Melatonin Application Modifies Antioxidant Defense and Induces Endoreplication in Maize Seeds Exposed to Chilling Stress.

The aim of the study was to demonstrate the biostimulating effect of exogenous melatonin (MEL) applied to seeds via hydroconditioning. It was indicated that only well-chosen application technique and MEL dose guarantees success concerning seed germination and young seedlings growth under stress conditions. For maize seed, 50 µM of MEL appeared to be the optimal dose. It improved seed germination and embryonic axes growth especially during chilling stress (5 °C/14 days) and during regeneration after its subsided. Unfortunately, MEL overdosing lowered IAA level in dry seeds and could disrupt the ROS-dependent signal transduction pathways. Very effective antioxidant MEL action was confirmed by low level of protein oxidative damage and smaller quantity of lipid oxidation products in embryonic axes isolated from seeds pre-treated with MEL and then exposed to cold. The stimulatory effects of MEL on antioxidant enzymes: SOD, APX and GSH-PX and on GST-a detoxifying enzyme, was also demonstrated. It was indicated for the first time, that MEL induced defence strategies against stress at the cytological level, as appearing endoreplication in embryonic axes cells even in the seeds germinating under optimal conditions (preventive action), but very intensively in those germinating under chilling stress conditions (intervention action), and after stress removal, to improve regeneration.

Melatonin in Medicinal and Food Plants: Occurrence, Bioavailability, and Health Potential for Humans.

Melatonin is a widespread molecule among living organisms involved in multiple biological, hormonal, and physiological processes at cellular, tissue, and organic levels. It is well-known for its ability to cross the blood-brain barrier, and renowned antioxidant effects, acting as a free radical scavenger, up-regulating antioxidant enzymes, reducing mitochondrial electron leakage, and interfering with proinflammatory signaling pathways. Detected in various medicinal and food plants, its concentration is widely variable. Plant generative organs (e.g., flowers, fruits), and especially seeds, have been proposed as having the highest melatonin concentrations, markedly higher than those found in vertebrate tissues. In addition, seeds are also rich in other substances (lipids, sugars, and proteins), constituting the energetic reserve for a potentially growing seedling and beneficial for the human diet. Thus, given that dietary melatonin is absorbed in the gastrointestinal tract and transported into the bloodstream, the ingestion of medicinal and plant foods by mammals as a source of melatonin may be conceived as a key step in serum melatonin modulation and, consequently, health promotion.

Melatonin redirects carbohydrates metabolism during sugar starvation in plant cells.

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 Protects Cultured Tobacco Cells against Lead-Induced Cell Death via Inhibition of Cytochrome c Translocation.

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.

Melatonin Improves the Photosynthetic Apparatus in Pea Leaves Stressed by Paraquat via Chlorophyll Breakdown Regulation and Its Accelerated de novo Synthesis.

The positive effect of melatonin on the function of the photosynthetic apparatus is known, but little is known about the specific mechanisms of melatonin's action in plants. The influence of melatonin on chlorophyll metabolism of 24-day-old Pisum sativum L. seedlings during paraquat (PQ)-induced oxidative stress was investigated in this study. Seeds were hydro-primed with water (H), 50 and 200 µM melatonin/water solutions (H-MEL50, H-MEL200), while non-primed seeds were used as controls (C). Increases in chlorophyllase activity (key enzyme in chlorophyll degradation) and 5-aminolevulinic acid contents (the first compound in the porphyrin synthesis pathway) were observed in H-MEL50 and H-MEL200 leaf disks. This suggests that melatonin may accelerate damaged chlorophyll breakdown and its de novo synthesis during the first hours of PQ treatment. Elevated level of pheophytin in control leaf disks following 24 h of PQ incubation probably was associated with an enhanced rate of chlorophyll degradation through formation of pheophytin as a chlorophyll derivative. This validates the hypothesis that chlorophyllide, considered for many years, as a first intermediate of chlorophyll breakdown is not. This is indicated by the almost unchanged chlorophyll to chlorophyllide ratio after 24 h of PQ treatment. However, prolonged effects of PQ-induced stress (48 h) revealed extensive discolouration of control and water-treated leaf disks, while melatonin treatment alleviated PQ-induced photobleaching. Also the ratio of chlorophyll to chlorophyllide and porphyrin contents were significantly higher in plants treated with melatonin, which may indicate that this indoleamine both retards chlorophyll breakdown and stimulates its de novo synthesis during extended stress. We concluded that melatonin added into the seeds enhances the ability of pea seedlings to accelerate chlorophyll breakdown and its de novo synthesis before stress appeared and for several hours after, while during prolonged PQ incubation melatonin delays chlorophyll degradation.

Melatonin Application to Pisum sativum L. Seeds Positively Influences the Function of the Photosynthetic Apparatus in Growing Seedlings during Paraquat-Induced Oxidative Stress.

Melatonin, due to its pleiotropic effects plays an important role improving tolerance to stresses. Plants increase endogenous melatonin synthesis when faced with harsh environments as well as exogenously applied melatonin limits stress injuries. Presented work demonstrated that single melatonin application into the seeds during pre-sowing priming improved oxidative stress tolerance of growing seedlings exposed to paraquat (PQ). PQ is a powerful herbicide which blocks the process of photosynthesis under light conditions due to free radicals excess production, when O2 is rapidly converted to [Formula: see text] and subsequently to other reactive oxygen species. The parameters of chlorophyll fluorescence [Fv/Fm, Fv/Fo, Rfd, ΦPSII, qP, and non-photochemical quenching (NPQ)] in all variants of pea leaves (derived from control non-treated seeds - C, and those hydroprimed with water - H, and hydroprimed with melatonin water solution 50 or 200 µM - H-MEL50 and H-MEL200, respectively) were analyzed as a tool for photosynthetic efficacy testing. Moreover stability of the photosynthetic pigments (chlorophylls a, b, and carotenoids) was also monitored under oxidative stress conditions. The results suggest that melatonin applied into the seed significantly enhances oxidative stress tolerance in growing seedlings. This beneficial effect was reflected in reduced accumulation of [Formula: see text] in leaf tissues, preservation of photosynthetic pigments, improved functioning of the photosynthetic apparatus and higher water content in the tissues during PQ-mediated stress. Our findings provide evidence for the physiological role of this molecule and serve as a platform for its possible applications in agricultural or related areas of research.

Melatonin restricts Pb-induced PCD by enhancing BI-1 expression in tobacco suspension cells.

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.

Biostimulators: A New Trend towards Solving an Old Problem.

Stresses provoked by adverse living conditions are inherent to a changing environment (climate change and anthropogenic influence) and they are basic factors that limit plant development and yields. Agriculture always struggled with this problem. The survey of non-toxic, natural, active substances useful in protection, and stimulation of plants growing under suboptimal and even harmful conditions, as well as searching for the most effective methods for their application, will direct our activities toward sustainable development and harmony with nature. It seems highly probable that boosting natural plant defense strategies by applying biostimulators will help to solve an old problem of poor yield in plant cultivation, by provoking their better growth and development even under suboptimal environmental conditions. This work is a concise review of such substances and methods of their application to plants.

Exogenous Melatonin Improves Antioxidant Defense in Cucumber Seeds (Cucumis sativus L.) Germinated under Chilling Stress.

The relationship between exogenous melatonin applied into cucumber seeds during osmopriming and modifications of their antioxidant defense was studied. Accumulation of hydrogen peroxide, antioxidant enzyme activities and glutathione pool were investigated in embryonic axes isolated from the control, osmoprimed, and osmoprimed with melatonin seeds. Germinating cucumber seeds are very sensitive to chilling. Temperature 10°C causes oxidative stress in young seedlings. Seed pre-treatment with melatonin seemed to limit H2O2 accumulation during germination under optimal condition as well as during chilling stress and recovery period. Melatonin affected superoxide dismutase (SOD) activity and its isoforms during stress and recovery period but did not influence CAT and POX activities. Thus it is possible that in cucumber this indoleamine could act mostly as a direct H2O2 scavenger, but superoxide anion combat via SOD stimulation. The GSH/GSSG ratio is considered as an indirect determinant of oxidative stress. When the cells are exposed to oxidative stress GSSG is accumulated and the ratio of GSH to GSSG decreases. In our research pre-sowing melatonin application into the cucumber seeds caused high beneficial value of GSH/GSSG ratio that could be helpful for stress countering. Glutathione reductase (GSSG-R) activity in the axes isolated from these seeds was two fold higher than in those isolated from the control and from the osmoprimed without melatonin ones. Additional isoforms of GSSG-R in melatonin treated seeds were also observed. It explains high and effective GSH pool restoration in the seeds pre-treated with melatonin. We confirmed that melatonin could protect cucumber seeds and young seedlings against oxidative stress directly and indirectly detoxifying ROS, thereby plants grown better even in harmful environmental conditions. This work is the first that investigated on plant in vivo model and documented melatonin influence on redox state during seed germination. This way we try to fill lack of information about melatonin-regulated pathways involved in antioxidant strategy of plant defense.

Exogenous melatonin improves corn (Zea mays L.) embryo proteome in seeds subjected to chilling stress.

Melatonin (MEL; N-acetyl-5-methoxytryptamine) plays an important role in plant stress defense. Various plant species rich in this indoleamine have shown a higher capacity for stress tolerance. Moreover, it has great potential for plant biostimulation, is biodegradable and non-toxic for the environment. All this indicates that our concept of seed enrichment with exogenous MEL is justified. This work concerns the effects of corn (Zea mays L.) seed pre-sowing treatments supplemented with MEL. Non-treated seeds (nt), and those hydroprimed with water (H) or with MEL solutions 50 and 500 µM (HMel50, HMel500) were compared. Positive effects of seed priming are particularly apparent during germination under suboptimal conditions. The impact of MEL applied by priming on seed protein profiles during imbibition/germination at low temperature has not been investigated to date. In order to identify changes in the corn seed proteome after applying hydropriming techniques, purified protein extracts of chilling stressed seed embryos (14 days, 5°C) were separated by two-dimensional electrophoresis. Then proteome maps were graphically and statistically compared and selected protein spots were qualitatively analyzed using mass spectrometry techniques and identified. This study aimed to analyze the priming-induced changes in maize embryo proteome and at identifying priming-associated and MEL-associated proteins in maize seeds subjected to chilling. We attempt to explain how MEL expands plant capacity for stress tolerance.

Red cabbage anthocyanins as inhibitors of lipopolysaccharide-induced oxidative stress in blood platelets.

LPS is a Gram-negative bacteria endotoxin, which is an important pro-inflammatory agent. Blood platelets take part both in inflammatory processes and in pathogenesis of septic shock following accumulation of LPS. As a platelet agonist LPS causes the intraplatelet overproduction of ROS/RNS that are responsible for adverse modifications in the structure of platelet compounds being associated with a development of platelet-dependent diseases. Existing evidence suggests that anthocyanins (ATH) are able to protect the circulatory system. The antioxidative properties of ATH are believed to be mainly responsible for their positive health effects. The main goal of the present in vitro study was to investigate the potential protective properties of red cabbage ATH against oxidative damage induced by LPS in blood platelets. Exposure of platelets to LPS resulted in carbonyl group increase, 3-nitrotyrosine formation, lipid peroxidation and O2(•-) generation. We have shown that ATH extract effectively decreased oxidative stress induced by LPSs. The in silico analysis demonstrated that both cyanin and LPS were located at the same region of human TLR4-MD-2 complex. Our findings suggest that there could be two-way ATH platelet protection mechanism, by their antioxidant properties and directly by binding with TLRs.

Melatonin applied to cucumber (Cucumis sativus L.) seeds improves germination during chilling stress.

The relationship between germination and melatonin applied during osmo- and hydropriming was studied in cucumber seeds. The proportion of nuclei with different DNA contents, the mean ploidy and the (2C + 4C = 8C)/2C ratio in unprimed and primed, dry and imbibed at 10 degrees C seeds were established by flow cytometry. Thiobarbituric acid reactive substances and protein oxidation were also estimated. Melatonin and indole-3-acetic acid (IAA) concentrations in the seeds were determined using high-performance liquid chromatography with electrochemical detection. Being sensitive to chilling stress, seeds that germinated well (99%) at 25 degrees C showed only 30% germination at 15 degrees C, and almost no germination (4%) at 10 degrees C. Hydropriming in water improved seed germination to 50-60% at 15 degrees C and the addition of melatonin (25-100 M) also increased the rate of germination. Osmopriming in polyethylene glycol increased germination at 15 degrees C to 78%, and 98% when combined with 50 M melatonin. Osmoprimed seeds germinated even at 10 degrees C and reached 43%, and 83% when 50 M melatonin was applied. None of the treatments induced DNA synthesis, although during the first 24 hr of imbibition at 10 degrees C the mean ploidy and the (2C + 4C = 8C)/2C ratio increased, which is indicative of the advanced Phase II of germination. Hydro- and osmopriming slightly decreased IAA content in the seeds in most of the cases; only hydropriming with 100 and 500 M melatonin increased it. Melatonin protected membrane structure against peroxidation during chilling, but excessive melatonin levels in cucumber seeds (approximately 4 microg/g fresh weight) provoked oxidative changes in proteins. There is still lack of information explained clearly the role of melatonin in plant physiology. This molecule acts multidirectionally and usually is alliged to other compounds.

Red cabbage anthocyanin extract alleviates copper-induced cytological disturbances in plant meristematic tissue and human lymphocytes.

Red cabbage is a source of health beneficial substances with antioxidant and antigenotoxic properties. HPLC analysis specifying the content of the investigated extract indicated that mainly anthocyanins (ATH) were responsible for its abilities. Cytological research was conducted with two experimental models: plant tissues--meristematic cells of Vicia faba, and animal tissue elements--human lymphocytes. Positive influence of ATH extract on mitotic activity of Vicia cells exposed to Cu(2+) stress, and inhibitory effect of ATH on cytotoxic actions of Cu(2+) on lymphocytes were demonstrated. In all experimental series with ATH application in combinations with Cu(2+), mitotic index (MI) were higher than those obtained for only Cu(2+) stressed tissues. Preincubation in ATH before Cu(2+) stress had the best effect. Similarly, after ATH applications in all tested series decrease in frequency of micronuclei (MN) appearance was noticed in comparison with only Cu(2+) stressed material. In the case of Vicia cells ATH acted effectively even applied after Cu(2+) stress. It suggests that this ATH mixture not only prevents and limits but also heals the cytological injury caused by Cu(2+) stress.

Presowing seed treatment with melatonin protects red cabbage seedlings against toxic copper ion concentrations.

One of the targets of modern plant physiology is to identify tools for improving seed germination and plant growth under unfavorable environmental conditions. Seeds of Brassica oleracea rubrum were pretreated with melatonin at concentrations: 1, 10, and 100 microM using a hydropriming method. Air-dried seeds of each experimental variants that were nonpretreated (control), hydroprimed (H) or hydroprimed with melatonin (HM1, HM10, and HM100) were germinated in darkness for 3 days at 25 degrees C. Young seedlings were then transferred to the light and grown for an additional 5 days. Both germination and growth tests were performed in water and in CuSO(4) water solutions in concentrations of 0.5 and 1 mM. H, HM1 and HM10 improved seed germination both in water and in the presence of Cu(2+). One or 10 microM melatonin eliminated the inhibitory effect of the 0.5 mM metal concentration on the fresh weight of seedlings. HM100 had a negative effect; thus seed germination was lower and seedlings had poor establishment. The toxic effect of Cu(2+) manifested by membrane peroxidation and DNA endoreplication blocking in the seedlings grown from nontreated (control) and H seeds was not observed in the seedlings grown from HM1 and HM10 seeds; in contrast, HM100 enhanced the toxic effect of Cu(2+).

Antioxidant enzymes and isoflavonoids in chilled soybean (Glycine max (L.) Merr.) seedlings.

Changes of activity antioxidant enzymes and of levels of isoflavonoids were studied in the roots and hypocotyls of the etiolated soybean (Glycine max (L.) Merr. var. Essor) seedlings, submitted to cold. Prolonged exposure to 1 degrees C inhibited hypocotyl and root elongation and limited their growth after seedlings were transferred to 25 degrees C. Roots were more sensitive to chilling than hypocotyls. At 1 degrees C a gradual increase in MDA concentration in roots but not in hypocotyls was observed. An increase in catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) activity in hypocotyls was observed both at 1 degrees C and after transfer of plants to 25 degrees C. In roots, CAT activity increased after 4 days of chilling, while SOD activity only after rewarming. L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity decreased in roots of chilled seedlings, but did not change in hypocotyls until activity increased after transfer to 25 degrees C. The content of genistein and daidzein increased after 24 h of treatment by low temperature and then decreased with prolonged chilling in hypocotyls and remained high in roots. However, it should be noted that genistin level (genistein glucoside) in chilled hypocotyls is 10 times higher than in roots, despite falling tendency. The role of antioxidant enzymes and isoflavonoids in preventing chilling injury in hypocotyls and roots of soybean seedlings is discussed.