and freezing temperatures. Taken together, our data suggest that DsDBF1 could be a promising gene candidate to improve stress tolerance in crop plants, and the characterization of TFs of a stress tolerant moss such as D. scoparium provides a better understanding of plant adaptation mechanisms.

Lichens often grow in microhabitats where they experience severe abiotic stresses. Some species respond to high UV radiation by synthesizing dark brown melanic pigments in the upper cortex. However, unlike the melanized structures of non-lichenized fungi, the morphology of the melanic layer in lichens remains unstudied. Here, we analyzed the morphology, ultrastructure, and elemental composition of the melanized layer in UV-exposed thalli of the lichen Lobaria pulmonaria (L.) Hoffm. Using light microscopy, we detected a pigmented layer sensitive to staining with 3,4-L-dihydroxyphenylalanine, a precursor of eumelanin, in the upper cortex of melanized thalli. Analysis of cross-sections of melanized thalli using scanning electron microscopy revealed that melanin-like granules are deposited into the hyphal lumens. Melanized thalli also possessed thicker hyphal cell walls compared to pale thalli. Energy-dispersive X-ray spectroscopy analysis of the elemental composition of the hyphal walls and extracted melanin indicated that the type of melanin synthesized by L. pulmonaria is eumelanin. Transmission electron microscopy was used to show that during melanization melanosome-like dark vesicles are transported to the cell surface and secreted into the cell walls of the fungal hyphae. Results from this study provide new insights into the effects of melanin synthesis on the microstructure of lichen thalli., Competing Interests: The authors declare no conflicts of interest.

Lichens are symbiotic photosynthesizing organisms with thalli formed by fungi and algae/cyanobacteria that possess high stress tolerance. One of the factors that contributes to the ability of a lichen to tolerate harsh environmental conditions is the presence of unique metabolites, including high-molecular-weight dark pigments termed melanins. The chemical composition and structure of lichen melanins remain poorly studied. We analyzed the elemental composition, the main functional groups, and the physicochemical properties of melanin extracted from Cetraria islandica and Pseudevernia furfuracea lichens. Based on the C/N ratio, this pigment is allomelanin. We also identified functional groups that provide photoprotective and antioxidant properties of melanin. Melanin synthesis might be an essential defense mechanism contributing to the survival of lichens under exposure to UV radiation.

In the present work, the APX gene encoding ascorbate peroxidase in the moss Dicranum scoparium was for the first time cloned and sequenced, and a high homology of APX with ascorbate peroxidase genes of the mosses Grimmia pilifera and Physcomitrella patens was shown. The structure of the protein was characterized using bioinfomatics approach, and the activity of the enzyme under abiotic stresses was studied. An increase in the activity of ascorbate peroxidase was detected during desiccation of D. scoparium shoots. When exposed to heat shock, a decrease in the activity of ascorbate peroxidase correlated with a decrease in the expression of APX. Conserved elements, which were found in the structure of ascorbate peroxidase gene and protein, indicate that these sequences are conserved in the plant genome during evolution, in support of the importance of this enzyme in maintaining cellular redox status.

Cold stress can significantly alter the composition and functioning of the major membrane lipids in plants. However, the roles of the sterol component of plant membranes in stress tolerance remain unclear. In the work presented here we investigated the role of sterols in the response of wheat to cold stress. Initial experiments demonstrated that the roots and leaves of wheat seedlings are differentially sensitive to low positive temperatures. In the roots, cold stress induced disturbance of membrane integrity and accumulation of ROS followed by the induction of autophagy. The absence of such changes in leaves suggests that in wheat, the roots are more sensitive to cold than the leaves. The roots display a time-dependent parabolic pattern of cold stress response, characterized by raised levels of sterols and markers of oxidative stress during short-term treatment, and a decline of these parameters after prolonged treatment. MβCD-induced sterol depletion aggravated the negative effects of cold on the roots. In the leaves the changes also displayed parabolic patterns, with significant changes occurring in 24-ethyl sterols and major PLs. Constitutively high levels of sterols, glycolipids and PLs, and up-regulation of TaSMTs in the leaves may provide membrane stability and cold tolerance. Taken together, results suggest that sterols play important roles in the response of wheat seedlings to cold stress., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Maternal high levels of the redox active amino acid homocysteine-called hyperhomocysteinemia (hHCY)-can affect the health state of the progeny. The effects of hydrogen sulfide (H 2 S) treatment on rats with maternal hHCY remain unknown. In the present study, we characterized the physical development, reflex ontogeny, locomotion and exploratory activity, muscle strength, motor coordination, and brain redox state of pups with maternal hHCY and tested potential beneficial action of the H 2 S donor-sodium hydrosulfide (NaHS)-on these parameters. Our results indicate a significant decrease in litter size and body weight of pups from dams fed with methionine-rich diet. In hHCY pups, a delay in the formation of sensory-motor reflexes was observed. Locomotor activity tested in the open field by head rearings, crossed squares, and rearings of hHCY pups at all studied ages (P8, P16, and P26) was diminished. Exploratory activity was decreased, and emotionality was higher in rats with hHCY. Prenatal hHCY resulted in reduced muscle strength and motor coordination assessed by the paw grip endurance test and rotarod test. Remarkably, administration of NaHS to pregnant rats with hHCY prevented the observed deleterious effects of high homocysteine on fetus development. In rats with prenatal hHCY, the endogenous generation of H 2 S brain tissues was lower compared to control and NaHS administration restored the H 2 S level to control values. Moreover, using redox signaling assays, we found an increased level of malondialdehyde (MDA), the end product of lipid peroxidation, and decreased activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the brain tissues of rats of the hHCY group. Notably, NaHS treatment restored the level of MDA and the activity of SOD and GPx. Our data suggest that H 2 S has neuroprotective/antioxidant effects against homocysteine-induced neurotoxicity providing a potential strategy for the prevention of developmental impairments in newborns.

This is the first study to show that polyamine spermine, a low-molecular-weight nitrogen-containing compound, can induce autophagy in plants. This process is accompanied by an increased generation of reactive oxygen species and nitric oxide, which play a signal role and are required for triggering autophagy.

Some free-living Ascomycetes and white and brown rot Basidiomycetes can generate hydroxyl radicals using extracellular redox cycling. However, the mechanisms of hydroxyl radical production differ between white and brown rot Basidiomycetes, and are unknown for Ascomycetes. Here, we present a survey of extracellular hydroxyl radical production by a range of lichenized Ascomycetes. Results show that given a quinone and chelated ferric ions, many lichens can readily produce hydroxyl radicals, and this is accompanied by the reduction of Fe 3 + to Fe 2+ . In white rot fungi, extracellular redox enzymes have been proposed to be involved in hydroxyl radical generation. However, a survey of a wide range of lichens suggests that in these fungi hydroxyl radical production does not directly correlate with the activity of laccases and peroxidases. Rather, radicals are probably produced by a mechanism like that proposed for brown rot fungi. Potential roles of hydroxyl radicals produced by lichens include the breakdown of lignocellulosic residues in the soil which may allow lichens to live a partially saprotrophic existence, the breakdown of toxic soil chemicals and the formation of an 'oxidative burst' to deter potential pathogens., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Three homeologous copies of the TaSMT1 gene for C24-sterol methyltransferase, which are located on chromosomes A, B, and D of Triticum aestivum hexaploid genome, were discovered. The bioinformatic analysis of the structure of these genes and sequencing de novo promoter sequences revealed differential expression of homeologous TaSMT1 genes in leaves and roots of wheat seedlings under normal conditions and in stress.

Sterols, which are isoprenoid derivatives, are structural components of biological membranes. Special attention is now being given not only to their structure and function, but also to their regulatory roles in plants. Plant sterols have diverse composition; they exist as free sterols, sterol esters with higher fatty acids, sterol glycosides, and acylsterol glycosides, which are absent in animal cells. This diversity of types of phytosterols determines a wide spectrum of functions they play in plant life. Sterols are precursors of a group of plant hormones, the brassinosteroids, which regulate plant growth and development. Furthermore, sterols participate in transmembrane signal transduction by forming lipid microdomains. The predominant sterols in plants are β-sitosterol, campesterol, and stigmasterol. These sterols differ in the presence of a methyl or an ethyl group in the side chain at the 24th carbon atom and are named methylsterols or ethylsterols, respectively. The balance between 24-methylsterols and 24-ethylsterols is specific for individual plant species. The present review focuses on the key stages of plant sterol biosynthesis that determine the ratios between the different types of sterols, and the crosstalk between the sterol and sphingolipid pathways. The main enzymes involved in plant sterol biosynthesis are 3-hydroxy-3-methylglutaryl-CoA reductase, C24-sterol methyltransferase, and C22-sterol desaturase. These enzymes are responsible for maintaining the optimal balance between sterols. Regulation of the ratios between the different types of sterols and sterols/sphingolipids can be of crucial importance in the responses of plants to stresses.

Autophagy is an efficient way of degradation and removal of unwanted or damaged intracellular components in plant cells. It plays an important role in recycling of intracellular structures (during starvation, removal of cell components formed during plant development or damaged by various stress factors) and in programmed cell death. Morphologically, autophagy is characterized by the formation of double-membrane vesicles called autophagosomes, which are essential for the isolation and degradation of cytoplasmic components. Among autophagic (ATG) proteins, ATG8 from the ubiquitin-like protein family plays a key role in autophagosome formation. ATG8 is also involved in selective autophagy, fusion of autophagosome with the vacuole, and some other intracellular processes not associated with autophagy. In contrast to yeasts that carry a single ATG8 gene, plants have multigene ATG8 families. The reason for such great ATG8 diversity in plants remains unclear. It is also unknown whether all members of the ATG8 family are involved in the formation and functioning of autophagosomes. To answer these questions, the identification of the structure and the possible functions of plant proteins from ATG8 family is required. In this review, we analyze the structures of ATG8 proteins from plants and their homologs from yeast and animal cells, interactions of ATG8 proteins with functional ligands, and involvement of ATG8 proteins in different metabolic processes in eukaryotes.

Anthocerotophyta (hornworts) belong to a group of ancient nonvascular plants and originate from a common ancestor with contemporary vascular plants. Hornworts represent a unique model for investigating mechanisms of formation of stress resistance in higher plants due to their high tolerance to the action of adverse environmental factors. In this work, we demonstrate that the thallus of Anthoceros natalensis exhibits high redox activity changing under stress. Dehydration of the thallus is accompanied by the decrease in activities of intracellular peroxidases, DOPA-peroxidases, and tyrosinases, while catalase activity increases. Subsequent rehydration results in the increase in peroxidase and catalase activities. Kinetic features of peroxidases and tyrosinases were characterized as well as the peroxidase isoenzyme composition of different fractions of the hornwort cell wall proteins. It was shown that the hornwort peroxidases are functionally similar to peroxidases of higher vascular plants including their ability to form superoxide anion-radical. The biochemical mechanism was elucidated, supporting the possible participation of peroxidases in the formation of reactive oxygen species (ROS) via substrate-substrate interactions in the hornwort thallus. It has been suggested that the ROS formation by peroxidases is an evolutionarily ancient process that emerged as a protective mechanism for enhancing adaptive responses of higher land plants and their adaptation to changing environmental conditions and successful colonization of various ecological niches.

Plant surfaces form the barrier between a plant and its environment. Upon damage, the wound healing process begins immediately and is accompanied by a rapid production of extracellular reactive oxygen species (ROS), essential in deterring pathogens, signalling responses and cell wall restructuring. Although many enzymes produce extracellular ROS, it is unclear if ROS-producing enzymes act synergistically. We characterised the oxidative burst of superoxide (O2(·-)) and hydrogen peroxide (H2O2) that follows wounding in pea (Pisum sativum L.) seedlings. Rates of ROS production were manipulated by exogenous application of enzyme substrates and inhibitors. The results indicate significant roles for di-amine oxidases (DAO) and peroxidases (Prx) rather than NADPH oxidase. The burst of O2(·-) was strongly dependent on the presence of H2O2 produced by DAO. Potential substrates released from wounded seedlings included linoleic acid that, upon exogenous application, strongly stimulated catalase-sensitive O2(·-) production. Moreover, a 65kD plasma membrane (PM) guaiacol Prx was found in the secretome of wounded seedlings and showed dependence on linoleic acid for O2(·-) production. Lipoxygenases are suggested to modulate O2(·-) production by consuming polyunsaturated fatty acids in the apoplast. Overall, a O2(·-)-producing mechanism involving H2O2-derived from DAO, linoleic acid and a PM-associated Prx is proposed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Nitrate reductase (NR) and peroxidase (POX) are important enzymes involved in the metabolism of reactive oxygen (ROS) and nitrogen species in leaves of wheat (Triticum aestivum L.) seedlings. It has been confirmed that NR activity in wheat leaves depends on the light conditions and the presence of nitrates during the cultivation of the seedlings, and it is regulated by the molybdenum cofactor and phosphorylation. In the present study, confocal microscopy and EPR spectroscopy studies showed that the addition of nitrite, a product of NR, increased the level of nitric oxide (NO). This increase was prevented by the addition of sodium azide, an inhibitor of NR. The results suggest that in wheat leaves one of the key functions of NR is the formation of the signaling NO molecule. Cultivation of green plants under conditions of prolonged (4 days) darkness, a strong stress factor for photosynthesizing cells, decreased the activity of NR. Moreover, darkness induced significant elevation of the POX activity that was prevented by the addition of nitrate to the growth medium. It is proposed that the changes in light conditions result in the competition between nitrate- and ROS-metabolizing activities of POX in leaves, and a possible interaction between NR and POX controls the levels of NO and ROS in the leaf tissue.

Lichens belonging to the order Peltigerales display strong activity of multi-copper oxidases (e.g. tyrosinase) as well as heme-containing peroxidases. The lichen peroxidase was purified to homogeneity from the thallus of Leptogium saturninum (LsaPOX) by fast protein liquid chromatography and then partially characterized. The oligomeric protein occurs as both 79 kDa dimeric and 42 kDa monomeric forms, and displayed broad substrate specificity. In addition to an ability to oxidize classic peroxidase substrates (e.g. 2,6-dimethoxyphenol), the enzyme could convert recalcitrant compounds such as synthetic dyes (e.g. Azure B and Reactive Blue 5), 4-nitrophenol and non-phenolic methoxylated aromatics (e.g. veratryl alcohol). Comparing LsaPOX with a basidiomycete dye-decolorizing (DyP)-type peroxidase from Auricularia auricula-judae showed that the lichen enzyme has a high-redox potential, with oxidation capabilities ranging between those of known plant and fungal peroxidases. Internal peptide fragments show homology (up to 60%) with putative proteins from free-living ascomycetes (e.g. Penicillium marneffei and Neosartorya fischeri), but not to sequences of algal or cyanobacterial peptides or to known fungal, bacterial or plant peroxidases. LsaPOX is the first heme peroxidase purified from an ascomyceteous lichen that may help the organism to successfully exploit the extreme micro-environments in which they often grow., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Plant sterols are important multifunctional lipids, which are involved in determining membrane properties. Biophysical characteristics of model lipid and isolated animal membranes with altered sterol component have been intensively studied. In plants however, the precise mechanisms of involvement of sterols in membrane functioning remain unclear. In present work the possible interactions between sterols and other membrane lipids in plant cells were studied. A useful experimental approach for elucidating the roles of sterols in membrane activity is to use agents that specifically bind with endogenous sterols, for example the antibiotic nystatin. Membrane characteristics and the composition of membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with nystatin were analyzed. The application of nystatin greatly increased the permeability of the plasma membrane for ions and SH-containing molecules and decreased the total sterol level mainly as a consequence of a reduction in the amount of β-sitosterol and campesterol. Dynamic light-scattering was used to confirm the in vitro formation of stable complexes between nystatin and β-sitosterol or cholesterol. Sterol depletion was accompanied by a significant rise in total glycoceramide (GlCer) content after 2h treatment with nystatin. Analysis of the GlCer composition using mass spectrometry with electrospray ionization demonstrated that nystatin induced changes in the ratio of molecular species of GlCer. Our results suggest that changes in the sphingolipid composition can contribute to the changes in plasma membrane functioning induced by sterol depletion., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

'Stresses' that impact upon seeds can affect plant reproduction and productivity, and, hence, agriculture and biodiversity. In the absence of a clear definition of plant stress, we relate concepts from physics, medicine and psychology to stresses that are specific to seeds. Potential 'eustresses' that enhance function and 'distresses' that have harmful effects are considered in relation to the seed life cycle. Taking a triphasic biomedical stress concept published in 1936, the 'General Adaptation Syndrome', to the molecular level, the 'alarm' response is defined by post-translational modifications and stress signalling through cross-talk between reactive oxygen and nitrogen species, and seed hormones, that result in modifications to the transcriptome. Protection, repair, acclimation and adaptation are viewed as the 'building blocks' of the 'resistance' response, which, in seeds, are the basis for their longevity over centuries. When protection and repair mechanisms eventually fail, depending on dose and time of exposure to stress, cell death and, ultimately, seed death are the result, corresponding to 'exhaustion'. This proposed seed stress concept may have wider applicability to plants in general., (© The Authors (2010). Journal compilation © New Phytologist Trust (2010).)

Extracellularly produced reactive oxygen species (ROS) play key roles in plant development, but their significance for seed germination and seedling establishment is poorly understood. Here we report on the characteristics of extracellular ROS production during seed germination and early seedling development in Pisum sativum. Extracellular superoxide (O2(.-)) and hydrogen peroxide (H2O2) production and the activity of extracellular peroxidases (ECPOX) were determined spectrophotometrically, and O2(.-) was identified by electron paramagnetic resonance. Cell wall fractionation of cotyledons, seed coats and radicles was used in conjunction with polyacrylamide gel electrophoresis to investigate substrate specificity and molecular masses of O2(.-)-producing enzymes, and the forces that bind them to the cell wall. Seed imbibition was accompanied by an immediate, transient burst of redox activity that involved O2(.-) and other substances capable of oxidizing epinephrine, and also H2O2. At the final stages of germination, coinciding with radicle elongation, a second increase in O2(.-) but not H2O2 production occurred and was correlated with an increase in extracellular ECPOX activity. Electrophoretic analyses of cell wall fractions demonstrated the presence of enzymes capable of O2(.-) production. The significance of extracellular ROS production during seed germination and early seedling development, and also during seed aging, is discussed., (2010 Elsevier GmbH. All rights reserved.)

The present work was devoted to the exploration of the role of sterols in the functioning of membranes in root cells. Membrane characteristics and composition of the membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with exogenous cholesterol and antibiotic nystatin, which specifically binds with endogenous sterols, were analyzed. Cholesterol caused a fall of membrane potential, acidification of the incubation medium, decrease in potassium leakage of roots, and increase in the level of exogenous superoxide radical. Similarly to cholesterol, the application of nystatin also induced the depolarization of the plasma membrane, but in contrast with cholesterol it was accompanied by alkalinization of the incubation medium and decrease in the level of exogenous superoxide radical. Analysis of membrane lipids showed that following nystatin treatment the total sterol content in roots did not change, while the level of complex sphingolipids represented mainly by glycoceramides became higher. Using mass spectrometry with electrospray ionization ((+)ESI-MS) for the analysis of the glycoceramide composition, we showed that nystatin induced changes in the ratios of molecular species of glycoceramides. It was suggested that the modification of the sterol component of plasma membrane could influence membrane functioning by changing the sphingolipid composition of lipid rafts.

In our earlier work, we showed that the liverwort Dumortiera hirsuta produces an extracellular oxidative burst of superoxide radicals during rehydration following desiccation stress. The oxidative burst is a common early response of organisms to biotic and abiotic stresses, with suggested roles in signal transduction, formation of protective substances such as suberin, melanin and lignin and defense against pathogens. To discover which enzymes are responsible for the extracellular superoxide production, we isolated apoplastic fractions from D. hirsuta, surveyed for the presence of potential redox enzymes, and performed non-denaturing polyacrylamide gel electrophoresis activity stains. Various isoforms of peroxidase (EC 1.11.1.7) and tyrosinase (o-diphenolase) (EC 1.10.3.1) were present at significant levels in the apoplast. In-gel activity staining revealed that some peroxidases isoforms could produce superoxide, while tryosinases could readily metabolize 3,4-dihydroxy phenyl l-alanine (l-dopa) into melanins. Interestingly, some peroxidase isoforms could oxidize the native tyrosinase substrate l-dopa at significant levels, even in the absence of hydrogen peroxide, while others could do so only in the presence of hydrogen peroxide. In D. hirsuta, peroxidases may play an important role in melanin formation. Possible functions for these diverse oxidases in liverwort biology are discussed.

Reactive oxygen species (ROS) are implicated in seed death following dehydration in desiccation-intolerant 'recalcitrant' seeds. However, it is unknown if and how ROS are produced in the apoplast and if they play a role in stress signalling during desiccation. We studied intracellular damage and extracellular superoxide (O(2)(.-)) production upon desiccation in Castanea sativa seeds, mechanisms of O(2)(.-) production and the effect of exogenously supplied ROS. A transient increase in extracellular O(2)(.-) production by the embryonic axes preceded significant desiccation-induced viability loss. Thereafter, progressively more oxidizing intracellular conditions, as indicated by a significant shift in glutathione half-cell reduction potential, accompanied cell and axis death, coinciding with the disruption of nuclear membranes. Most hydrogen peroxide (H(2)O(2))-dependent O(2)(.-) production was found in a cell wall fraction that contained extracellular peroxidases (ECPOX) with molecular masses of approximately 50 kDa. Cinnamic acid was identified as a potential reductant required for ECPOX-mediated O(2)(.-) production. H(2)O(2), applied exogenously to mimic the transient ROS burst at the onset of desiccation, counteracted viability loss of sub-lethally desiccation-stressed seeds and of excised embryonic axes grown in tissue culture. Hence, extracellular ROS produced by embryonic axes appear to be important signalling components involved in wound response, regeneration and growth.

Competitive and complimentary relationships of various peroxidase substrates were studied to elucidate the enzymatic mechanisms underlying production of reactive oxygen species in plant cell apoplast. Dianisidine peroxidase released from wheat seedling roots was inhibited by ferulate and coniferol, while ferulic and coniferyl peroxidases were activated by o-dianisidine. Both ferulate and coniferol, when added together with hydrogen peroxide, stimulated superoxide production by extracellular peroxidase. We suggest that substrate-substrate activation of extracellular peroxidases is important for stress-induced oxidative burst in plant cells.

Recalcitrant seeds are intolerant of desiccation and cannot be stored in conventional seed banks. Cryopreservation allows storage of the germplasm of some recalcitrant seeded species, but application to a wide range of plant diversity is still limited. The present work aimed at understanding the stresses that accompany the first steps in cryopreservation protocols, wounding and desiccation, both of which are likely to lead to the formation of reactive oxygen species (ROS). Extracellular ROS production was studied in isolated embryonic axes of sweet chestnut (Castanea sativa). Axis excision was accompanied by a burst of superoxide (O(2)(*-)), demonstrated by a colorimetric assay using epinephrine, electron spin resonance and staining with nitroblue tetrazolium. Superoxide was immediately produced on the cut surface after isolation of the axis from the seed, with an initial 'burst' in the first 5 min. Isolated axes subjected to variable levels of desiccation stress showed a decrease in viability and vigour and increased electrolyte leakage, indicative of impaired membrane integrity. The pattern of O(2)(*-) production showed a typical Gaussian pattern in response to increasing desiccation stress. The results indicate a complex interaction between excision and subsequent drying and are discussed with a view of manipulating ROS production for optimisation of cryopreservation protocols.

Background and Aims: Following previous findings of high extracellular redox activity in lichens and the presence of laccases in lichen cell walls, the work presented here additionally demonstrates the presence of tyrosinases. Tests were made for the presence of tyrosinases in 40 species of lichens, and from selected species their cellular location and molecular weights were determined. The effects of stress and inhibitors on enzyme activity were also studied., Methods: Tyrosinase and laccase activities were assayed spectrophotometrically using a variety of substrates. The molecular mass of the enzymes was estimated using polyacrylamide gel electrophoresis., Key Results: Extracellular tyrosinase and laccase activity was measured in 40 species of lichens from different taxonomic groupings and contrasting habitats. Out of 20 species tested from the sub-order Peltigerineae, all displayed significant tyrosinase and laccase activity, while activity was low or absent in other species tested. Representatives from both groups of lichens displayed low peroxidase activities. Identification of the enzymes as tyrosinases was confirmed by the ability of lichen thalli or leachates derived by shaking lichens in distilled water to metabolize substrates such as L-dihydroxyphenylalanine (DOPA), tyrosine and epinephrine readily in the absence of hydrogen peroxide, the sensitivity of the enzymes to the inhibitors cyanide, azide and hexylresorcinol, activation by SDS and having typical tyrosinase molecular masses of approx. 60 kDa. Comparing different species within the Peltigerineae showed that the activities of tyrosinases and laccase were correlated to each other. Desiccation and wounding stimulated laccase activity, while only wounding stimulated tyrosinase activity., Conclusions: Cell walls of lichens in sub-order Peltigerineae have much higher activities and a greater diversity of cell wall redox enzymes compared with other lichens. Possible roles of tyrosinases include melanization, removal of toxic phenols or quinones, and production of herbivore deterrents.

Following our previous findings of high extracellular redox activity in lichens, the results of the work presented here identify the enzymes involved as laccases. Despite numerous data on laccases in fungi and flowering plants, this is the first report of the occurrence of laccases in lichenized ascomycetes. Extracellular laccase activity was measured in 40 species of lichens from different taxonomic groupings and contrasting habitats. Out of 20 species tested from suborder Peltigerineae, 18 displayed laccase activity, while activity was absent in species tested from other lichen groups. Identification of the enzymes as laccases was confirmed by the ability of lichen leachates to readily metabolize substrates such as 2,2'-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS), syringaldazine and o-tolidine in the absence of hydrogen peroxide, sensitivity of the enzymes to cyanide and azide, the enzymes having typical laccase pH and temperature optima, and an absorption spectrum with a peak at 614nm. Desiccation and wounding stimulated laccase activity. Laccase activity was not increased after treatment with normal inducers of laccase synthesis, suggesting that they are constitutively expressed. Electrophoresis showed that the active form of laccase from Peltigera malacea was a tetramer with an unusually high molecular mass of 340kDa and an isoelectric point (pI) of 4.7. The finding of abundant extracellular redox enzymes known to actively produce reactive oxygen species suggest that their roles may include increasing nutrient supply to lichens by delignification, and deterring pathogens by contributing to the oxidative burst. Furthermore, once released into the environment, they may participate in the carbon cycle by facilitating the breakdown or formation of humic substances.

Background and Aims: The ability of partial dehydration and abscisic acid pretreatments to increase desiccation tolerance in the cyanobacterial lichen Peltigera polydactylon was tested. *, Methods: Net photosynthesis and respiration were measured using infrared gas analysis during a drying and rehydration cycle. At the same time, the efficiency of photosystem two was measured using chlorophyll fluorescence, and the concentrations of chlorophyll a were spectrophotometrically assayed. Heat production was also measured during a shorter drying and rehydration cycle using differential dark microcalorimetry. *, Key Results: Pretreating lichens by dehydrating them to a relative water content of approx. 0.65 for 3 d, followed by storing thalli hydrated for 1 d in the light, significantly improved their ability to recover net photosynthesis during rehydration after desiccation for 15 but not 30 d. Abscisic acid pretreatment could substitute for partial dehydration. The improved rates of photosynthesis during the rehydration of pretreated material were not accompanied by preservation of photosystem two activity or chlorophyll a concentrations compared with untreated lichens. Partial dehydration and ABA pretreatments appeared to have little direct effect on the desiccation tolerance of the mycobiont, because the bursts of respiration and heat production that occurred during rehydration were similar in control and pretreated lichens. *, Conclusions: Results indicate that the photobiont of P. polydactylon possesses inducible tolerance mechanisms that reduce desiccation-induced damage to carbon fixation, and will therefore improve the supply of carbohydrates to the whole thallus following stress. In this lichen, ABA is involved in signal transduction pathways that increase tolerance of the photobiont.

Extracellular peroxidase has been shown to contribute to superoxide production in wounded wheat (Triticum aestivum L. cv. Ljuba) root cells. The superoxide-synthesizing system of root cells was considerably inhibited by KCN and NaN3 and activated by MnCl2 and H2O2. Treatment of roots with salicylic acid and a range of di- and tri-carbonic acids (malic, citric, malonic, fumaric, and succinic acids) stimulated superoxide production in both root cells and extracellular solution. The H2O2-stimulated superoxide production in the extracellular solution was much higher when roots were preincubated with salicylic or succinic acid. Exogenous acids enhanced peroxidase activity in the extracellular solution. Pretreatment of root cells with the detergents trypsin and sodium dodecyl sulfate had similar effects on the peroxidase activity. Significant inhibition of both superoxide production and peroxidase activity by diphenylene iodonium suggests that the specificity of the latter as an inhibitor of NADPH oxidase is doubtful. Results obtained indicate that extra-cellular peroxidase is involved in the superoxide production in wheat root cells. The mobile form of peroxidase can be readily secreted to the apoplastic solution and serve as an emergency enzyme involved in plant wound response.