Agnieszka Kobylińska

Many studies have shown that melatonin (an indoleamine) is an important molecule in plant physiology. It is known that this indoleamine is crucial during plant stress responses, especially by counteracting secondary oxidative stress (efficient direct and indirect antioxidant) and switching on different defense plant strategies. In this report, we present exogenous melatonin’s potential to protect lipid profile modification and membrane integrity in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) cell culture exposed to lead. There are some reports of the positive effect of melatonin on animal cell membranes; ours is the first to report changes in the lipid profile in plant cells. The experiments were performed in the following variants: LS: cells cultured on unmodified LS medium—control; (ii) MEL: BY-2 cells cultured on LS medium with melatonin added from the beginning of culture; (iii) Pb: BY-2 cells cultured on LS medium with Pb²⁺ added on the 4th day of culture; (iv) MEL+Pb: BY-2 cells cultured on LS medium with melatonin added from the start of culture and stressed with Pb²⁺ added on the 4th day of culture. Lipidomic analysis of BY-2 cells revealed the presence of 40 different phospholipids. Exposing cells to lead led to the overproduction of ROS, altered fatty acid composition and increased PLD activity and subsequently elevated the level of phosphatidic acid at the cost of dropping the phosphatidylcholine. In the presence of lead, double-bond index elevation, mainly by higher quantities of linoleic (C18:2) and linolenic (C18:3) acids in the log phase of growth, was observed. In contrast, cells exposed to heavy metal but primed with melatonin showed more similarities with the control. Surprisingly, the overproduction of ROS caused of lipid peroxidation only in the stationary phase of growth, although considerable changes in lipid profiles were observed in the log phase of growth—just 4 h after lead administration. Our results indicate that the pretreatment of BY-2 with exogenous melatonin protected tobacco cells against membrane dysfunctions caused by oxidative stress (lipid oxidation), but also findings on a molecular level suggest the possible role of this indoleamine in the safeguarding of the membrane lipid composition that limited lead-provoked cell death. The presented research indicates a new mechanism of the defense strategy of plant cells generated by melatonin.

Recent studies have shown that melatonin is an important molecule in plant physiology. It seems that the most important is that melatonin effectively eliminates oxidative stress (direct and indirect antioxidant) and switches on different defence strategies (preventive and interventive actions) during environmental stresses. In the presented report, exogenous melatonin potential to protect Nicotiana tabacum L. line Bright Yellow 2 (BY-2) exposed to lead against death was examined. Analyses of cell proliferation and viability, the level of intracellular calcium, changes in mitochondrial membrane potential (ΔΨm) as well as possible translocation of cytochrome c from mitochondria to cytosol and subsequent caspase-like proteolytic activity were conducted. Our results indicate that pretreatment BY-2 with melatonin protected tobacco cells against mitochondrial dysfunction and caspase-like activation caused by lead. The findings suggest the possible role of this indoleamine in the molecular mechanism of mitochondria, safeguarding against potential collapse and cytochrome c release. Thus, it seems that applied melatonin acted as an effective factor, promoting survival and increasing plant tolerance to lead.

Mosses are mainly the object of ecological and taxonomic research. This group of plants are still underestimated by scientists in other aspects of research. Recent research has shown that these plants contain remarkable and unique substances with high biological activity. Five species of mosses from a large urban ecosystem were identified for present study. In order to determine their biological potential, multifaceted studies were carried out, including: total phenolics content, antioxidant activity, antimicrobial and antifungal study, cytotoxicity evaluation, and scratch assay to assess pro-regenerative effect in the context of their possible use as the ingredients of biologically active cosmetics. Additionally, determination of individual phenolic compounds in selected extracts of the tested mosses was made. Research showed that Ceratodon purpureus and Dryptodon pulvinatus extracts had the greatest potential as antioxidants and antimicrobial activity. The cytotoxicity assessment indicated that the extracts from Dryptodon pulvinatus and Rhytidiadelphus squarossus exerted the strongest negative effect on mouse fibroblast line L929 viability at higher concentrations. While, the extract from Tortulla muralis best stimulated human foreskin fibroblast line HFF-1 proliferation and wound healing. The research on individual phenolic compounds content in the extracts tested indicated over 20 peaks on UPLC chromatograms. The conducted study has shown that mosses, especially so far unexplored species of open ecosystems, and e.g. epilytic habitats, may be a valuable source of biologically active substances and thus may constitute important medical and cosmetic possibilities.

Programmed cell death (PCD) is a physiological process in which infected or unnecessary cells due to their suicidal death capability can be selectively eliminated. Pro- and antiapoptotic proteins play an important role in the induction or inhibition of this process. Presented article shows property of Bax-1 (BI-1) inhibitor which is one of the conservative protein associated with the endoplasmic reticulum (ER) as well as its cytoprotective role in the regulation of cellular processes. It was shown that: 1) BI-1 is a small protein consisting of 237 amino acids (human protein - 36 kDa) and has 6 (in animals) and 7 (in plants) α-helical transmembrane domains, 2) BI-1 is expressed in all organisms and in most tissues, moreover its level depends on the functional condition of cells and it is involved in the development or reaction to biotic and abiotic stresses, 3) BI-1 forms a pH-dependent Ca2+ channel enabling release of these ions from the ER, 4) cytoprotective effects of BI-1 requires a whole, unchanged C-terminus, 5) BI-1 can interact directly with numerous other proteins, BI-1 protein affects numerous cellular processes, including: counteracting ER stress, oxidative stress, loss of cellular Ca2+ homeostasis as well as this protein influences on sphingolipid metabolism, autophagy, actin polymerization, lysosomal activity and cell proliferation. Studies of BI-1 functions will allow understanding the mechanisms of anticancer therapy or increases the knowledge of crop tolerance to environmental stresses.

Recent studies have shown that melatonin is an important molecule in plant physiology. It seems that the most important is that melatonin efficacy eliminates oxidative stress (direct and indirect antioxidant) and moreover induce plant stress reaction and switch on different defence strategies (preventively and interventively actions). In this report, the impact of exogenous melatonin on carbohydrate metabolism in Nicotiana tabacum L. line Bright Yellow 2 (BY‐2) suspension cells during sugar starvation was examined. We analysed starch concentration, α‐amylase and PEPCK activity as well as proteolytic activity in culture media. It has been shown that BY‐2 cell treatment with 200 nM of melatonin improved viability of sugar‐starved cells. It was correlated with higher starch content and phosphoenolpyruvate carboxykinase (PEPCK) activity. The obtained results revealed that exogenous melatonin under specific conditions (stress) can play regulatory role in sugar metabolism, and it may modulate carbohydrate concentration in etiolated BY‐2 cells. Moreover, our results confirmed the hypothesis that if the starch is synthesised even in sugar‐starved cells, it is highly probable that melatonin shifts the BY‐2 cell metabolism on gluconeogenesis pathway and allows for synthesis of carbohydrates from nonsugar precursors, that is amino acids. These points to another defence strategy that was induced by exogenous melatonin applied in plants to overcome adverse environmental conditions.

Melatonin was discovered in plants more than two decades ago and, especially in the last decade, it has captured the interests of plant biologists. Beyond its possible participation in photoperiod processes and its role as a direct free radical scavenger as well as an indirect antioxidant, melatonin is also involved in plant defense strategies/reactions. However, the mechanisms that this indoleamine activates to improve plant stress tolerance still require identification and clarification. In the present report, the ability of exogenous melatonin to protect Nicotiana tabacum L. line Bright Yellow 2 (BY-2) suspension cells against the toxic exposure to lead was examined. Studies related to cell proliferation and viability, DNA fragmentation, possible translocation of cytochrome c from mitochondria to cytosol, cell morphology after fluorescence staining and also the in situ accumulation of superoxide radicals measured via the nitro blue tetrazolium reducing test, were conducted. This work establishes a novel finding by correcting the inhibition of release of mitochondrial ctytocrome c in to the cytoplasm with the high accumulation of superoxide radicals. The results show that pretreatment with 200 nm of melatonin protected tobacco cells from DNA damage caused by lead. Melatonin, as an efficacious antioxidant, limited superoxide radical accumulation as well as cytochrome c release thereby, it likely prevents the activation of the cascade of processes leading to cell death. Fluorescence staining with acridine orange and ethidium bromide documented that lead-stressed cells additionally treated with melatonin displayed intact nuclei. The results revealed that melatonin at proper dosage could significantly increase BY-2 cell proliferation and protected them against death. It was proved that melatonin could function as an effective priming agent to promote survival of tobacco cells under harmful lead-induced stress conditions.

Programmed cell death (PCD) is a cellular suicide process, commonly found in organisms, that is important for elimination unnecessary and damaged cells during development and adaptation to abiotic and biotic environmental stresses. PCD is a complex and precise, genetically controlled cellular process, in opposite to non-programmed death, necrosis, in which cells are "killed" by strong abiotic factors. This article shows: the occurrence of PCD during animals and plants ontogenesis, classification of cell death types in these organisms with description of autophagy, apoptosis and necrotic cell death and with discussion on plant cell death by apoptosis. The role of Bcl-2 protein and other proteins involved in the regulation of apoptosis induction and detection in the plant's (whose genomes do not encode these proteins) proteins of analogous function is also discussed. The paper also presents the effects of the expression of animals pro- and anti-apoptotic genes transformed into yeast and plants, and the use of transformed yeast as model to identify in cDNA libraries animal and plant genes involved in regulation of the induction and course of the PCD.

Melatonin is a conserved substance, which was discovered in the evolutionary distant organisms like bacteria, plants, invertebrates and vertebrates. Recent studies have shown that melatonin despite its possible role in photoperiod processes, has been found to be a direct free radical scavenger and an indirect antioxidant. In this report the impact of exogenous melatonin on the Bax inhibitor-1 (BI-1) expression level in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) suspension cells exposed to lead was examined. BI-1 is a well-conserved protein in plants and animals that serves as the inhibitor of mammalian proapoptotic proteins as well as plant ROS-induced cell death. Our results showed that pretreatment with 200 nm melatonin, expressing BI-1 and fortified tobacco suspension cells against damages induced by lead. The obtained results revealed, that melatonin significantly increases BY-2 cells proliferation and protects BY-2 cells against death. Moreover, the conducted analyses showed for the first time that the protective effect of melatonin may be connected not only with its antioxidant properties but also with its direct impact on elevating BI-1 expression and lead-induced programmed cell death (PCD) restriction.

Quercetin is a plant flavonoid phytochemical exhibiting a broad spectrum of properties i.a. antioxidant, anti-inflammatory and immunomodulatory. However, the effect of quercetin is not clear. This compound at low concentrations can stimulate proliferation of human cells, so it can be a potential drug in the treatment of neurodegenerative diseases and in high concentrations, it induces apoptosis thereby eliminating the infected or abnormal cells and can serve as a potential anticancer drug with wide clinical application. Action of quercetin can be explained by its interference with cellular enzymes, receptors, transporters and signalling system. Due to its widespread occurrence in the plant world, it is an integral component of the human diet. The dietary quercetin occurs most often in the form of β-glycosides connected mostly with rutinose, rhamnose and glucose. Depending on the nutritional habits, the daily intake of flavonoids, including quercetin, ranges from 3 to 70 mg. Epidemiological studies confirm an inverse correlation between the consumption of flavonoids and the incidence of lifestyle diseases and tumor formation. Published data indicate that consumption of foods rich in flavonoids reduces the risk of coronary heart disease. Thus, flavonoids - including quercetin - seem to be an interesting pro-health agent.

cAMP is a second messenger which plays a regulatory role in a wide variety of biological processes in organisms ranging from prokaryotes to higher eukaryotes, but knowledge of its role in macroalgae and vascular plants is limited. We modified cAMP levels in the macroalga Chara vulgaris thallus and studied the effects on thallus growth and gametangia development: db-cAMP (permeable analog of cAMP), adenylate cyclase (AC) activator, forskolin and theophylline (cAMP phosphodiesterase (PDE) inhibitor) were used to elevate cAMP levels, and the AC inhibitors 2'-dAdo and 2'-d3'-AMP were used to decrease them. The results suggest that in Chara vulgaris the cAMP pathway may regulate both vegetative thallus growth and gametangia development, and that these effects may depend on this second-messenger level. Elevated cAMP stimulated thallus growth and delayed gametangia development; decreased cAMP inhibited thallus growth and accelerated maturation of both antheridia and oogonia. These results suggest that the cAMP pathway participates in regulation of developmental processes in Chara vulgaris and that thallus growth and gametangia development require different cAMP levels in cells.