Biljana Balen

The effects of cadmium-zinc interactions on biochemical responses in tobacco seedlings and adult plants.

The objective of the present study was to investigate the effects of cadmium-zinc (Cd-Zn) interactions on their uptake, oxidative damage of cell macromolecules (lipids, proteins, DNA) and activities of antioxidative enzymes in tobacco seedlings as well as roots and leaves of adult plants. Seedlings and plants were exposed to Cd (10 µM and 15 µM) and Zn (25 µM and 50 µM) as well as their combinations (10 µM or 15 µM Cd with either 25 µM or 50 µM Zn). Measurement of metal accumulation exhibited that Zn had mostly positive effect on Cd uptake in roots and seedlings, while Cd had antagonistic effect on Zn uptake in leaves and roots. According to examined oxidative stress parameters, in seedlings and roots individual Cd treatments induced oxidative damage, which was less prominent in combined treatments, indicating that the presence of Zn alleviates oxidative stress. However, DNA damage found in seedlings, and lower glutathione reductase (GR) and superoxide dismutase (SOD) activity recorded in both seedlings and roots, after individual Zn treatments, indicate that Zn accumulation could impose toxic effects. In leaves, oxidative stress was found after exposure to Cd either alone or in combination with Zn, thus implying that in this tissue Zn did not have alleviating effects. In conclusion, results obtained in different tobacco tissues suggest tissue-dependent Cd-Zn interactions, which resulted in activation of different mechanisms involved in the protection against metal stress.

Isoenzymes of peroxidase and esterase related to morphogenesis in Mammillaria gracillis Pfeiff. tissue culture

In vitro propagated plants of the cactus Mammillaria gracillis Pfeiff. (Cactaceae) spontaneously produced callus. The habituated callus regenerated normal and hyperhydric shoots without the addition of grown regulators. Tumours were obtained by infecting cactus explants with Agrobacterium tumefaciens; the wild strain B6S3 (tumour TW) or with the rooty mutant GV3101 (tumour TR). Both tumour lines grew vigorously, never expressing any morphogenic potential. In this study, cactus shoots, callus, normal and hyperhydric regenerants and TW and TR tumours were compared with regard to peroxidase (EC 1.11.1.7) and esterase activity, and isoenzyme patterns. Guaiacol peroxidase activity was the lowest in the cactus shoots and in the normal regenerants. Callus, hyperhydric regenerants and tumours had peroxidase activity of 6 to 7 times higher. Esterase activity was measured with 1- and 2-naphthylacetate as broad-spectrum substrates. The highest esterase activity was determined in tumours with both substrates. All tissues, except the TR tumour, had higher esterase activity for 2-compared to 1-naphtylacetate. Peroxidase and esterase isoenzyme patterns were not completely identical among the investigated tissues.

Toxicity of silver ions and differently coated silver nanoparticles in Allium cepa roots

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.

Effect of Copper on the Toxicity and Genotoxicity of Cadmium in Duckweed (Lemna minor L.)

Effect of Copper on the Toxicity and Genotoxicity of Cadmium in Duckweed (Lemna Minor L.) We investigated interactions between copper (in the concentrations of 2.5 μmol L-1 and 5 μmol L-1) and cadmium (5 μmol L-1) in common duckweed (Lemna minor L.) by exposing it to either metal or to their combinations for four or seven days. Their uptake increased with time, but it was lower in plants treated with combinations of metals than in plants treated with either metal given alone. In separate treatments, either metal increased malondialdehyde (MDA) level and catalase and peroxidase activity. Both induced DNA damage, but copper did it only after 7 days of treatment. On day 4, the combination of cadmium and 5 μmol L-1 copper additionally increased MDA as well as catalase and peroxidase activity. In contrast, on day 7, MDA dropped in plants treated with combinations of metals, and especially with 2.5 μmol L-1 copper plus cadmium. In these plants, catalase activity was higher than in copper treated plants. Peroxidase activity increased after treatment with cadmium and 2.5 μmol L-1 copper but decreased in plants treated with cadmium and 5 μmol L-1 copper. Compared to copper alone, combinations of metals enhanced DNA damage after 4 days of treatment but it dropped on day 7. In conclusion, either metal given alone was toxic/genotoxic and caused oxidative stress. On day 4 of combined treatment, the higher copper concentration was more toxic than either metal alone. In contrast, on day 7 of combined treatment, the lower copper concentration showed lower oxidative and DNA damage. These complex interactions can not be explained by simple antagonism and/or synergism. Further studies should go in that direction. Učinak bakra na toksičnost i genotoksičnost kadmija u vodenoj leći (Lemna minor L.) U svrhu istraživanja interakcija između bakra kao esencijalnog elementa te kadmija kao neesencijalnog i toksičnog metala, vodenu leću Lemna minor L. uzgajali smo na podlogama s kadmijem (5 μmol L-1) odnosno s bakrom (2,5 μmol L-1 i 5 μmol L-1) te s njihovim kombinacijama. Unos metala u biljke povećavao se s trajanjem pokusa, a kod kombinacije metala u biljkama je izmjerena niža količina kadmija nego u onima uzgajanima samo na kadmiju. U biljkama tretiranim pojedinačnim metalom došlo je do povećanja sadržaja malondialdehida (MDA) te aktivnosti katalaze i peroksidaze u odnosu na kontrolne biljke. Također, primijećeno je oštećenje DNA iako kod bakra tek sedmog dana tretmana. Količina MDA i aktivnost obaju enzima dodatno se povećala na tretmanu kombinacijom kadmija i bakra (5 μmol L-1) nakon četvrtog dana pokusa, dok se količina MDA smanjila nakon sedmog dana kod kombinacije kadmija i 2,5 μmol L-1 bakra. U tim biljkama primijećena je i veća aktivnost katalaze, dok je aktivnost peroksidaze porasla na tretmanu kadmijem i 2,5 μmol L-1 bakrom, ali se smanjila na tretmanu kadmijem i 5 μmol L-1 bakrom. Oštećenje DNA koje je bilo veće kod kombinacije metala nakon četvrtog dana, osobito u usporedbi sa samim bakrom, smanjilo se nakon sedmog dana pokusa. Iz ovih rezultata može se zaključiti da su oba metala u istraživanim koncentracijama toksična i genotoksična za vodenu leću i da uzrokuju oksidacijski stres. Kadmij u kombinaciji s bakrom više koncentracije bio je toksičniji od pojedinačnih metala nakon četvrtog dana pokusa, dok su u biljaka tretiranih kombinacijom kadmija i bakra niže koncentracije toksični učinci bili manji. Budući da su primijećene interakcije vrlo kompleksne i ne uključuju samo antagonizam odnosno sinergizam potrebna su daljnja istraživanja.

Proteomics of heavy metal toxicity in plants

Summary Plants endure a variety of abiotic and biotic stresses, all of which cause major limitations to production. Among abiotic stressors, heavy metal contamination represents a global environmental problem endangering humans, animals, and plants. Exposure to heavy metals has been documented to induce changes in the expression of plant proteins. Proteins are macromolecules directly responsible for most biological processes in a living cell, while protein function is directly influenced by posttranslational modifications, which cannot be identified through genome studies. Therefore, it is necessary to conduct proteomic studies, which enable the elucidation of the presence and role of proteins under specific environmental conditions. This review attempts to present current knowledge on proteomic techniques developed with an aim to detect the response of plant to heavy metal stress. Significant contributions to a better understanding of the complex mechanisms of plant acclimation to metal stress are also discussed. Sažetak Biljke su neprestano izložene različitim čimbenicima abiotičkog i biotičkog stresa koji nepovoljno utječu na njihovu produktivnost. Teški metali kao česti zagađivači okoliša vrlo nepovoljno djeluju na sva živa bića, uključujući biljke, životinje i ljude. Poznato je da teški metali mogu mijenjati ekspresiju biljnih proteina. Proteine ubrajamo u biološki vrlo važne makromolekule čija je aktivnost u stanici izravno ovisna o posttranslacijskim modifikacijama, koje nije moguće pratiti na razini genoma. Stoga je nužno provoditi proteomska istraživanja kako bi se razotkrila prisutnost i uloga proteina u različitim vrstama okolišnog stresa. U ovom radu sažete su različite tehnike i metode istraživanja učinaka teških metala na biljni proteom, uključujući i sažet osvrt na složene mehanizme odgovora biljke na stres izazvan teškim metalima.

Cadmium and Zinc Induced Similar Changes in Protein and Glycoprotein Patterns in Tobacco (Nicotiana tabacum L.) Seedlings and Plants

The effects of 10 μmol L-1 and 15 μmol L-1 cadmium (Cd), a nonessential toxic element and 25 μmol L-1 and 50 μmol L-1 zinc (Zn), an essential micronutrient, on proteins and glycoproteins of Nicotiana tabacum L. seedlings and plants were investigated after exposure to each metal alone or to their combinations. Changes in only few polypeptides related to heavy metal treatments were observed in tobacco seedlings and leaves of adult plants, while the greatest change in total soluble protein pattern was observed in plant roots. Differences between control and treated tobacco tissues were more pronounced in the glycoprotein pattern, which was analysed by application of different lectins. The majority of the detected glycoproteins in leaves and roots of adult plants can be considered as a result of enhanced glycosylation due to heavy metal stress. The difference in glycoproteins between Cd and Zn application on tobacco seedlings and adult plants could not be determined since enhanced glycosylation was noticed after treatment with either metal alone or in combination. Therefore, it can be concluded that both metals induced N- and Oglycosylation as a result of changed environmental conditions.

Dynamics of heat-shock induced DNA damage and repair in senescent tobacco plants

Oxidative stress plays an important role in plant ageing and in response to different stresses. Oxidative DNA damage, unless repaired, may have detrimental consequences and increase genetic instability. Therefore, we determined the role of heat-shock induced oxidative stress on induction and repair of DNA damage in relation to oxidative stress tolerance in senescent tobacco plants. One-month-old (young) and three-month-old (senescent) plants were exposed to 42 °C for 2 and 4 h and left to recover at 26 °C for 24 and 72 h. The progression of senescence was characterized by the lower soluble protein and malondialdehyde content compared to young plants. Immediately after the heat shock, an increase in lipid peroxidation and guaiacol peroxidase activity, as well as DNA damage measured by the Comet assay were induced to higher extent in the young plants than in the senescent ones compared to their respective controls. Moreover, after 24-h recovery, the DNA damage further increased in the young plants whereas tendency of DNA repair was observed in the senescent plants. Upon 72-h recovery, no significant differences were noticed in all parameters studied (regardless of plant age) compared to the controls. The random amplified polymorphic DNA (RAPD) analysis confirmed genetic stability of the tobacco plants during the heat-shock exposures as well as the subsequent recovery periods.

Spine micromorphology of normal and hyperhydric Mammillaria gracilis Pfeiff. (Cactaceae) shoots.

Artificial conditions of tissue culture affect growth and physiology of crassulacean acid metabolism plants which often results in formation of hyperhydric shoots. In in vitro conditions Mammillaria gracilis Pfeiff. (Cactaceae) growth switches from organized to unorganized way, producing a habituated organogenic callus which simultaneously regenerates morphologically normal as well as altered hyperhydric shoots. In this study, influence of tissue culture conditions on morphology of cactus spines of normal and hyperhydric shoots was investigated. Spines of pot‐grown Mammillaria plants and of in vitro regenerated shoots were examined with stereo microscope and scanning electron microscope. The pot‐grown plants had 16–17 spines per areole. In vitro grown normal shoots, even though they kept typical shoot morphology, had lower number of spines (11–12) and altered spine morphology. This difference was even more pronounced in spine number (six to seven) and morphology of the hyperhydric shoots. Scanning electron microscopy analysis revealed remarkable differences in micromorphology of spine surface between pot‐grown and in vitro grown shoots. Spines of in vitro grown normal shoots showed numerous long trichomes, which were more elongated on spines of the hyperhydric shoots; the corresponding structures on spine surface of pot‐grown plants were noticed only as small protrusions. Scanning electron microscopy morphometric studies showed that the spines of pot‐grown plants were significantly longer compared to the spines of shoots grown in tissue culture. Moreover, transverse section shape varies from elliptical in pot‐grown plants to circular in normal and hyperhydric shoots grown in vitro. Cluster and correspondence analyses performed on the scanning electron microscope obtained results suggest great variability among spines of pot‐grown plants. Spines of in vitro grown normal and hyperhydric shoots showed low level of morphological variation among themselves despite the significant difference in shoot morphology.

Formation of embryogenic callus in hairy roots of pumpkin (Cucurbita pepo L.)

SummaryExcised cotyledons from 8-d-old pumpkin (Cucurbita pepo L.) seedlings were inoculated with Agrobacterium rhizogenes and cultured on hormone-free Murashige and Skoog medium. At the site of inoculation, transformed hairy roots were successfully induced by using wild strains 8196 (mannopine-type) and 15834 (agropine-type). After a subsequent transfer on a solid MS medium without hormones, roots obtained by transformation with strain 15834 failed to form stable hairy root cultures, while several hairy root lines were established with strain 8196. Three hairy root lines, Cp1, Cp2, and Cp31, have spontaneously generated callus with embryo-like structures after more than 3 yr of growth on the solid medium. The callus proliferation was more frequent when the autoclaving of nutrient medium, pH 5.7, was prolonged to 30 min. Separated calluses continued to proliferate and generated embryos with abnormal morphology. The combination of indole-3-acetic acid and benzyladenine had a favorable influence on embryogenesis and organogenesis in the Cp31 callus line. The Southern analysis of Cp31 root and embryo DNA confirmed the presence of the T-DNA of Agrobacterium rhizogenes.

In vitro conditions affect photosynthetic performance and crassulacean acid metabolism in Mammillaria gracilis Pfeiff. tissues

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 M. 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 β subunit of ATP synthase (β 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.