Melanin from the Lichens Cetraria islandica and Pseudevernia furfuracea: Structural Features and Physicochemical Properties.

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.

Ascorbate Peroxidase of Moss Dicranum scoparium: Gene Identification and Enzyme Activity.

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.

Hydrogen Sulfide Ameliorates Developmental Impairments of Rat Offspring with Prenatal Hyperhomocysteinemia.

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 (H2S) 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 H2S 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 H2S brain tissues was lower compared to control and NaHS administration restored the H2S 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 H2S has neuroprotective/antioxidant effects against homocysteine-induced neurotoxicity providing a potential strategy for the prevention of developmental impairments in newborns.

Spermine Induces Autophagy in Plants: Possible Role of NO and Reactive Oxygen Species.

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.

Characterization of the homeologous genes of C24-sterol methyltransferase in Triticum aestivum L.

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.

Plant Sterols: Diversity, Biosynthesis, and Physiological Functions.

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 ß-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.

Molecular Mechanisms of Autophagy in Plants: Role of ATG8 Proteins in Formation and Functioning of Autophagosomes.

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.

Activity of Redox Enzymes in the Thallus of Anthoceros natalensis.

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.

Nitrate reductase from Triticum aestivum leaves: regulation of activity and possible role in production of nitric oxide.

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.

Binding of sterols affects membrane functioning and sphingolipid composition in wheat roots.

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.

Effect of exogenous phenols on superoxide production by extracellular peroxidase from wheat seedling roots.

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.

Role of extracellular peroxidase in the superoxide production by wheat root cells.

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.