Pristupačnost
Ena Dumančić (Supervisor: Prof. Biljana Balen)
IMPACT OF SILVER NITRATE ON ANTIOXIDANT ENZYMES IN TOBACCO PLANTS (Nicotiana tabacum L.)
Summary: Silver nitrate (AgNO3) is a silver compound that is often found in fertilizers and protection products of industrially important plants such as tobacco (Nicotiana tabacum L.). AgNO3 often enters ecosystem trough industrial and mining activities. Due to ubiquity of silver in industry and agriculture, it also
indirectly affects people, so it is important to widen our current knowledge about its potentially negative impact on plants. In this study, the effects of 25, 50 and 100 μM AgNO3 applied either alone or in combination with cysteine (a well-known silver chelator) as well as cysteine itself, on the activity and expression of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and pyrogallol peroxidase (PPX) in roots and leaves of adult tobacco plants were investigated and compared. After spectrophotometric measurements of enzyme activities, in-gel detection of isoforms after electrophoresis in native conditions and immunodetection with specific antibodies, changes in the activity and expression of antioxidant enzymes were determined. The SOD exhibited the highest and the most significant changes in activity in both roots and leaves, while the activity of the enzymes CAT, APX and PPX was less pronounced after the exposure to the treatments. The application of cysteine had a mitigating effect on the activity of antioxidant enzymes compared to treatments with AgNO3 alone, in both roots and leaves. Root tissue exhibited greater changes in protein expression and enzyme activities compared to leaf, which was expected and can be explained by the direct contact of the root cells with the exposure solution.
Lucija Kotarski (Supervisor: Prof. Biljana Balen)
IMPACT OF SILVER NANOPARTICLES STABILIZED BY DIFFERENT COATINGS ON ACTIVITY AND EXPRESSION OF ANTIOXIDANT ENZYMES IN TOBACCO SEEDLINGS (Nicotiana tabacum L.)
Summary: Silver nanoparticles (AgNP) are one of the most frequently used nanomaterials, due to their antimicrobial properties, which provide them great market potential and wide application. Numerous toxicological studies indicate the importance of investigating and understanding potentially harmful effects of AgNP on the environment. This work is focused on toxicity testing of 100 μM AgNP stabilized with polyvinylpyrrolidone (AgNP-PVP) and cetyltrimethylammonium bromide (AgNP- CTAB) coatings and 100 μM AgNO3, single and in combination with 500 μM cysteine, applied to tobacco seedlings (Nicotiana tabacum L.). Spectrophotometric analysis of AgNP stability in ½ MS nutrient medium showed higher stability of AgNP-PVP compared to AgNP-CTAB, while cysteine addition disturbed AgNP-PVP stability and prolonged the stability of AgNP-CTAB in the short term. Spectrophotometric measurement of activity of antioxidant enzymes (superoxide dismutase, catalase, ascorbate and pyrogallol peroxidase), detection of their isoforms by native electrophoresis and immunochemical method showed changes in their activity and expression. Ascorbate peroxidase proved to be the most sensitive enzyme. Comparison of AgNP and AgNO3 treatments revealed higher toxicity of AgNP-CTAB and lower toxicity of AgNP-PVP compared to AgNO3, according to the majority of the parameters. Addition of cysteine did not affect AgNP-PVP phytotoxicity, but partially alleviated toxic effects of AgNP-CTAB, leading to the conclusion that phytotoxicity of AgNP-PVP comes predominantly from the nanoparticles, while AgNP-CTAB phytotoxicity results from the released Ag+ ions or CTAB coating.
Bruno Komazec (supervisor: Dr. Petra Peharec Štefanić, Asst. Prof.), Graduation Thesis
THE EFFECT OF SILVER NANOPARTICLES AND IONS ON THE AUXIN ACCUMULATION AND TRANSPORT IN Arabidopsis thaliana ROOT
Summary: Silver nanoparticles (AgNPs) are used for their antimicrobial properties in many commercially available products, including pesticides, but they can pose a risk to the environment due to their toxicity, reactivity and accumulation in plants. Due to their large surface area to volume ratio, AgNPs are highly reactive and susceptible to the release of silver ions, which potentially increases their toxicity. Plant hormones are largely responsible for the normal growth of plants, and auxin is one of the most important phytohormones that play a role in many metabolically important plant processes. The Arabidopsis thaliana is an established model organism in molecular and cellular biology. In this thesis, the effects of PVP-AgNPs and silver ions (AgNO3) on Arabidopsis seedlings after one, three, five, seven, nine, and eleven days of treatment were investigated. The seeds were germinated for four days on a solid MS medium and afterwards on a modified solid ½ MS medium supplemented with PVP-AgNPs or silver ions at final concentrations of 25, 50 and 100 μM. Control seedlings were transferred to silver-free solid medium. In AgNO3-supplemented solid medium, the formation of AgNPs was noticed which were stabilized on the third day. After the third day and until the eleventh day, the absorbance of the newly formed AgNPs in medium slowly decreased. In contrast, PVP-AgNPs were stabilized in the solid medium around the eighth day. PVP-AgNPs and newly formed AgNPs affected the root length and were localized in the root cap and in the intercellular space and cell wall of the root tip. No significant changes in root and cotyledon structures were observed, but changes in chloroplast ultrastructure were observed on treatment with 100 μM AgNO3 after the eleventh day. A change in the accumulation and transport of auxin was observed in all treatments, which depended on the concentration and length of treatment, indicating that the plant adjusted to the AgNPs stress.
Katarina Drobac (Supervisors: prof. Ana-Marija Domijan and prof. Biljana Balen)
Summary: Previous research has shown that silver nanoparticles (AgNP) can be toxic to various ecosystem participants, which can consequently affect humans as well. The aim of this study was to investigate whether polyvinylpyrrolidone coated AgNPs (AgNP-PVP) cause changes in the protein concentration and concentration of the oxidative stress parameters, malondialdehyde and protein carbonyls in tobacco seedlings (Nicotiana tabacum). The question whether the potentially toxic effect of AgNP-PVP could be prevented by
the addition of cysteine, a good chelator of silver ions that, if released, also contribute to toxicity of AgNPPVP, was also posed. Tobacco seedlings were exposed to AgNP-PVP for one week at concentrations of 25, 50 and 100 μM. A part of the seedlings was exposed to AgNP-PVP in combination with cysteine (AgNP-PVP + cysteine: 25 + 125 μM, 50 + 250 μM, 100 + 500 μM) and cysteine alone at concentrations of 125, 250 and 500 μM. One part of the plants remained untreated, as a control group. The results showed that the parameters of oxidative stress did not significantly increase with AgNP-PVP treatment. Also, there were no significant differences after treatments with AgNP-PVP and AgNP-PVP in combination with cysteine. From the obtained results it can be concluded that under the mentioned experimental conditions (model organism, concentration and length of treatment) AgNP-PVP did not cause oxidative stress. Given that no difference was observed in the treatment with AgNP-PVP and AgNP-PVP in combination with cysteine, it can be concluded that PVP is a coating that contributes to the stability of AgNP and it prevents release of silver ions.
Dajana Matić (Supervisor: Assoc. Prof. Biljana Balen)
Phytotoxic effects of differently coated silver nanoparticles on tobacco (Nicotiana tabacum L.)
Summary: Due to their unique and beneficial properties, silver nanoparticles (AgNP) have been increasingly used in various industrial and scientific fields and commercial products. That makes environmental exposure to nanoparticles inevitable with unknown consequences on living organisms. Although the mechanisms of AgNP toxicity have not been fully elucidated, it is evident that physicochemical properties of AgNP affect their biological impacts. In this work, the effects of AgNP with three different coatings (citrate, PVP or CTAB), pure coatings and ionic silver (AgNO3) on germination and growth of tobacco (Nicotiana tabacum L.) seedlings as well as on the appearance of oxidative stress were investigated. The most prominent negative effect on tobacco seedling growth was detected in plants treated with AgNP-CTAB and CTAB coating, whereas AgNO3 treatments had no significant impact on the germination and seedling growth. Observed changes in the parameters of oxidative stress and activity of antioxidant enzymes demonstrated that both AgNP and AgNO3 induce oxidative stress, although by different mechanisms. The obtained results indicate that the toxicity of AgNP is greatly influenced by coatings used for their stabilization and that nanoparticles themselves account for their toxicity rather than released Ag+ ions.
Martina Jarnević (Supervisor: Assoc. Prof. Biljana Balen)
The impact of silver nanoparticles and ions on tobacco (Nicotiana tabacum L.) proteome
Summary: Due to its antimicrobial and antifungal properties, silver nanoparticles (AgNPs) are increasingly applied in consumer products as well as in various industries. However, because of their large surface area to volume ratio, AgNPs are highly reactive and subjected to silver ions (Ag+) release, which potentially increases their toxicity. So far, phyto-, cyto-and genotoxic effects of AgNPs to the various plant and animal organisms were observed. In this graduation thesis, the impact of 100 µM citrate-stabilized AgNPs and silver nitrate (AgNO3) on expression of total soluble proteins of tobacco (Nicotiana tabacum L.) seedlings and adult plants was examined. Two-dimensional gel electrophoresis was applied to analyse changes in protein profiles of whole tobacco seedlings as well as roots and leaves of adult plants, while mass spectrometry was used to identify proteins with different expression levels. It was revealed that AgNP and AgNO3 induced similar changes in the protein profiles of the certain tissue, while the expression level was found to be dependent on the developmental stage. Most of the proteins with altered expression are involved in the metabolism of carbohydrates, photosynthesis, response to stress and response to oxidative stress. The results showed that AgNP and AgNO3 mostly induced up-regulation of identified proteins in tobacco seedlings, while the protein expression was mainly reduced in the roots and leaves of adult plants. From the obtained results it can be concluded that the tobacco seedlings cope better with the stress induced with 100 µM AgNP and AgNO3 treatments compared to adult plants..
Željka Bajan (Supervisor: Assoc. Prof. Biljana Balen)
Oxidative stress in tobacco plants (Nicotiana tabacum L.) exposed to silver nanoparticles
Summary: Silver nanoparticles (AgNP) dominate among the types of available nanomaterials because of the well-known antibacterial and antifungal effects of silver. Therefore, their impact on the environment and living organisms intensively studied. This diploma thesis investigates the impact of AgNPs on the appereance of oxidative stress in adult tobacco plants (Nicotiana tabacum L.). The effects of nanosilver are compared with the effects of ionic silver (AgNO3), applied in the same concentration. AgNPs didn't induce oxidative stress since no changes were recorded in the content of reactive oxygen species (ROS), malondialdehyde, protein carbonyls and DNA damage in the roots and leaves of tobacco plants. However oxidative stress was recorded after the AgNO3 treatments, in the roots according to all tested parameters, and in the leaves according to majority of investigated parameters, which was particularly pronounced at the highest applied concentrations. Greater changes in the activity and expression of antioxidant enzymes SOD, PPX, APX and CAT were measured in the roots and leaves of tobacco after exposure to treatments with AgNO3 in relation to AgNP. Ionic silver proved to be more toxic to the tobacco plants compared to nanosilver and its stronger effects have been found in the roots compared to the leaves.
Renata Biba (Supervisor: Assoc. Prof. Biljana Balen)
The effect of silver nanoparticles on appearance of oxidative stress and protein expression in tobacco seedlings (Nicotiana tabacum)
Summary: Silver nanoparticles (AgNP) are excellent antibacterial and antifungal agents found in various consumer products. Due to their potential toxicity numerous studies are being conducted to determine whether they pose a threat to ecosystems and human health. In this research, the effects of AgNP on tobacco seedlings (Nicotiana tabacum) were analysed and compared with the effects of ionic silver (AgNO3) of the same concentrations. AgNP was toxic when applied in higher concentrations, while AgNO3 showed harmful effects even in lower concentrations. The measured increase of reactive oxygen species indicated appearance of oxidative stress, which was proven by the increase in contents of malondialdehyde and protein carbonyls. Both silver forms caused damage to DNA molecules, which was more pronounced in AgNO3 treatments. Antioxidative enzymes showed a change in activity and expression; the activity of SOD and APX increased, while PPX and CAT activity decreased in all treatments with AgNP and AgNO3. Change in protein patterns was also observed, with emphasis on the expression of stress protein Hsp70. Results indicate that AgNO3 is more toxic for plants than AgNP; however, higher AgNP concentrations also induce oxidative stress and affect protein expression, making them potential environmental hazards.