Małgorzata M. Posmyk

Antioxidant enzymes activity and phenolic compounds content in red cabbage seedlings exposed to copper stress.

The phenolics: anthocyanin (ATH), sinapoyl esters and activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POX), ascorbate peroxidase (APX), glutathione peroxidase (GPX) and glutathione reductase (GR), in red cabbage seedlings subjected to Cu2+ stress were investigated. Cu2+ at low doses (0.5 mM), increased the levels of ATH and sinapoyl derivatives in red cabbage. High Cu2+ concentration (2.5 mM) provoked oxidative stress and enhanced thiobarbituric acid reactive substances (TBARS) content in tissues. A lower level of TBARS was correlated with high ATH content. It seems that synthesis of these isoflavonoids is an effective strategy against reactive oxygen species (ROS). The analysis of the antioxidant enzymes activity suggested that peroxidases were the most active enzymes in red cabbage seedlings exposed to Cu2+ stress. It could results from the fact that phenolic compounds (PhC), which could be also substrates for different peroxidases, were the first line of defence against metal stress.

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

Abstract:  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°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°C showed only 30% germination at 15°C, and almost no germination (4%) at 10°C. Hydropriming in water improved seed germination to 50–60% at 15°C and the addition of melatonin (25–100 m) also increased the rate of germination. Osmopriming in polyethylene glycol increased germination at 15°C to 78%, and 98% when combined with 50 m melatonin. Osmoprimed seeds germinated even at 10°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°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 (∼4 μg/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.

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

Abstract:  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 μm 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°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 CuSO4 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 Cu2+. One or 10 μm 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 Cu2+ 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 Cu2+.

Melatonin in plants

A widespread occurrence of melatonin (MEL) in plant kingdom has been reported. MEL is a highly conserved molecule occurring in evolutionary distant organisms. Its role in plants seems to be similar to that in animals. Although MEL function in plants is not well known, yet a hypothesis can be put forward that it probably functions as a night signal, coordinating responses to diurnal and photoperiodic environmental cues. It has also been suggested that MEL is an independent plant growth regulator, probably its action is analogous to IAA and it may mediate the actions of other plant growth regulators. Due to its antioxidant properties MEL may also stabilize cell red-ox status and protect them against reactive oxygen species (ROS) and other harmful environmental influence.

Constitutive elevated accumulation of phenylpropanoids in soybean roots at low temperature

Abstract Changes in phenolic acid and isoflavonoid contents in soybean (Glycine max. (L.) Merr) roots after 24-h of low temperature cultivation were investigated. The level of total phenolic acids (represented by the sum of derivatives of benzoic and cinnamic acids) and genistein, daidzein and genistin (glucosyl conjugate of genistein) increased after 24 h of exposure to low temperature. In chill-treated roots the highest increase in genistin was observed. Application of 2-aminoindan-2-phenylphoshonic acid (AIP), a competitive inhibitor of l -phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) resulted in an increased level of endogenous free phenylalanine. An increase in the content of free phenylalanine is regarded as an indication for the inhibition of PAL in vivo. Chilling with AIP decreased valine level. AIP applied in combination with chilling decreased phenolic acids but did not effect the isoflavonoids content. These findings may indicate that the increase in the phenolics level comes from the pre-formed conjugates of phenylpropanoids.

Melatonin, an underestimated natural substance with great potential for agricultural application

Abstract Melatonin (MEL) was thought to be only a neurotransmitter found in vertebrates until its detection in other organisms including plants. Although the number of publications on MEL function in plants is expanding, the knowledge of this subject is still insufficient. Among many functions which MEL performs in plants, its role as an antioxidant and a growth promoter is most supported by experimental evidence. This compound is an independent plant growth regulator and it may mediate the activities of other plant growth regulators. Due to its antioxidant properties MEL may also stabilize cell redox status and protect tissues against reactive oxygen and nitrogen species which accumulated under stressful environment. Some researchers propose that MEL could be used to improve the phytoremediation efficiency of plants against different pollutants. In this paper we show that exogenous MEL applied into the seeds could be a good biostimulator improving not only seed germination, seedling/plant growth but also crop production especially under stress conditions. We also believe that this compound can increase food quality (the aspect of functional food) and may improve human health. Since MEL is inexpensive and safe for animals and humans its application as a biostimulator could be a good, feasible and cost-effective method useful in agriculture.

The impact of copper ions on growth, lipid peroxidation, and phenolic compound accumulation and localization in lentil (Lens culinaris Medic.) seedlings

Changes in phenolics (PhC) measured as UV-absorbing compounds (UVAC) and their localization as well as growth, lipid peroxidation (TBARS level), H(2)O(2) and Cu accumulation and other ions content in roots of two lentil cultivars (cv. Krak and cv. Tina) contrasting in Cu sensitivity were examined. The aim of this study was to reveal the importance of PhC in Cu tolerance. During Cu treatment, inhibition of growth and increases in lipid peroxidation in roots of both cultivars were observed, but the effects were more pronounced in cv. Tina (more sensitive) than in cv. Krak (less sensitive). Cu at 0.5 mM caused higher Cu and H(2)O(2) accumulation, but lower K(+) content and UVAC levels in the root tips of cv. Tina. Opposite changes were recorded in cv. Krak. Fluorescence microscopic analyses confirmed greater PhC accumulation in cv. Krak (less sensitive) than in cv. Tina (more sensitive) after Cu treatment and showed that these compounds accumulated particularly in vacuoles and the cell wall. Taken together, these results show that, in spite of the high concentration of Cu-stimulated PhC accumulation in cv. Krak, it was not sufficient to counteract the amount of ROS generated by the metal. The role of PhC in different reactions to Cu stress in lentil roots is discussed.

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.

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 O2•– 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 O2•– 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.

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.