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Silver nanoparticles (AgNPs) have been implemented in a wide range of commercial products, resulting in their unregulated release into aquatic as well as terrestrial systems. This raises concerns over their impending environmental effects. Once released into the environment, they are prone to various transformation processes that modify their reactivity. In order to increase AgNP stability, different stabilizing coatings are applied during their synthesis. However, coating agents determine particle size and shape and influence their solubility, reactivity, and overall stability as well as their behavior and transformations in the biological medium. In this review, we attempt to give an overview on how the employment of different stabilizing coatings can modulate AgNP-induced phytotoxicity with respect to growth, physiology, and gene and protein expression in terrestrial and aquatic plants and freshwater algae.

The harmful effects of silver nanoparticles (AgNPs) have been confirmed in many organisms, but the mechanism of their toxicity is not yet fully understood. In biological systems, AgNPs tend to aggregate and dissolve, so they are often stabilized by coatings that influence their physico-chemical properties. In this study, the effects of AgNPs with different coatings [polyvinylpyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB)] on oxidative stress appearance and proteome changes in tobacco (

Rising temperatures and pronounced drought are significantly affecting biodiversity worldwide and reducing yields and quality of Brassica crops. To elucidate the mechanisms of tolerance, 33 kale accessions (B. oleracea var. acephala) were evaluated for individual (osmotic and elevated temperature stress) and combined stress (osmotic + temperature). Using root growth, biomass and proline content as reliable markers, accessions were evaluated for stress responses. Four representatives were selected for further investigation (photosynthetic performance, biochemical markers, sugar content, specialized metabolites, transcription level of transcription factors NAC, HSF, DREB and expression of heat shock proteins HSP70 and HSP90): very sensitive (392), moderately sensitive (395), tolerant (404) and most tolerant (411). Accessions more tolerant to stress conditions were characterized by higher basal content of proline, total sugars, glucosinolates and higher transcription of NAC and DREB. Under all stress conditions, 392 was characterized by a significant decrease in biomass, root growth, photosynthesis performance, fructan content, especially under osmotic and combined stress, a significant increase in HSF transcription and HSP accumulation under temperature stress and a significant decrease in NAC transcription under all stresses. The most tolerant accession under all applied stresses, 411 showed the least changes in all analyzed parameters compared with the other accessions.

Rising temperatures and pronounced drought are significantly affecting biodiversity worldwide and reducing yields and quality of Brassica crops. To elucidate the mechanisms of tolerance, 33 kale accessions (

Extensive commercialization of silver nanoparticles (AgNPs) raises the risk of their accumulation in the soil-plant system. Once released into the environment, AgNPs are prone to chemical transformations, which make it hard to determine whether their phytotoxic effects are purely NP-related or a consequence of released Ag+ ions. In this study the effects of 25, 50, 75, 100 and 150 μM AgNPs and AgNO3 on seed germination and early growth of tobacco (Nicotiana tabacum L.) seedlings were compared. Additionally, the effects on photosynthetic performance and pigment content were investigated. Germination rate and index values indicated delayed and slower germination in some AgNP treatments. Lower AgNP concentrations stimulated root growth, but induced a prominent reduction in fresh weight. By contrast, all AgNO3 concentrations inhibited root growth but only the higher ones decreased fresh weight. Obtained results imply that the observed AgNP toxicity could be ascribed to NP form and can be correlated with high AgNP stability in the solid medium. On the other hand, the majority of AgNP and AgNO3 treatments induced an increase in chlorophyll content that was accompanied by significantly lower values of relative electron transport rate and coefficient of photochemical quenching, implying an inhibition of the electron transport chain. A similar impact of AgNPs and AgNO3 on photosynthesis can be correlated with lower stability of AgNPs in a liquid medium, resulting in AgNP aggregation and dissolution of Ag+ ions.

Klimatske promjene ozbiljno utječu na biološku raznolikost i poljoprivrednu proizvodnju diljem svijeta. U nadolazećim godinama stres uzrokovan sušom i povišenom temperaturom postat će glavni problem u uzgoju mnogih poljoprivredno važnih biljaka, a odabir sorti tolerantnijih na sušu i povišenu temperaturu, te njihova šira uporaba u poljoprivrednoj proizvodnji mogli bi smanjiti rizike i gubitke koji se očekuju zbog nepovoljnih vremenskih uvjeta. Kako bi rasvijetlili molekularne mehanizme tolerancije raštike (B. oleracea var. acephala) na stres uzrokovan sušom i toplinom, 33 ekopopulacije iz Hrvatske i Bosne i Hercegovine bile su izložene spomenutim stresnim čimbenicima. Na temelju promjena u biomasi, prirasta korijena te sadržaja vode i prolina odabrana su dva tolerantnija i dva osjetljivija ekotipa za detaljnije analize. Nakon tretmana izmjerena je fotosintetska učinkovitost, te su analizirani markeri oksidacijskog stresa, šećeri, ukupni fenoli, flavonoidi i glukozinolati. Također, kvantificirana je ekspresija gena transkripcijskih faktora vezanih uz sušu/toplinski stres, te ekspresija proteina HSP. U osjetljivim ekopopulacijama stres je uzrokovao značajnije promjene mjerenih molekularnih biljega nego u tolerantnijim ekopopulacijama čiji je odgovor bio umjereniji

Laccase activity reduction in the chestnut blight fungus Cryphonectria parasitica usually accompanies the hypovirulence caused by the infection of fungus with Cryphonectria hypovirus 1 (CHV1). However, the different methods utilized for assessing this phenomenon has produced varied and often conflicting results. Furthermore, the majority of experimental setups included only one prototypic system, further confounding the results. Considering the diversity of fungal isolates, viral strains, and variability of their effects on the phytopathogenic process observed in nature, our goal was to ascertain if laccase activity variability is affected by (1) different C. parasitica isolates infected with several CHV1 strains, and (2) growth conditions. We have demonstrated that some CHV1 strains, contrary to previous assumptions, increase the activity of C. parasitica laccases. The specific fungal isolates used in the experiments and culture conditions also affected the results. Furthermore, we showed that two commonly used laccase substrates, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 2,4-dimethoxyphenol, cannot be used interchangeably in C. parasitica laccase activity measurements. Our results illustrate the importance of conducting this type of study in experimental systems and culture conditions that resemble natural conditions as much as possible to be able to infer the most relevant conclusions applicable to natural populations.

Widespread application of silver nanoparticles (AgNPs), due to their antibacterial and antifungal properties, increases their release into the environment and potential detrimental impact on living organisms. Plants may serve as a potential pathway for AgNPs bioaccumulation and a route into the food chain, hence investigation of AgNP phytotoxic effects are of particular importance. Since proteins are directly involved in stress response, studies of their abundance changes can help elucidate the mechanism of the AgNP-mediated phytotoxicity. In this study, we investigated proteomic changes in tobacco (Nicotiana tabacum) exposed to AgNPs and ionic silver (AgNO3). A high overlap of differently abundant proteins was found in root after exposure to both treatments, while in leaf, almost a half of the proteins exhibited different abundance level between treatments, indicating tissue-specific responses. Majority of the identified proteins were down-regulated in both tissues after exposure to either AgNPs or AgNO3; in roots, the most affected proteins were those involved in response to abiotic and biotic stimuli and oxidative stress, while in leaf, both treatments had the most prominent effect on photosynthesis-related proteins. However, since AgNPs induced higher suppression of protein abundance than AgNO3, we conclude that AgNP effects can, at least partially, be attributed to nanoparticle form.

The production and utilization of nanoparticles (NPs) is a rapidly growing field of industry. Among different types of NPs, silver nanoparticles (AgNPs) are of particular interest because of their well-known antimicrobial properties, which make them very interesting for potential application in agriculture and food production. However, increasing usage and unregulated release of AgNPs into aquatic and terrestrial systems bring concerns about their impact on the environment and living organisms, among which plants, as sessile organisms, are particularly affected. In this chapter, we summarize the novel findings on AgNP stability in biological media applied for plant exposure. Moreover, we also present AgNP roles in the promotion of cytotoxic and genotoxic effects, as well as in changes in gene and protein expression, to reveal the molecular response triggered by AgNPs in plants and assess their potential phytotoxicity mechanisms.

Exposure to radiofrequency electromagnetic fields (RF-EMF) at the operating frequencies of different communication devices can cause various biological effects. However, there is a lack of studies on the oxidative stress response and genotoxicity in the honey bee (Apis mellifera) after exposure to RF-EMF. In this study, we investigated the oxidative stress and DNA damage in honey bee larvae situated in waxcomb cells, exposed to modulated RF-EMF 23 Vm-1. The glutathione S-transferase activity decreased, whereas the catalase activity increased significantly in the honey bee larvae upon RF-EMF exposure. Superoxide dismutase activity, the level of lipid peroxidation, and DNA damage were not statistically altered in exposed honey bee larvae when compared to the control group. These results suggest that the biological effects of modulated RF-EMF in honey bee larvae depend on the exposure design.

Silver nanoparticles (AgNPs) are used in a wide range of consumer products because of their excellent antimicrobial properties. AgNPs released into the environment are prone to transformations such as aggregation, oxidation, or dissolution so they are often stabilised by coatings that affect their physico-chemical properties and change their effect on living organisms. In this study we investigated the stability of polyvinylpyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB) coated AgNPs in an exposure medium, as well as their effect on tobacco germination and early growth. AgNP-CTAB was found to be more stable in the solid Murashige and Skoog (MS) medium compared to AgNP-PVP. The uptake and accumulation of silver in seedlings was equally efficient after exposure to both types of AgNPs. However, AgNP-PVP induced only mild toxicity on seedlings growth, while AgNP-CTAB caused severe negative effects on all parameters, even compared to AgNO3. Moreover, CTAB coating itself exerted negative effects on growth. Cysteine addition generally alleviated AgNP-PVP-induced negative effects, while it failed to improve germination and growth parameters after exposure to AgNP-CTAB. These results suggest that the toxic effects of AgNP-PVP are mainly a consequence of release of Ag+ ions, while phytotoxicity of AgNP-CTAB can rather be ascribed to surface coating itself.

The small size of nanoparticles (NPs), with dimensions between 1 and 100 nm, results in unique chemical and physical characteristics, which is why they are implemented in various consumer products. Therefore, an important concern is the potential detrimental impact of NPs on the environment. As plants are a vital part of ecosystem, investigation of the phytotoxic effects of NPs is particularly interesting. This study investigated the potential phytotoxicity of silver nanoparticles (AgNPs) on tobacco (Nicotiana tabacum) plants and compared it with the effects of the same AgNO3 concentrations. Accumulation of silver in roots and leaves was equally efficient after both AgNP and AgNO3 treatment, with predominant Ag levels found in the roots. Exposure to AgNPs did not result in elevated values of oxidative stress parameters either in roots or in leaves, while AgNO3 induced oxidative stress in both plant tissues. In the presence of both AgNPs and AgNO3, root meristem cells became highly vacuolated, which indicates that vacuoles might be the primary storage target for accumulated Ag. Direct AgNP uptake by root cells was confirmed. Leaf ultrastructural studies revealed changes mainly in the size of chloroplasts of AgNP-treated and AgNO3- treated plants. All of these findings indicate that nano form of silver is less toxic to tobacco plants than silver ions.

Exposure of different animal species to radiofrequency electromagnetic fields (RF-EMF) could cause various biological effects such as oxidative stress, genotoxic effects and dysfunction of the immune system. However, there are a lack of results on oxidative stress response and genotoxicity in the honey bee (Apis mellifera) after exposure to RF-EMF. This study was performed to investigate the effects of exposure to RF-EMF on the activity of catalase, superoxide dismutase, glutathione S- transferase, lipid peroxidation level and DNA damage in honey bee larvae. Honey bee larvae were exposed to RF-EMF at 900 MHz and field levels of 10, 23, 41 and 120 V m−1 for 2 h. At a field level of 23 V m−1 the effect of 80% AM 1 kHz sinusoidal and 217 Hz modulation was investigated as well. Catalase activity and the lipid peroxidation level decreased significantly in the honey bee larvae exposed to the unmodulated field at 10 V m−1 compared to the control. Superoxide dismutase and glutathione S-transferase activity in the honey bee larvae exposed to unmodulated fields were not statistically different compared to the control. DNA damage increased significantly in honey bee larvae exposed to modulated (80% AM 1 kHz sinus) field at 23 V m−1 compared to the control and all other exposure groups. These results suggest that RF-EMF effects in honey bee larvae appeared only after exposure to a certain EMF conditions. The increase of the field level did not cause a linear dose- response in any of the measured parameters. Modulated RF-EMF produced more negative effects than the corresponding unmodulated field. Although honey bees in nature would not be exposed to such high field levels as used in our experiments, our results show the need for further intensive research in all stages of honey bee development.

Eisenia fetida earthworms were exposed to electromagnetic field (EMF) at a mobile phone frequency (900 MHz) and at field levels ranging from 10 to 120 V m-1 for a period of two hours (corresponding to specific absorption rates ranging from 0.13 to 9.33 mW kg-1). Potential effects of longer exposure (four hours), field modulation, and a recovery period of 24 h after two hours of exposure were addressed at the field level of 23 V m-1. All exposure treatments induced significant DNA modifications as assessed by a quantitative random amplified polymorphic DNA-PCR. Even after 24 h of recovery following a two hour-exposure, the number of probe hybridisation sites displayed a significant two-fold decrease as compared to untreated control earthworms, implying a loss of hybridisation sites and a persistent genotoxic effect of EMF. Expression of genes involved in the response to general stress (HSP70 encoding the 70 kDa heat shock protein, and MEKK1 involved in signal transduction), oxidative stress (CAT, encoding catalase), and chemical and immune defence (LYS, encoding lysenin, and MYD, encoding a myeloid differentiation factor) were up-regulated after exposure to 10 and modulated 23 V m-1 field levels. Western blots showing an increased quantity of HSP70 and MTCO1 proteins confirmed this stress response. HSP70 and LYS genes were up-regulated after 24 h of recovery following a two hour-exposure, meaning that the effect of EMF exposure lasted for hours.

Silver nanoparticles (AgNPs) are the dominating nanomaterial in consumer products due to their well-known antibacterial and antifungal properties. To enhance their properties, different surface coatings may be used, which affect physico-chemical properties of AgNPs. Due to their wide application, there has been concern about possible environmental and health consequences. Since plants play a significant role in accumulation and biodistribution of many environmentally released substances, they are also very likely to be influenced by AgNPs. In this study we investigated the toxicity of AgNO3 and three types of laboratory-synthesized AgNPs with different surface coatings [citrate, polyvinylpyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB)] on Allium cepa roots. Ionic form of Ag was confirmed to be more toxic than any of the AgNPs applied. All tested AgNPs caused oxidative stress and exhibited toxicity only when applied in higher concentrations. The highest toxicity was recorded for AgNPs-CTAB, which resulted with increased Ag uptake in the roots, consequently leading to strong reduction of the root growth and oxidative damage. The weakest impact was found for AgNPs-citrate, much bigger, negatively charged NPs, which also aggregated to larger particles. Therefore, we can conclude that the toxicity of AgNPs is directly correlated with their size, overall surface charge and/or surface coating.

Lichens are one of the most common air quality bioindicators. Airborne heavy metal pollution causes various physiological changes in lichens, but sensitivity to metal pollution is species specific. In this research, three lichen species (Parmelia sulcata, Flavoparmelia caperata and Evernia prunastri) were exposed to cadmium (50 mg L−1) in laboratory conditions. Photosynthetic efficiency of photosystem II and content of secondary metabolites were determined after one, three and eight days of exposure. In all investigated species treatment of lichen thalli with cadmium significantly changed Fv/Fm and RFd only after eight days of exposure. Quantification of metabolites showed a decreased content of the medullary depsidones salazinic acid (in P. sulcata) and protocetraric acid (in F. caperata) but increased content of cortical depside atranorin (in P. sulcata) and dibenzofurane usnic acid (in F. caperata) after cadmium exposure. However, no changes in secondary metabolites were found in E. prunastri. Results show that investigated species are relatively resistant to short-term cadmium-exposure and that secondary metabolites could have an important role in the protection of primary metabolism from negative cadmium impacts, at least in some species.

Sarraceniais a genus of carnivorous plants characterised by leaves modified into pitchers which lure, trap and digest insects. The aim of this study was to analyse the photochemical efficiency and contents of photosynthetic pigments and phenolic compounds in different pitcher parts – operculum, wing, pitchertube upper part and pitcher-tube lower part of three morphologically differentSarraceniahybrids. The photochemical efficiency of the operculum and the pitcher-tube upper part was lower than that of the pitcher-tube lower part and wing, especially in hybrid B. In all hybrids, the wing had higher amount of chlorophyllathan other pitcher parts. In contrast, a higher amount of phenolic compounds, in particular anthocyanins, was measured in the operculum and the pitcher-tube upper part, parts which are red-coloured and participate in luring and trapping insects. Although there were some differences among the hybrids, the results show that amount of phenolic compounds and photosynthetic pigments as well as photochemical efficiency are related to the function of the pitcher part analysed.

Salinity is an abiotic factor that has a negative impact on plant growth and seed germination, leading to limited crop production. The effect of salt stress (100 and 200 mM NaCl) on the expression of dehydrins (DHNs), heat-shock protein 70 (HSP70), superoxide dismutase (Cu/Zn SOD), and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was investigated in leaves of tobacco (Nicotiana tabacum L.) F1 hybrid DH10 and four dihaploid lines (207B, 238C, 239K, 244B) under in vitro conditions. After 33 d, the dihaploid lines as well as DH10 showed enhanced expression of 70-kDa DHN in response to 200 mM NaCl stress. This was especially evident in line 207B, which previously proved to be tolerant to salt stress, suggesting that DHNs have an important role in salt stress tolerance. Also, line 207B as well as lines 238C and 244B responded to 200 mM NaCl stress by increased expression of Cu/Zn SOD, indicating its possible role in providing salt stress tolerance. On the other hand, salt stress induced in line 239K resulted in a significantly higher expression of RuBisCO, indicating that differential susceptibility of dihaploids to increased levels of NaCl might be the result of their different genetic traits. Decreased expression of HSP70 in salt-treated tobacco dihaploids indicates that HSP70 is not included in protection against salt stress.

In the eastern Adriatic, Podarcis siculus, an invasive species, competitively excludes the native Podarcis melisellensis. Monoamine neurotransmitters—serotonin (5HT), dopamine (DA), and noradrenaline (NA)—are implicated in social behavior, and could lie at the basis of the direct behavioral interference of P. siculus with P. melisellensis. To understand the relationship between social behavior and monoamines, as well as the differences in behavior between P. siculus and P. melisellensis, we developed a high-performance liquid chromatography (UV/VIS detection) method with which we were able to reliably measure concentrations of 5HT, DA, and NA in 32 brains of the two lizard species. We observed no statistically significant influence of species, sex, or their interaction on brain NA and 5HT concentrations. Statistically significant influence of species on dopamine levels were recorded, with P. siculus having twice as much dopamine in their brains. Taking into account that a significant aggressive relationship, with P. siculus dominating over P. melisellensis, has been previously observed, and that dopamine directly influences this behavior, the observed differences in dopamine levels could represent a trait in these species and may contribute to the competitive exclusion of P. melisellensis by P. siculus in the eastern Adriatic.

Among different types of nanoparticles, particularly interesting are silver nanoparticles (AgNPs) because of their antibacterial and antifungal properties, due to which they have been implemented in various commercial products. However, unique properties allow them to interact with living organisms. Plants can serve as a potential pathway for AgNP transport and bioaccumulation into food chains and any negative effects of AgNPs on plants could cause significant changes in the ecosystem, potentially causing irreversible damage. Understanding the nature of plant interactions with nanoparticles is crucial for assessing their uptake and distribution as well as AgNP-induced changes in plant morphology and physiology. This chapter reviews the current knowledge on phytotoxicity assessment and interactions of AgNPs with plants at the whole plant and organ as well as cellular and molecular level and highlights issues that still need to be explored in order to improve our knowledge in this field.

Invasive species, especially plant pathogens, have a potential to completely eradicate native plant species and remodel landscapes. Tripartite interactions among sweet chestnut (Castanea sativa), chestnut blight-causing invasive fungus Cryphonectria parasitica, and hyperparasitic virus Cryphonectria hypovirus 1 (CHV1) were studied in two populations. The number of different vegetative compatibility (vc) types of C. parasitica more than doubled over the 10 years, while the hypovirulence incidence dropped in one population and slightly increased in the other one. Over the course of our 3-year monitoring experiment, the prevalence of hypovirulent isolates obtained from monitored cankers increased slowly (i.e., more hypovirulent isolates were being obtained from the same cankers over time). Within studied cankers, considerable changes in vc type and CHV1 presence were observed, indicating a highly dynamic system in which virulent and hypovirulent mycelia, sometimes of discordant vc types, often appeared together. The increase in hypovirulence prevalence did not have any observable curative effect on the cankers and, occasionally, reactivation of healed cankers by new, virulent C. parasitica isolates was observed. Both short- and long-term observations and revalidation of the infected plant populations are necessary to accurately estimate disease progress and formulate an adequate disease management strategy.

Metals have a variety of negative outcomes on plants, essential components of any ecosystem. The effects of CdCl2 (5 μmol L-1), ZnCl2 (25 or 50 μmol L-1), and CuCl2 (2.5 or 5 μmol L-1) and combinations of CdCl2 with either ZnCl2 or CuCl2 on the growth, photosynthetic pigments, and photosystem II (PSII) efficiency of duckweed (Lemna minor L.) were investigated. All of the treatments caused growth inhibition and remarkable metal accumulation in plant tissue after 4 and 7 days. In the combined treatments, the accumulation of each metal applied was lesser in comparison to treatments with single metals. After 4 days, all of the treatments generally diminished chlorophyll a content and decreased the maximum quantum yield (Fv/Fm) and effective quantum yield (ΔF/F’m) of PSII. However, after 7 days of exposure to a combination of Cd and Zn, pigment content and PSII activity recovered to control levels. A higher concentration of Cu (5 μmol L-1) as well as Cd in combination with Cu had a prolonged inhibitory effect on photosynthetic features. Our results suggest that growth inhibition was due to the toxic effect of absolute metal quantity in plant tissue. Zn counteracted Cd uptake, as seen from the recovery of pigment content and PSII efficiency in plants exposed for 7 days to the Cd and Zn combination. Cu-induced oxidative stress led to a prolonged inhibitory effect in plants treated both with a higher concentration of Cu (5 μmol L-1) and simultaneously with Cd and Cu. Our findings could contribute to general knowledge on anthropogenic and environmental contaminants that endanger plant communities and significantly disrupt the sensitive balance of an ecosystem by influencing photosynthetic mechanisms.

Salt and drought stress are important abiotic factors that negatively affect plant growth and productivity. Defense mechanisms, which plants have developed to cope with stress, are followed by alterations in a genome expression profile that in turn result in qualitative and quantitative change of the proteome. Although proteomic-based approach for studies of plant responses to salinity and drought has already been successfully employed in several plants, for cactus species such analyses have not been done so far. Therefore, in this study we have performed proteomic analysis of Mammillaria gracilis Pfeiff. in vitro grown cultures, callus and tumor, exposed to iso-osmotic NaCl and mannitol. Obtained results differed among analyzed tissues. The higher number of differentially expressed proteins after either salt or mannitol treatment was revealed in tumor compared to callus. According to classification to different functional categories, majority of the identified callus responsive proteins belongs to protein synthesis and processing category, while the highest number of identified tumor proteins belongs to category of metabolism, which suggest that the mechanisms that mediate responses to salt- and mannitol-induced stress in cactus callus and tumor are dependent on tissue type. Down-regulation of proteins involved in cell protection suggests the inability of tumor to activate protective processes against salinity and osmotic stress.

Since silver nanoparticles (AgNPs) are a dominant nanomaterial in consumer products, there is growing concern about their impact on the environment. Although numerous studies on the effects of AgNPs on living organisms have been conducted, the interaction of AgNPs with plants has not been fully clarified. To reveal the plant mechanisms activated after exposure to AgNPs and to differentiate between effects specific to nanoparticles and ionic silver, we investigated the physiological, ultrastructural and proteomic changes in seedlings of tobacco (Nicotiana tabacum) exposed to commercial AgNPs and ionic silver (AgNO3) from the seed stage. A higher Ag content was measured in seedlings exposed to AgNPs than in those exposed to the same concentration of AgNO3. However, the results on oxidative stress parameters obtained revealed that, in general, higher toxicity was recorded in AgNO3-treated seedlings than in those exposed to nanosilver. Ultrastructural analysis of root cells confirmed the presence of silver in the form of nanoparticles, which may explain the lower toxicity of AgNPs. However, the ultrastructural changes of chloroplasts as well as proteomic study showed that both AgNPs and AgNO3 can affect photosynthesis. Moreover, the majority of the proteins involved in the primary metabolism were up-regulated after both types of treatments, indicating that enhanced energy production, which can be used to reinforce defensive mechanisms, enables plants to cope with silver-induced toxicity.

Salinity is an important abiotic factor that limits plant growth and development. The influence of salt stress induced by sodium chloride on plant growth, proline content, level of lipid peroxidation and activities of antioxidative enzymes was studied in F1 hybrid DH10 and four dihaploid lines (207B, 238C, 239K, 244B) of tobacco (Nicotiana tabacum L.). Dihaploids were obtained from anther-derived haploids of hybrid DH10 and were previously proved to be tolerant to Potato virus Y (PVY). In our study plants were grown in vitro and exposed to NaCl (100 and 200 mM) for 33 days. All dihaploids and hybrid DH10 showed reduced growth after NaCl treatment. They accumulated significant amounts of sodium and proline in response to salt stress as have already been observed in tobacco and other plant species. In tobacco exposed to NaCl the lipid peroxidation level did not increase and activities of superoxide dismutase (SOD), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT) mostly did not change significantly. The exception was line 239K where salt induced higher activities of SOD, CAT and POD. Two (238C and 244B) out of four dihaploids appeared more susceptible to salt stress as they showed weak growth in correlation with high proline and sodium content. Therefore, it seems that salt tolerance is not associated with tolerance to PVY. Variations in malondialdehyde and proline content as well as in enzymes activities observed among tobacco lines imply that dihaploids have different genetic properties which might result in different sensitivity to NaCl.

Effects of iso-osmotic concentrations of NaCl and mannitol were studied in Mammilaria gracilis (Cactaceae) in both calli and tumors grown in vitro. In both tissues, relative growth rates were reduced under osmotic stress, which were accompanied by a decrease in both tissue water and K+ content. However, growth was inhibited to a lesser extent after exposure to NaCl, when accumulation of Na+ ions was observed. In calli, only salinity increased proline content, whereas with tumors proline accumulated after both osmotic stresses. Osmotic stresses also induced oxidative damage in both cactus tissues, although higher oxidative injury was caused by mannitol in calli and by salt in tumors. Low iso-osmotic concentrations of NaCl (75 mM) and mannitol (150 mM) increased peroxidase, ascorbate peroxidase, and esterase activities, whereas elevated catalase activity was recorded only after mannitol treatment in both tissues. High osmotic stress generally decreased enzymatic activities. However, in calli, esterase activity increased in response to high salinity, whereas ascorbate peroxidase activity was enhanced after high mannitol stress. In conclusion, both in vitro-grown cactus tissues were found to be sensitive to osmotic stress caused by either mannitol or NaCl, but accumulation of Na+ ions in response to salt somewhat contributed to osmotic adjustment. However, more prominent oxidative damage induced by NaCl compared to mannitol in tumor could be related to ion toxicity. The mechanisms that mediate responses to salt- and mannitol-induced osmotic stresses differed and were dependent on tissue type.

Eisenia fetida earthworms were exposed to electromagnetic field (EMF) at a mobile phone frequency (900 MHz) and at field levels ranging from 10 to 120 V m-1 for a period of two hours (corresponding to specific absorption rates ranging from 0.13 to 9.33 mW kg-1). Potential effects of longer exposure (four hours), field modulation, and a recovery period of 24 h after two hours of exposure were addressed at the field level of 23 V m-1. All exposure treatments induced significant DNA modifications as assessed by a quantitative random amplified polymorphic DNA-PCR. Even after 24 h of recovery following a two hour-exposure, the number of probe hybridisation sites displayed a significant two-fold decrease as compared to untreated control earthworms, implying a loss of hybridisation sites and a persistent genotoxic effect of EMF. Expression of genes involved in the response to general stress (HSP70 encoding the 70 kDa heat shock protein, and MEKK1 involved in signal transduction), oxidative stress (CAT, encoding catalase), and chemical and immune defence (LYS, encoding lysenin, and MYD, encoding a myeloid differentiation factor) were up-regulated after exposure to 10 and modulated 23 V m-1 field levels. Western blots showing an increased quantity of HSP70 and MTCO1 proteins confirmed this stress response. HSP70 and LYS genes were up-regulated after 24 h of recovery following a two hour-exposure, meaning that the effect of EMF exposure lasted for hours., U ovom istraživanju gujavice vrste Eisenia fetida bile su izložene elektromagnetskom polju (EMP) na frekvenciji mobilnih telefona (900 MHz) te poljima jačine 10 do 120 V m-1 u dvosatnom razdoblju (što odgovara specifičnim ratama apsorpcije od 0,13 do 9,33 mW kg-1). Utjecaj dužeg izlaganja (4 sata), modulacije polja te vrijeme oporavka od 24 sata nakon dva sata izlaganja proučavan je pri jačini polja od 23 V m-1. Metoda kvantitativne nasumično umnožene polimorfne DNA (engl. quantitative random amplified polymorphic DNA – qRAPD) otkrila je značajne modifikacije DNA na svim proučavanim tretmanima. Čak i nakon 24-satnog oporavka broj hibridizacijskih mjesta bio je dvostruko manji u odnosu na broj zabilježen u kontrolnim gujavicama, što upozorava na gubitak hibridizacijskih mjesta i na dugoročan utjecaj EMP-a. Ekspresija gena uključenih u odgovor na stres (HSP70: kodira za 70kDa heat shock protein i MEKK1: uključen u provođenje signala), oksidacijski stres (CAT: kodira za katalazu) te kemijsku i imunosnu obranu (LYS: kodira za lysenin i MYD: kodira za faktor mijeloidne diferencijacije) bila je povišena nakon izlaganja polju jačine 10 V m-1 te moduliranome polju jačine 23 V m-1. Western blot analiza potvrdila je odgovor na stres detekcijom povišene količine HSP70 i MTCO1 proteina. HSP70 i LYS geni imali su povišenu ekspresiju i nakon razdoblja oporavka, što upućuje na dugotrajan utjecaj EMP-a.

Salinity is a common abiotic factor that affects plant growth and development. Seedlings of tobacco (Nicotiana tabacum L.) F1 hybrid DH10 and three dihaploid lines (207B, 238C and 239K) obtained by diploidization of anther-derived haploids of hybrid DH10 were subjected to 0, 100 and 200 mM NaCl in in vitro conditions for 33 days and the effect on roots was evaluated. In all lines and in the hybrid DH10 exposed to 200 mM NaCl evident root growth inhibition and increased proline content were noticed. However, in some cases lines differed in the activity of antioxidative enzymes, which could account for differences in their salinity tolerance. Increased activity of catalase and peroxidase in roots of line 239K could contribute to the more pronounced salinity tolerance previously reported for shoots of this line.

Heavy metals are generally known to induce oxidative stress, but are rarely strategically studied in an embracive manner, taking into account interplay between their various effects. Furthermore, although metals in the environment are present in mixtures and interact with each other, their combined effects to organisms have been much less studied in comparison to individual effects. Here we present a complete comprehensive study of Cd/Cu oxidative stress interactions in Nicotiana tabacum seedlings and adult plants. Plants were treated with Cd (10 µM and 15 µM), Cu (2.5 µM and 5 µM) and their combinations ; seedlings during one month-period and adult plants during the period of 7 days. Metal accumulation measurements showed that Cu stimulates Cd uptake in roots, while reducing Cd translocation to shoots. We employ the power of extensive principal component analysis (PCA) to decipher differences and similarities in antioxidative response to these two metals. PCA analysis showed that although indicators of oxidative damage to biomolecules highly correlate across tissues, seedlings show greater sensitivity in terms of DNA, while adult plants in terms of protein and lipid oxidation. Majority of toxic effects were caused by Cd alone, with addition of Cu often resulting in damage alleviation. However, mixture of high concentrations of both Cd and Cu induced most adverse effects. In conclusion, our results indicate that copper in lower concentration has antagonistic effect to cadmium toxicity, while in higher concentration these metals interact synergistically in tobacco.

Mammillaria gracilis Pfeiff. plants cultivated in the pot (pot plants, PP), as well as in vitro- grown normal shoots (NS), habituated callus (HC), hyperhydric shoots (HS), and tumour tissue (TT) were investigated in order to reveal the influence of in vitro culture on functionality of the photosynthetic apparatus and CAM photosynthesis in cactus Mammillaria gracilis Pfeiff. Photosynthetic pigments content as well as maximum (Fv/Fm) and effective (ΦPSII) quantum yield of photosystem II (PSII) decreased in all in vitro-grown tissues in comparison to PP. The decrease observed in hyperhydric HC, HS and TT correlated with a low expression of Rubisco large subunit (RbcL) and  subunit of ATP synthase ( ATP synt) and almost undetectable levels of protein D1, light- harvesting chlorophyll a/b-binding protein (LHCII) and cytochrome f protein of thylakoid Cyt b6/f- complex (Cyt f) found in these tissues. As for crassulacean acid metabolism (CAM) pattern, PP and NS expressed diurnal acid fluctuation, while HC, HS and TT failed to show it. Nevertheless, all M. gracilis tissues exhibited diurnal changes of phosphoenolpyruvate carboxylase (PEPC) activity indicating the typical CAM physiology. In conclusion, the photosynthesis was down regulated in all in vitro-grown tissues. NS maintained typical CAM photosynthesis, while HC, HS and TT withheld PEPC activity, but not acid accumulation specific for CAM. Minor changes observed in NS in comparison to PP could be attributed to the sugar supplementation while the more prominent deviations found in HC, HS and TT could be correlated with hyperhydricity and the loss of characteristic tissue organisation pattern.

The effects of exposure to radiofrequency electromagnetic fields (RF-EMFs) on seed germination, primary root growth as well as mitotic activity and mitotic aberrations in root meristematic cells were examined in Allium cepa L. cv. Srebrnjak Majski. Seeds were exposed for 2h to EMFs of 400 and 900MHz at field strengths of 10, 23, 41 and 120Vm(-1). The effect of longer exposure time (4h) and field modulation was investigated at 23Vm(-1) as well. Germination rate and root length did not change significantly after exposure to radiofrequency fields under any of the treatment conditions. At 900MHz, exposures to EMFs of higher field strengths (41 and 120Vm(-1)) or to modulated fields showed a significant increase of the mitotic index compared with corresponding controls, while the percentage of mitotic abnormalities increased after all exposure treatments. On the other hand, at 400MHz the mitotic index increased only after exposure to modulated EMF. At this frequency, compared with the control higher numbers of mitotic abnormalities were found after exposure to modulated EMF as well as after exposure to EMFs of higher strengths (41 and 120Vm(-1)). The types of aberration induced by the EMFs of both frequencies were quite similar, mainly consisting of lagging chromosomes, vagrants, disturbed anaphases and chromosome stickiness. Our results show that non-thermal exposure to the radiofrequency fields investigated here can induce mitotic aberrations in root meristematic cells of A. cepa. The observed effects were markedly dependent on the field frequencies applied as well as on field strength and modulation. Our findings also indicate that mitotic effects of RF-EMF could be due to impairment of the mitotic spindle.

Effects of residual wastewater sludge, amended with zeolites, on the early growth of perennial ryegrass (Lolium perenne L.) were investigated. For that purpose growth parameters, content of photosynthetic pigments, substrate characteristics, levels of macro- and micronutrients, as well as toxic elements in plant tissue, plant inorganic matter and substrates, sewage sludge and soil, were determined. Among the growth indicators, only protein content and inorganic matter in plant tissue were found to be influenced by the type and composition of the substrate. Sewage sludge was found to contain elevated levels of Mo, Ni, Pb, Sb, Se, Sr and Zn, while Cu and Sn were found in an order of magnitude higher levels compared to the control sample. Application of sewage sludge, however, modified only the content of Cu and Zn in the plant tissue, inducing accumulation of these elements by a factor of 2. Despite the fact that all other measured elements (Al, As, Ba, Ca, Cd, Co, Cr, Cs, Fe, K, Li, Mg, Mn, Na, Ti, U and V) were found in plant tissue at similar level regardless of the substrate metal loading, accumulation indices point to overall lower bioavailability of elements from digested sludge in comparison with control substrate. Moreover, metal concentrations in all analyzed plant samples were below legally established limits.

The discharge of untreated electroplating wastewaters directly into the environment is a certain source of heavy metals in surface waters. Even though heavy metal discharge is regulated by environmental laws many small-scale electroplating facilities do not apply adequate protective measures. Electroplating wastewaters contain large amounts of various heavy metals (the composition depending on the facility) and the pH value often bellow 2. Such pollution diminishes the biodiversity of aquatic ecosystems and also endangers human health. The aim of our study was to observe/measure the toxic effects induced by a mixture of seven heavy metals on a bioindicator species Lemna minor L. Since artificial laboratory metal mixtures cannot entirely predict behaviour of metal mixtures nor provide us with informations relating to the specific conditions in the realistic environment we have used an actual electroplating wastewater sample discharged from a small electroplating facility. In order to obtain three more samples with the same composition of heavy metals but at different concentrations, the original electroplating wastewater sample has undergone a purification process. The purification process used was developed by Orescanin et al. [Orescanin V, Mikelić L, Lulić S, Nad K, Rubcić M, Pavlović G. Purification of electroplating wastewaters utilizing waste by-product ferrous sulphate and wood fly ash. J Environ Sci Health A 2004; 39 (9): 2437-2446.] in order to remove the heavy metals and adjust the pH value to acceptable values for discharge into the environment. Studies involving plants and multielemental waters are very rare because of the difficulty in explaining interactions of the combined toxicities. Regardless of the complexity in interpretation, Lemna bioassay can be efficiently used to assess combined effects of multimetal samples. Such realistic samples should not be avoided because they can provide us with a wide range of information which can help explain many different interactions of metals on plant growth and metabolism. In this study we have primarily evaluated classical toxicity endpoints (relative growth rate, Nfronds/Ncolonies ratio, dry to fresh weight ratio and frond area) and measured guaiacol peroxidase (GPX) activity as early indicator of oxidative stress. Also, we have measured metal accumulation in plants treated with waste ash water sample with EDXRF analysis and have used toxic unit (TU) approach to predict which metal will contribute the most to the general toxicity of the tested samples.

Round-leaved sundew (Drosera rotundifolia L.) is a carnivorous plant which inhabits nutrient-poor, moist and sun exposed areas such as peat bogs and sandpits. These habitats are threatened by succession which could lead to substantial shading of sundews. Nevertheless, D. rotundifolia can also grow in shaded environment within a layer of dwarf shrubs, indicating great photosynthetic plasticity whose mechanism still remains largely unknown. Therefore, the aim of this experiment was to investigate physiological responses involved in D. rotundifolia acclimation to different light levels. Photosynthetic pigment content, photochemical efficiency and content of phenolic compounds were studied in plants acclimated to sunlight conditions (outdoor-growing plants) as well as in those acclimated to low-light conditions (indoor-growing plants) before and after sudden exposure to high-intensity solar radiation for five hours per day during 7-day period. Outdoor-growing plants were larger in size, had higher Fv/F0 ratio, reddish leaves and they flowered. Also, they had higher content of phenolic compounds including flavonoids and anthocyanins as well as higher Car/Chl ratio, while indoor-growing plants showed higher chlorophyll a and b content. There was no significant difference in the photochemical efficiency of PSII between indoor- and outdoor-growing plants. The indoor-growing plants that were exposed to solar radiation showed initial photoinhibition of photosynthesis. However, after 7-day exposure, the chlorophyll content significantly decreased while contents of carotenoids, flavonoids and anthocyanins as well as Car/Chl ratio increased, leading to the restoration of PSII activity. In conclusion, our results revealed that sundews can successfully acclimate to both low- and high-light intensities by changing content and composition of the photosynthetic pigments and phenolic compounds.

Increased use of radio and microwave frequencies requires investigations of their effects on living organisms. Duckweed (Lemna minor L.) has been commonly used as a model plant for environmental monitoring. In the present study, duckweed growth and peroxidase activity was evaluated after exposure in a Gigahertz Transversal Electromagnetic (GTEM) cell to electric fields of frequencies 400, 900, and 1900 MHz. The growth of plants exposed for 2 h to the 23 V/m electric field of 900 MHz significantly decreased in comparison with the control, while an electric field of the same strength but at 400 MHz did not have such effect. A modulated field at 900 MHz strongly inhibited the growth, while at 400 MHz modulation did not influence the growth significantly. At both frequencies a longer exposure mostly decreased the growth and the highest electric field (390 V/m) strongly inhibited the growth. Exposure of plants to lower field strength (10 V/m) for 14 h caused significant decrease at 400 and 1900 MHz while 900 MHz did not influence the growth. Peroxidase activity in exposed plants varied, depending on the exposure characteristics. Observed changes were mostly small, except in plants exposed for 2 h to 41 V/m at 900 MHz where a significant increase (41%) was found. Our results suggest that investigated electromagnetic fields (EMFs) might influence plant growth and, to some extent, peroxidase activity. However, the effects of EMFs strongly depended on the characteristics of the field exposure. Bioelectromagnetics 26:185–193, 2005. © 2005 Wiley-Liss, Inc.

Metals have a variety of negative outcomes on plants, essential components of any ecosystem. The effects of CdCl2 (5 μmol L-1), ZnCl2 (25 or 50 μmol L-1), and CuCl2 (2.5 or 5 μmol L-1) and combinations of CdCl2 with either ZnCl2 or CuCl2 on the growth, photosynthetic pigments, and photosystem II (PSII) efficiency of duckweed (Lemna minor L.) were investigated. All of the treatments caused growth inhibition and remarkable metal accumulation in plant tissue after 4 and 7 days. In the combined treatments, the accumulation of each metal applied was lesser in comparison to treatments with single metals. After 4 days, all of the treatments generally diminished chlorophyll a content and decreased the maximum quantum yield (Fv/Fm) and effective quantum yield (ΔF/F’m) of PSII. However, after 7 days of exposure to a combination of Cd and Zn, pigment content and PSII activity recovered to control levels. A higher concentration of Cu (5 μmol L-1) as well as Cd in combination with Cu had a prolonged inhibitory effect on photosynthetic features. Our results suggest that growth inhibition was due to the toxic effect of absolute metal quantity in plant tissue. Zn counteracted Cd uptake, as seen from the recovery of pigment content and PSII efficiency in plants exposed for 7 days to the Cd and Zn combination. Cu-induced oxidative stress led to a prolonged inhibitory effect in plants treated both with a higher concentration of Cu (5 μmol L-1) and simultaneously with Cd and Cu. Our findings could contribute to general knowledge on anthropogenic and environmental contaminants that endanger plant communities and significantly disrupt the sensitive balance of an ecosystem by influencing photosynthetic mechanisms., Izloženost metalima može izazvati različite štetne učinke u biljaka. Vodene leće izložili smo solima teških metala CdCl2 (5 μmol L 1), ZnCl2 (25 μmol L 1 ili 50 μmol L 1) i CuCl2 (2,5 μmol L 1 ili 5 μmol L 1) te kombinaciji CdCl2 sa svakom od navedenih koncentracija ZnCl2 i CuCl2. Rast biljaka, količina fotosintetskih pigmenata i učinkovitost fotosistema II (PSII) mjereni su nakon četiri i sedam dana tretmana. Utvrđeno je da su svi tretmani uzrokovali značajnu inhibiciju rasta te akumulaciju metala u biljci. U biljaka koje su bile izložene kombinacijama teških metala količina pojedinog metala u tkivu bila je niža u odnosu na količinu istog metala u biljaka izloženih samo tom metalu. Nakon četiri dana tretmana sva su tri metala, neovisno o tome jesu li bila primijenjena zasebno ili u kombinacijama, uzrokovala smanjenje količine klorofila a i pad vrijednosti maksimalnog (Fv/Fm) i efektivnog (ΔF/F’m) prinosa PSII. Međutim, u biljaka koje su bile istovremeno izlagane kadmiju i cinku, vrijednosti količine pigmenata i učinkovitost PSII vratile su se nakon sedam dana na kontrolnu razinu, a bakar u koncentraciji 5 μmol L 1 te kombinacija kadmija i bakra i dalje su imali inhibitorni učinak. Budući da smanjeno primanje pojedinog metala uočeno u biljaka izloženih kombiniranim tretmanima nije ublažilo inhibitorni učinak na rast, možemo zaključiti da je inhibicija rasta uzrokovana apsolutnom količinom metala primljenog u tkivo. Povećanje količine fotosintetskih pigmenata i učinkovitosti PSII nakon sedam dana tretmana kadmijem i cinkom upućuje na oporavak biljaka, što se može objasniti ublažavajućim djelovanjem cinka na učinak kadmija uslijed smanjenog primanja kadmija u biljku. Suprotno tome, dugotrajni inhibitorni učinak istovremenog tretmana biljaka kadmijem i bakrom te samim bakrom u koncentraciji 5 μmol L 1 može se objasniti oksidacijskim stresom uzrokovanim bakrom. Rezultati ovoga istraživanja pridonose saznanjima o štetnim učincima antropogenih i okolišnih onečišćivača, koji narušavanjem mehanizama fotosinteze ugrožavaju ne samo biljke i njihove zajednice nego i osjetljivu ravnotežu ekosustava.

Metals have a variety of negative outcomes on plants, essential components of any ecosystem. The effects of CdCl2 (5 μmol L-1), ZnCl2 (25 or 50 μmol L-1), and CuCl2 (2.5 or 5 μmol L-1) and combinations of CdCl2 with either ZnCl2 or CuCl2 on the growth, photosynthetic pigments, and photosystem II (PSII) efficiency of duckweed (Lemna minor L.) were investigated. All of the treatments caused growth inhibition and remarkable metal accumulation in plant tissue after 4 and 7 days. In the combined treatments, the accumulation of each metal applied was lesser in comparison to treatments with single metals. After 4 days, all of the treatments generally diminished chlorophyll a content and decreased the maximum quantum yield (Fv/Fm) and effective quantum yield (ΔF/F'm) of PSII. However, after 7 days of exposure to a combination of Cd and Zn, pigment content and PSII activity recovered to control levels. A higher concentration of Cu (5 μmol L-1) as well as Cd in combination with Cu had a prolonged inhibitory effect on photosynthetic features. Our results suggest that growth inhibition was due to the toxic effect of absolute metal quantity in plant tissue. Zn counteracted Cd uptake, as seen from the recovery of pigment content and PSII efficiency in plants exposed for 7 days to the Cd and Zn combination. Cu-induced oxidative stress led to a prolonged inhibitory effect in plants treated both with a higher concentration of Cu (5 μmol L-1) and simultaneously with Cd and Cu. Our findings could contribute to general knowledge on anthropogenic and environmental contaminants that endanger plant communities and significantly disrupt the sensitive balance of an ecosystem by influencing photosynthetic mechanisms.