An improved HPLC-DAD method for simultaneously measuring phenolics in the leaves of Tilia platyphyllos and Ailanthus altissima

Phenolic compounds are one of the most important groups of secondary metabolites in plants, with various physiological functions. Their diverse chemical structure and susceptibility to auto-oxidation, and their ability to act as both antioxidants and prooxidants in the presence of metal ions, are some of the main reasons why it is difficult to measure phenolic groups in plant tissues accurately. We present an optimized extraction and hydrolysis procedure which preserves the original chemical structure of phenolics. The presented HPLC method was improved to enable the simultaneous separation and quantification of 39 compounds from different phenolic subclasses (benzoic acids, hydroxycinnamic acids, flavones, flavonols, flavanones, flavanols, isoflavones, anthocyanidins; aglycones and glycosides). Recovery after extraction and complete hydrolysis of glycosides was more than 95% and 84%, respectively. The method was applied to the analysis and comparison of phenolic profiles in the leaves of two species, Ailanthus altissima (Mill.) Swingle and Tilia platyphyllos Scop. from an urban forest park and busy traffic area. The presence of cyanidin glycosides in A. altissima leaves was reported for the first time. Results indicated higher accumulation of phenolics, with two hydroxyl groups in the ortho- position, than flavonoids, with a monohydroxy substitution in the leaves of both species from a busy traffic area

Induction of peroxidase isoforms in the roots of two Verbascum thapsus L. populations is involved in adaptive responses to excess Zn2+ and Cu2+

To investigate metal specific responses of root class III peroxidases (POD, EC 1.11.1.7), two populations of Verbascum thapsus L. were exposed to excess Zn2+ or Cu2+ for three weeks in hydroponic culture. One population originating from an uncontaminated area (NMET) and one from an industrial disposal area for jarosite residues from zinc refining (MET) were chosen to test the capacity of V. thapsus to adapt to excess metal in the soil. Exposure to 60 μM Zn2+ led to increased levels of protein carbonyl groups only in the roots of NMET, which was accompanied by higher increase of POD activity and NADH-oxidase activity compared with MET plants. New anionic and cationic POD isoforms were induced in the roots of both populations in response to Zn2+ treatment, while IAA-oxidase activity decreased. On the other hand, root growth was more affected by Cu2+ than Zn2+ in both populations, which was correlated with increased auxin-oxidase (IAA-oxidase) activity. Cu2+ induced an increased activity of anionic POD isoforms in the roots of both populations, yet the ratio of NADH-oxidative to peroxidative POD activity remained higher in NMET than in MET plants. Overall results show differential effects of Zn2+ and Cu2+ on POD activity in the roots of V. thapsus L. In addition, higher tolerance to Zn2+ in MET plants than in NMET indicated that these plants have developed an adaptive mechanism to cope with Zn2+ excess

Induction of peroxidase isoforms in the roots of two Verbascum thapsus L. populations is involved in adaptive responses to excess Zn2+ and Cu2+

To investigate metal specific responses of root class III peroxidases (POD, EC 1.11.1.7), two populations of Verbascum thapsus L. were exposed to excess Zn2+ or Cu2+ for three weeks in hydroponic culture. One population originating from an uncontaminated area (NMET) and one from an industrial disposal area for jarosite residues from zinc refining (MET) were chosen to test the capacity of V. thapsus to adapt to excess metal in the soil. Exposure to 60 μM Zn2+ led to increased levels of protein carbonyl groups only in the roots of NMET, which was accompanied by higher increase of POD activity and NADH-oxidase activity compared with MET plants. New anionic and cationic POD isoforms were induced in the roots of both populations in response to Zn2+ treatment, while IAA-oxidase activity decreased. On the other hand, root growth was more affected by Cu2+ than Zn2+ in both populations, which was correlated with increased auxin-oxidase (IAA-oxidase) activity. Cu2+ induced an increased activity of anionic POD isoforms in the roots of both populations, yet the ratio of NADH-oxidative to peroxidative POD activity remained higher in NMET than in MET plants. Overall results show differential effects of Zn2+ and Cu2+ on POD activity in the roots of V. thapsus L. In addition, higher tolerance to Zn2+ in MET plants than in NMET indicated that these plants have developed an adaptive mechanism to cope with Zn2+ excess

Peroxidase, phenolics, and antioxidative capacity of common mullein (verbascum thapsus l.) Grown in a zinc excess

Common mullein (Verbascum thapsus L.) is the dominant plant species at a disposal site polluted with metal from the hydrometallurgical jarosite zinc production process. Seeds collected at the site were germinated and plants were grown hydroponically under controlled conditions in a excess of Zn. Induction of total soluble POD activity in the root occurred at 1, 5, and 10 mM Zn, indicating Zn accumulation within the root. Accumulation of Zn in leaves was not accompanied by changes in POD activity, but resulted in gradual increase of total antioxidative capacity, which could be partly attributed to accumulation of soluble phenolics. The role of the phenolics/POD system in defense of V thapsus against zinc is discussed

Genetic variability of verbascum populations from metal polluted and unpolluted sites

Mullein (Verbascum) plants have extensive distribution and can grow in variable environmental conditions. Seed was collected from mullein plants grown at 4 locations, two metals contaminated and two metal uncontaminated areas. Genetic variability of progeny was examined. Populations collected from unpolluted areas were genetically more similar than those collected from polluted areas as revealed by RAPD and SSR markers and UPGMA analysis. The results indicate that there is genetic differentiation between examined populations and therefore they represent suitable material for further investigation of plant adaptation mechanisms to increased metal content

Sun as a stressor and/or regulator of plant metabolism: responses to UV radiation and high light

In their natural environment, plants are constantly exposed to dynamic changes of solar radiation, which mainly consists of infrared (IR, >700 nm), photosynthetically active radiation (PAR, 400-700 nm) and minor portion of ultraviolet (UV) radiation (UV-B, 290-315 nm and UV-A, 315-400 nm). Sunlight is not only the primary source of energy in photosynthesis, it is also an important signal which regulates plant growth and development. During the period from the 1970s to 1990s, investigations on UV‐B effects on organisms were in the centre of attention due to alarming depletion of stratospheric ozone layer and increased UV‐B radiation reaching the Earth’s surface. UV-B radiation has been perceived only as a stressor. A decade later, new data obtained using realistic UV-B doses and realistic UV-B:UV-A:PAR ratio, clearly show that UV-B is very important environmental cue and regulator of plant metabolism, rather than a stressor. In the recent years, great progress has been made in understanding the mechanisms of light signals’ perception. However, the complications arise from the overlapping of the acclimative responses to UV‐B radiation and high PAR intensity, imposing cross‐tolerance to different components of solar radiation. Moreover, information on other constituents involved in the UV‐B response, such as reactive oxygen species in relation to their tissue- and subcellular-localization is scarce. Our latest findings using leaf variegation as a model with metabolically contrasting tissues show specific responses to UV-B radiation and high light in relation to antioxidative metabolism, photosynthesis, carbohydrate metabolism, and distribution of phenolics.Abstract: Serbian Biochemical Society Seventh Conference with international participation , Faculty of Chemistry, University of Belgrade 10.11.2017. Belgrade, Serbia “Biochemistry of Control in Life and Technology

Effects of mixed saline and alkaline stress on the morphology and anatomy of pisum sativum l.: the role of peroxidase and ascorbate oxidase in growth regulation

The effects of hyperalkaline, thermo-mineral water from Slatina on the morphology and anatomy of pea plants (Pisum sativum L.), were examined after eleven days of treatment with a mixture of tap water and Slatina water in 3:1 (T1) and 1:1 ratios (T2). Complete growth arrest of seedlings was observed in the Slatina water (T3). The alkalinity of external media was recovered to pH 8 within four days only in T1 and T2. Analysis of morphological parameters (the length of the main root, root application zone, number of lateral roots) indicated that the thermo-mineral water either promoted (T1) or inhibited (T2) the formation of lateral roots and plant growth. Comparative histological and anatomical analyses showed that inhibition of lateral roots was accompanied by an increase in the xylem and phloem. These changes in root morphology were accompanied by an increase in the activity of superoxide dismutase (SOD: E.C. 1.15.1.1) and peroxidase (POD: E.C 1.1.1.17) in the soluble fraction, whereas the activities of ascorbate oxidase (AAO: E.C. 1.10.3.3) bound to the cell wall and ionic POD decreased. The lower ratio of Slatina water in the hydroponic solution contributed to a more developed mesophyll with significantly higher AAO activity in the leaves and the induction of ionic POD isoforms. Besides alkalinity and excess NaCl, we suggest that a specific combination of metals (e.g. Ca and Mg) might be responsible for subtle changes in the cell area and xylem development, leading to dramatic changes in root anatomy

Antioxidative response in variegated Pelargonium zonale leaves and generation of extracellular H2O2 in (peri)vascular tissue induced by sunlight and paraquat

In this study we exposed variegated leaves of Pelargonium zonale to strong sunlight ( gt 1100 mu mol m(-2) s(-1) of photosynthetically active radiation) with and without paraquat (Pq), with the aim to elucidate the mechanisms of H2O2 regulation in green and white tissues with respect to the photosynthetically-dependent generation of reactive oxygen species (ROS). Sunlight induced marked accumulation of H2O2 in the apoplast of vascular and (peri)vascular tissues only in green sectors. This effect was enhanced by the addition of Pq. In the presence of diphenyl iodide, an NADPH oxidase inhibitor, H2O2 accumulation was abolished. Distinct light-induced responses were observed: in photosynthetic cells, sunlight rapidly provoked ascorbate (Asc) biosynthesis and an increase of glutathione reductase (GR) and catalase activities, while in non-photosynthetic cells, early up-regulation of soluble ascorbate peroxidase, dehydroascorbate reductase (DHAR) and GR activities was observed. Paraquat addition stimulated DHAR and GR activities in green sectors, while in white sectors activities of monodehydroascorbate reductase, DHAR and class III peroxidases, as well as Asc content rapidly increased. Differential antioxidative responses in the two tissues in the frame of their contrasting metabolisms, and the possible role of (peri)vascular H2O2 in signaling were discussed.This is the peer reviewed version of the paper: Vidović, M., Morina, F., Prokić, L., Milić-Komić, S., Živanović, B., & Jovanović, S. V. (2016). Antioxidative response in variegated Pelargonium zonale leaves and generation of extracellular H2O2 in (peri)vascular tissue induced by sunlight and paraquat. Journal of Plant Physiology, 206, 25–39. [https://doi.org/10.1016/j.jplph.2016.07.017

Contents of phenolics and carotenoids in tomato grown under polytunnels with different UV-transmission rates

Tomato (Solanum lycopersicum L.) is among the economically most important vegetables in Europe, valued for its bioactive properties due to significant contents of vitamins, carotenoids, and phenolic compounds. In this study, the tomato cultivar Big Beef F1 was grown in the open field (OF) and under polytunnels in central Serbia during 3 years. Polytunnels were covered with two foils (both with 57% reduced photosynthetic active radiation, PAR) differing in UV-A and UV-B transmittance. The aim of our work was to determine the influence of light conditions on accumulation of phytonutrients (carotenoids and phenolics) in the peel and flesh of ripe tomato fruits. The amount of effective antioxidants, caffeic acid, and quercetin (phenolics with ortho-dihydroxy substitution) in the peel was the highest in tomato fruits grown in the OF (maximal PAR and UV-A and UV-B radiation). Moreover, the content of leaf epidermal flavonoids was the highest in the OF. The content of lycopene and beta-carotene in the flesh of tomato fruit was higher under the polytunnel with higher UV-transmittance. Our results showed that selection of the right light conditions (quality and intensity) for tomato production has a significant effect on the accumulation of beneficial phenolics and carotenoids