Katarzyna Szafrańska

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.

Changes in the nature of phenolic deposits after re-warming as a result of melatonin pre-sowing treatment of Vigna radiata seeds

Changes in phenolics (PhC - phenolic compounds) measured as UV-absorbing compounds (UVAC) and their localization as well as growth, lipid peroxidation (TBARS level) and proline (Pro) level in three-day-old roots of seedlings (T(o) stage) obtained from hydroprimed (H) and hydroprimed with melatonin (H-MEL) seeds after 2 days of chilling (5°C) and 2 days of re-warming were examined. H and H-MEL resulted in inhibition of root growth under optimal conditions, but after re-warming, a positive effect of MEL was noted. The results also showed a positive MEL impact on TBARS level already after chilling and especially after re-warming. Exposure of Vigna radiata seedlings to chilling caused a significant increase in Pro level, especially in H-MEL roots, but after re-warming it drastically decreased. Under chilling stress, accumulation of UVAC also decreased. However, after re-warming it returned to the level observed in the roots grown constantly at 25°C. Even if after re-warming of V. radiata seedlings only slight accumulation of total PhC was observed, phenolic deposits accumulating in the vacuoles of H-MEL roots were completely different from those in the vacuoles of the control and H roots. H-MEL application to the seeds resulted in a significant increase in small granular composite materials, while in the control and H roots, large oval deposits prevailed. Taken together, it is probable that all of these differences were connected with positive effects of MEL on chilled V. radiata seedlings after re-warming.

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.

Ameliorative effect of melatonin on meristematic cells of chilled and re-warmed Vigna radiata roots

Changes in ultrastructure of meristematic cells as well as growth and lipid peroxidation in roots of 3-d-old seedlings obtained from control (C), hydroprimed (H) and hydroprimed with melatonin (H-MEL) seeds after 2 d of incubation at 25 or 5 °C and 2 d of re-warming after chilling were investigated. Under 25 °C hydropriming (H and H-MEL) inhibited root growth, but after chilling and re-warming a positive MEL effect on root elongation was observed. The results show decreased lipid peroxidation in H-MEL roots already after chilling, but the significant extent of MEL impact was seen after re-warming. Similarly at the ultrastructural level, the protective effect of MEL at chilling was also visible, especially in plastids, and this effect maintained also after re-warming.

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.

Involvement of melatonin applied to Vigna radiata L. seeds in plant response to chilling stress

The impact of melatonin (50 µM L−1) applied to Vigna radiata seeds by hydro-priming on phenolic content, L-phenylalanine ammonialyase (PAL) activity, MEL level, antioxidant properties of ethanol extracts as well as electrolyte leakage from chilled and re-warmed Vigna radiata roots of seedlings were examined. Seedlings obtained from non-primed seeds, hydro-primed and hydro-primed with MEL were investigated after 2 days of chilling and 2 days of re-warming. At 25°C, the level of MEL in roots derived from seeds hydro-primed with MEL was 7-fold higher than in roots derived from non-primed seeds. However, the content of MEL significantly decreased in all variants investigated after re-warming, in contrast to PAL activity and phenolic levels, which reached the highest values. The antioxidant capacity of ethanol extracts from chilled and re-warmed roots, determined by ABTS+· assay, was correlated with phenolic content while the reducing ability of these extracts, determined by the FRAP method, correlated with PAL activity. However, both were the highest in rewarmed roots with applied MEL, which was accompanied by a significant decline in electrolyte leakage. Taken together, results may indicate that MEL can play a positive role in plant acclimation to stressful conditions and activation of phenolic pathway by this molecule.

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 melatonins 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.

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.

Influence of copper ions on the plant material obtained from the anther culture of carrot (Daucus carota L.).

Influence of Copper Ions on the Plant Material Obtained from the Anther Culture of Carrot (Daucus carota L.) The influence of copper ions on the regeneration of carrot (Daucus carota L.) androgenic embryos, accumulation of copper in rosettes, soluble esterbound phenolic acids and some parameters of oxidative stress were investigated. Two carrots: cv. Feria and 1014 breeding line were subjected to 1 μM, 10 μM and 100 μM Cu stress for 16 and 24 weeks. Under this stress, better growth, lower lipid peroxidation (TBARS level) and higher phenolic acid contents were observed in the cv. Feria. The rosettes of 1014 line accumulated less copper and produced smaller amount of TBARS after 24 weeks of incubation than after 16 weeks. Chlorogenic and caffeic acids were the main phenolic acids in both cultures. In the Feria rosettes the application of 10 μM Cu caused relatively high level of chlorogenic acid combined with low accumulation of copper in the tissues and unchanged levels of TBARS after both 16 and 24 weeks of incubation. On the other hand, despite the dose-dependent decline of chlorogenic acid in the rosettes of 1014 line, decrease in TBARS content was also observed after 24 weeks. The obtained results might suggest that the Feria carrot culture was able to develop more effective protection system/strategy against Cu excess in comparison to the 1014 line. Wpływ Jonów Miedzi na Materiał Roślinny Uzyskany z Zarodków Androgenetycznych Marchwi (Daucus carota L.) W niniejszej pracy zbadano wpływ różnych stężeń jonów miedzi na zdolności regeneracyjne zarodków androgenetycznych marchwi (Daucus carota L.), akumulację tego pierwiastka w rozetach, utlenianie lipidów błon komórkowych mierzonych stężeniem TBARS (substancje reagujące z kwasem tiobarbiturowym) oraz rozpuszczalnych estrów kwasów fenolowych. Do badań zastosowano pożywki B5 wg Gamborga i in. (1968), które zawierały 0,1 (kontrola); 1; 10 i 100 μM CuSO4 x 5 H2O. Materiał roślinny pasażowano na świeże pożywki i analizowano po 16 i 24 tygodniach. Odmiana Feria charakteryzowała się lepszym wzrostem, niższym stopniem peroksydacji lipidów błon oraz wyższą zawartością kwasów fenolowych w obecności Cu niż linia 1014. Po 24 tygodniach inkubacji rozety linii 1014 akumulowały mniej miedzi i TBARS niż po 16 tygodniach. Głównymi kwasami fenolowymi, zarówno w kulturze odmiany Feria jak i linii 1014, były kwas chlorogenowy i kawowy. W przypadku Ferii, rosnącej w obecności 10 μM Cu, stwierdzono relatywnie wysoką zawartość kwasu chlorogenowego a niską miedzi w tkankach oraz niezmieniony poziom TBARS zarówno po 16 jak i 24 tygodniach hodowli. Natomiast, w rozetach linii 1014 wraz ze wzrostem stężenia Cu w podłożu zaobserwowano spadek zawartości kwasu chlorogenowego oraz TBARS po 24 tygodniach inkubacji. Uzyskane wyniki mogą sugerować, że kultury marchwi odmiany Feria posiadają lepszą strategię obrony przed nadmiarem miedzi niż linia hodowlana 1014.