Marija Vidović

Zinc-induced oxidative stress in Verbascum thapsus is caused by an accumulation of reactive oxygen species and quinhydrone in the cell wall

Oxidative stress is one aspect of metal toxicity. Zinc, although unable to perform univalent oxido-reduction reactions, can induce the oxidative damage of cellular components and alter antioxidative systems. Verbascum thapsus L. plants that were grown hydroponically were exposed to 1 and 5 mM Zn²+. Reactive oxygen species (ROS) accumulation was demonstrated by the fluorescent probe H₂ DCFDA and EPR measurements. The extent of zinc-induced oxidative damage was assessed by measuring the level of protein carbonylation. Activities and isoform profiles of some antioxidant enzymes and the changes in ascorbate and total phenolic contents of leaves and roots were determined. Stunted growth because of zinc accumulation, preferentially in the roots, was accompanied by H₂O₂ production in the leaf and root apoplasts. Increased EPR signals of the endogenous oxidant quinhydrone, •CH₃ and •OH, were found in the cell walls of zinc-treated plants. The activities of the antioxidative enzymes ascorbate peroxidase (APX) (EC 1.11.1.11), soluble superoxide dismutase (SOD) (EC 1.15.1.1), peroxidase (POD), (EC 1.11.1.7) and monodehydroascorbate reductase (EC 1.6.5.4) were increased; those of glutathione reductase (EC 1.6.4.2), dehydroascorbate reductase (EC 1.8.5.1) and ascorbate oxidase (AAO) (EC 1.10.3.3) were decreased with zinc treatment. Zinc induced a cell-wall-bound SOD isoform in both organs. Leaves accumulated more ascorbate and phenolics in comparison to roots. We propose a mechanism for zinc-promoted oxidative stress in V. thapsus L. through the generation of charge transfer complexes and quinhydrone because of phenoxyl radical stabilisation by Zn²+ in the cell wall. Our results suggest that the SOD and APX responses are mediated by ROS accumulation in the apoplast. The importance of the POD/Phe/AA (ascorbic acid) scavenging system in the apoplast is also discussed.

Ultraviolet-B component of sunlight stimulates photosynthesis and flavonoid accumulation in variegated Plectranthus coleoides leaves depending on background light.

We used variegated Plectranthus coleoides as a model plant with the aim of clarifying whether the effects of realistic ultraviolet-B (UV-B) doses on phenolic metabolism in leaves are mediated by photosynthesis. Plants were exposed to UV-B radiation (0.90 W m(-2) ) combined with two photosynthetically active radiation (PAR) intensities [395 and 1350 μmol m(-2)  s(-1) , low light (LL) and high light (HL)] for 9 d in sun simulators. Our study indicates that UV-B component of sunlight stimulates CO2 assimilation and stomatal conductance, depending on background light. UV-B-specific induction of apigenin and cyanidin glycosides was observed in both green and white tissues. However, all the other phenolic subclasses were up to four times more abundant in green leaf tissue. Caffeic and rosmarinic acids, catechin and epicatechin, which are endogenous peroxidase substrates, were depleted at HL in green tissue. This was correlated with increased peroxidase and ascorbate peroxidase activities and increased ascorbate content. The UV-B supplement to HL attenuated antioxidative metabolism and partly recovered the phenolic pool indicating stimulation of the phenylpropanoid pathway. In summary, we propose that ortho-dihydroxy phenolics are involved in antioxidative defence in chlorophyllous tissue upon light excess, while apigenin and cyanidin in white tissue have preferentially UV-screening function.

UV responses of Lolium perenne raised along a latitudinal gradient across Europe: a filtration study

Lolium perenne (cv. AberDart) was grown at 14 locations along a latitudinal gradient across Europe (37-68°N) to study the impact of ultraviolet radiation (UV) and climate on aboveground growth and foliar UV-B absorbing compounds. At each location, plants were grown outdoors for 5 weeks in a replicated UV-B filtration experiment consisting of open, UV-B transparent (cellulose diacetate) and UV-B opaque (polyester) environments. Fourier transform-infrared spectroscopy was used to compare plant metabolite profiles in relation to treatment and location. UV radiation and climatic parameters were determined for each location from online sources and the data were assessed using a combination of anova and multiple regression analyses. Most of the variation in growth between the locations was attributable to the combination of climatic parameters, with minimum temperature identified as an important growth constraint. However, no single environmental parameter could consistently account for the variability in plant growth. Concentrations of foliar UV-B absorbing compounds showed a positive trend with solar UV across the latitudinal gradient; however, this relationship was not consistent in all treatments. The most striking experimental outcome from this study was the effect of presence or absence of filtration frames on UV-absorbing compounds. Overall, the study demonstrates the value of an European approach in studying the impacts of natural UV across a large latitudinal gradient. We have shown the feasibility of coordinated UV filtration at multiple sites but have also highlighted the need for open controls and careful interpretation of plant responses.

Characterisation of antioxidants in photosynthetic and non‐photosynthetic leaf tissues of variegated Pelargonium zonale plants

Hydrogen peroxide is an important signalling molecule, involved in regulation of numerous metabolic processes in plants. The most important sources of H2 O2 in photosynthetically active cells are chloroplasts and peroxisomes. Here we employed variegated Pelargonium zonale to characterise and compare enzymatic and non-enzymatic components of the antioxidative system in autotrophic and heterotrophic leaf tissues at (sub)cellular level under optimal growth conditions. The results revealed that both leaf tissues had specific strategies to regulate H2 O2 levels. In photosynthetic cells, the redox regulatory system was based on ascorbate, and on the activities of thylakoid-bound ascorbate peroxidase (tAPX) and catalase. In this leaf tissue, ascorbate was predominantly localised in the nucleus, peroxisomes, plastids and mitochondria. On the other hand, non-photosynthetic cells contained higher glutathione content, mostly located in mitochondria. The enzymatic antioxidative system in non-photosynthetic cells relied on the ascorbate-glutathione cycle and both Mn and Cu/Zn superoxide dismutase. Interestingly, higher content of ascorbate and glutathione, and higher activities of APX in the cytosol of non-photosynthetic leaf cells compared to the photosynthetic ones, suggest the importance of this compartment in H2 O2 regulation. Together, these results imply different regulation of processes linked with H2 O2 signalling at subcellular level. Thus, we propose green-white variegated leaves as an excellent system for examination of redox signal transduction and redox communication between two cell types, autotrophic and heterotrophic, within the same organ.

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 (>1100μmolm-2s-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.

Evaluation of procedures for assessing anti- and pro-oxidants in plant samples

Plants as well as other aerobic organisms constantly produce reactive oxygen species (ROS). At regulated low concentrations ROS may serve as signal molecules, while in excessive amounts these may cause oxidative damage to biomolecules. Actual cellular concentrations are controlled by a network of various antioxidants, and acclimation to stress conditions is achieved by a dynamic balance of ROS production and neutralization. Accordingly, plant stress physiology studies generally include an array of methods testing the occurrence of ROS as well as evaluating antioxidant capacities. The aim of the present work is to provide an overview of these methods, with special emphasis on avoiding errors that can possibly lead to either inaccurate data or misinterpretations of otherwise correct measurements.

Ascorbate as a Key Player in Plant Abiotic Stress Response and Tolerance

During their lifespan, plants are frequently exposed to adverse environmental conditions such as high solar irradiance, drought, heat, chilling, salinity, metal excess, and nutrient deficiency. The effects of these factors on plants are often interrelated and usually result in a decreased capacity of carbon fixation in photosynthesis, disturbed redox homeostasis, and growth arrest. Under severe conditions, increased excitation pressure in the chloroplasts exceeds the antioxidative capacity of plant cells leading to oxidative damage of cellular constituents. Although the plant ascorbate (Asc) level varies depending on external factors, developmental stage, diurnal rhythm, and light, its redox status is related to redox homeostasis in the cell. In chloroplasts, peroxisomes, and cytosol, Asc has a key role in hydrogen peroxide (H2O2) scavenging via Asc peroxidase and is efficiently recycled via the ascorbate-glutathione (Asc–GSH) cycle and directly by monodehydroascorbate reductase activity. In apoplast and vacuoles, Asc is the main reductant of phenolic radicals generated under oxidative stress. Besides its antioxidative role, Asc has an important role in a complex and well-orchestrated plant response network to environmental stress, performing multiple tasks in redox signalling, regulation of enzymatic activities, modulation of gene expression, biosynthesis of phytohormones, and growth regulation. The content of Asc and its redox state is tightly related to cellular compartments. Therefore, it is important to emphasize Asc cellular distribution, which has a great impact on reactive oxygen species regulation and signalling. Numerous studies on transgenic plants with altered endogenous Asc levels and redox status were done with the aim to influence plant growth and improve tolerance to various abiotic stressors. In this chapter, we discuss the current understanding of the involvement of Asc metabolism in abiotic stress response. Moreover, the improved resilience to stressors in transgenic plants with altered enzymes involved in Asc biosynthesis and recycling will be discussed.

Class III Peroxidases: Functions, Localization and Redox Regulation of Isoenzymes

Class III peroxidases (POXs; EC. 1.11.1.7), are secretory, multifunctional plant enzymes that catalyze the oxidation of a variety of substrates by hydrogen peroxide (H2O2). They show a remarkable diversity of isoenzymes, are encoded by a large number of paralogous genes, and are involved in a broad range of metabolic processes throughout plant growth and development. Peroxidases isoenzymes are located in the cell wall, apoplast and vacuole, and may be either soluble or ionically and covalently cell wall bound. They are involved in cell wall cross-linking and loosening, lignification and suberization, auxin catabolism and secondary metabolism. Due to their ability to control the levels of reactive oxygen species (ROS), POXs are efficient components of the antioxidative system induced in response to environmental stress, such as pathogen attack, metal excess, salinity, drought and high light intensity. In addition to the peroxidative function, POXs can catalyze H2O2 production in the oxidative cycle. Peroxidases are responsible either for cell elongation or cell wall stiffening, affecting carbon allocation, auxin level and redox homeostasis, which implicates their key role as being in the regulation of growth and defence under stress condition. This chapter will discuss novel insights into the functions of PODs with special emphasis on their localization, substrate specificity and the regulation of redox homeostasis.