Propagation and antioxidative enzymes of Campanula velebitica Borbás grown in vitro

Background and purpose: Micropropagation possibility for Campanula velebitica Borbás (Campanulaceae), a rare Croatian endemic plant was studied. Additionally, the activity of several antioxidative enzymes, as biomarkers of stress conditions, was investigated in plants grown in vitro. Materials and methods: Shoots from aseptically germinated seeds, collected from natural habitat,were used for culture initiation. Multiplication rate of shoots was monitored after six weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0, 0.2, 0.5 and 1.0 M m6-benzylaminopurine (BA). Rooting was induced on half-strength MS medium supplemented with 2.9 or 5.7 mM indole-3-acetic acid (IAA); or 2.5 or 4.9 mM indole-3-butyric acid (IBA). Activity of ascorbat (APX) and guaiacol (GPOD) peroxidases as well as catalase (CAT) was determined in shoot extracts spectrophotometrically and in gel. Results and conclusions: The highest multiplication rate, 7.7 shoots per explant, was achieved in the 3rd subculture on the medium containing 1.0 mMBA. Very good rooting (>90%) of excised shootswas achieved on media containing either indole-3-butyric acid (IBA) or indole-3-acetic acid (IAA). On the medium containing 4.9 mM IBA flowering was observed. Activities of APX and GPOD were significantly increased onmedia containing lower concentrations of BA (0.2 and 0.5 mM).Native PAGE analysis revealed one CAT, three GPOD and two APX isoenzymes. We conclude that C. velebitica is easily propagated in in vitro conditions, without substantial stress response. With further studies of plantlet acclimation, this endemic plant species could be efficiently preserved through culture in vitro

Photochemical efficiency, content of photosynthetic pigments and phenolic compounds in different pitcher parts of Sarracenia hybrids

Sarracenia is 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 effi ciency 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 different Sarracenia hybrids. 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 chlorophyll a than 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

Transgenerational stress memory in Arabidopsis thaliana (L.) Heynh.: antioxidative enzymes and HSP70

Transgenerational transmission of information about stress exposure is manifested as an increase in the somatic homologous recombination frequency in plants. Our aim was to investigate whether information about changes of antioxidative enzyme activities and protein HSP70 induction are also transmitted in response to stress caused by UV-C irradiation. These stress indicators were investigated in Arabidopsis thaliana plants exposed to UV-C irradiation (6 and 600 J m-2) and its non-irradiated progeny. The activity of catalase was significantly decreased in the irradiated plants in comparison to the non-irradiated control plants, while the activity of guaiacol peroxidase was increased. The ascorbate peroxidase activity was not significantly changed. In irradiated plants there was an induction of a new HSP70 protein isoform. In the non- irradiated progeny of irradiated plants, a significant decrease in catalase and ascorbate peroxidase activity was noticed in comparison to plants whose parents were not irradiated. There was no significant change in guaiacol peroxidase activity or induction of HSP70 isoforms in the progeny. The obtained results indicate that, besides the already known increase in frequency of somatic homologous recombination, transmission of information about stress exposure can also include changes in activities of antioxidative enzymes catalase and ascorbate peroxidase. The explanations for the observed changes and the mechanism by which they occur have to be established in further research

Effect of calcium chloride and calcium bromide on chloroplasts of Lemna minor L.

Saturated water solutions of CaCl2, CaBr, and a 1:1 mixture of them are commonly used as »high density brines« for pressure control in oil wells. To investigate the effect of these chemicals on chloroplast morphology and their sedimentation profile in a sucrose gradient, duckweed, Lemna minor L., was chosen as a test organism. By light microscopy, round to oval chloroplasts in control plants and in plants grown on media supplemented with 0.025 and 0.050 mol dm-3 of tested salts were observed. However, in plants grown on media containing higher concentrations of the salts ested (0.075 and 0.1 mol dm-3) most of the chloroplasts were of irregular shape, and bigger. The sedimentation profile of the control and the treated chloroplasts (based on chlorophyll determination in 200-pi fractions) also differed. Control samples showed three peaks, while treated samples showed an additional peak (fractions 5-8). One of the peaks obtained in the treated samples was sharper and more intensive than the peak present at the same position in the control extracts. The results obtained showed changes in chloroplast morphology and sedimentation profile after treatment with the higher concentrations (0.075 and 0.1 mol dm-3) of the salts tested that could be the consequence of starch accumulation in chloroplasts

The impact of cadmium on photosynthetic performance and secondary metabolites in the lichens Parmelia sulcata, Flavoparmelia caperata and Evernia prunastri

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 signifi cantly changed Fv/Fm and RFd only after eight days of exposure. Quantifi cation 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

The effect of salinity and osmotic stress on duckweed Lemna minor L.

The response of duckweed (Lemna minor L.) to salinity and osmotic stress was evaluated by monitoring growth and peroxidase activity every second day, for a period of 17 days. The nutrient medium was supplemented with isoosmolar concentrations of NaCl and mannitol (50 mM and 100 mM NaCl, 100 mM and 200 mM mannitol). Growth decreased markedly with increasing concentrations of NaCl and mannitol. The lower concentrations of NaCl (50 mM) and mannitol (100 mM) started to reduce growth significantly in the second week of the experiment, while the higher concentrations (100 and 200 mM) reduced growth from the beginning. Protein concentrations and peroxidase activity fluctuated during the growth period but, most of the time, they were increased in plants treated with NaCl and mannitol. These results suggest that Lemna minor is sensitive to both salinity and osmotic stress. However, the continued growth, and also the increase in peroxidase activity and protein concentration, during the NaCl and mannitol treatments, demonstrate its potential for adaptation to long-term stress. Although salinity and osmotic stress have similar overall effects on Lemna minor, consistent minor differences in growth, protein concentration and peroxidase activity between plants grown on NaCl and those grown on mannitol suggest overlapping, rather than identical, mechanisms of adaptation to salinity and osmotic stress

Response of dihaploid tobacco roots to salt stress

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

Antibacterial Activity of Pinus pinaster Bark Extract and its Components Against Multidrug-resistant Clinical Isolates of Acinetobacter baumannii

The aim of this research was to test the antibacterial activity of Pinus pinaster aqueous bark extract (PABE) and its basic components against multidrug-resistant isolates of Acinetobacter baumannii belonging to European clone I and II, isolated previously from the clinical outbreaks. The minimum bactericidal concentration of PABE against both clones of A. baumannii was 200 mg ml–1, while lower concentrations showed high antibacterial activity. After 24 h of treatment with 100, 50 or 10 mg ml–1 of extract, the reduction in the number of A. baumannii isolates belonging to European clone I and II was 85.8 ± 2.5 %, 78.5 ± 1.1 %, 66.3 ± 2.5 % and 90.2 ± 1.7 %, 78.6 ± 1.2 %, 69.8 ± 0.7 %, respectively. Several basic components: caffeic acid, catechin, epicatechin, gallic acid and vanillin, detected in the extract by high performance liquid chromatography, contributed to the antibacterial activity of the extract against both clones of A. baumannii. However, the antibacterial activity of extract was higher than that of each tested basic component suggesting that proanthocyanidins, which were present in quite a large amount in the extract, might have also contributed to the activity of the extract. Antibacterial activity of PABE against A. baumannii reveals that complex and inexpensive natural product might be useful in combat against naturally competent bacteria that easily acquire resistance against antibiotics

Response of Kalanchoe daigremontiana to wounding and infection with Agrobacterium tumefaciens

Background and Purpose: Transformation of plant tissue with Agrobacterium tumefaciens includes wounding of plant and subsequent infection by bacteria. Polyphenol oxidase activity and oxidative stress parameters – the content of H2O2, as well as activity and isoenzymes of antioxidative enzymes catalase, pyrogallol and guaiacol peroxidase were investigated as markers of plant response to wounding and infection. Materials and Methods: Five tissue types – healthy tissue, wounded tissue, tissue in immediate proximity of the wound, tumour tissue and tissue next to the tumourwere collected on 7th, 14th and 21st day afterwounding or infection. Activities of antioxidative enzymes were evaluated spectrophotometrically. Isoenzymes were separated electrophoretically in native conditions. H2O2 content was estimated by measuring the titanium hydro--peroxide complex. Results and Conclusion: All measured parameters were remarkably different in tumour in comparison to healthy tissue. Activities of peroxidases were significantly increased, accompanied with induction of new isoenzymes. After two weeks of the experiment catalase activity was also significantly increased in tumour tissue as well as in tissue next to the wound. H2O2 content was lower in tumour after three weeks. Aside from tumour tissue, polyphenol oxidase activity was significantly increased in tissue next to the tumour. Remarkably increased peroxidase and polyphenol oxidase activity in tumour tissue, additional isoenzymes of peroxidases as well as decreased H2O2 content, indicated changes in tissue organisation and induction of defence mechanisms against pathogen, in which these two groups of enzymes are involved.Wounded tissue, as well as tissue next to the wound or infection, did not show significant variation from healthy tissue

Oksidacijski stres u tkivu hrena (Armoracia lapathifolia Gilib.) uzgojenom u uvjetima in vitro

In a previous study it was reported that transformed tissue of horseradish (Armoracia lapathifolia Gilib.), obtained by infection of leaf explants with A. tumefaciens, developed two tumour lines with different morphology. One line grew as a completely unorganized tissue (TN – tumour tissue), while the other line grew as a partially organized teratogenous tumour with malformed hyperhydric shoots (TM – teratoma tissue), but did not regenerate the whole plant of normal morphology. The factor responsible for this problem could be the increased production of reactive oxygen species (ROS). Therefore, in this study a possible involvement of activated oxygen metabolism in dedifferentiation and hyperhydricity in TM and TN tissues is investigated. Elevated values of malondialdehyde and protein carbonyl contents found in TM and TN, in comparison with plantlet leaf, confirm the presence of oxidative stress. However, lower H2O2 content was measured in TM and TN. Lipoxygenase (LOX) activity was more pronounced in TM and especially in TN compared to leaf, which suggests that the LOX-dependent peroxidation of fatty acids might be one of the causes of oxidative damage. Moreover, significantly higher peroxidase (PRX) and ascorbate peroxidase (APX) activity as well as the increased number of their isoforms was found in transformed TM and TN in comparison with leaf. On the other hand, significantly lower superoxide dismutase (SOD) activity was found in TM and TN, which correlates with lower H2O2 content. High catalase (CAT) activity measured in leaf and partially organized TM is consistent with the role of CAT in growth and differentiation. In conclusion, in horseradish transformed tissues that underwent dedifferentiation and hyperhydricity, prominent oxidative damage was found. This result suggests that oxidative stress could be associated with the inability of partially organized teratogenous TM to regenerate plantlets with normal morphology.Dosadašnja istraživanja autora pokazala su da je transformirano tkivo hrena (Armoracia lapathifolia Gilib.), nakon infekcije bakterijom A. tumefaciens, razvilo dvije morfološki različite linije tumorskog tkiva. Jedna je linija rasla kao potpuno neorganizirano tumorsko tkivo, a druga kao djelomično organizirani teratogeni tumor s malformiranim hiperhidriranim izdancima (teratomska linija), ali bez mogućnosti regeneriranja biljke normalnih morfoloških karakteristika. Budući da bi povećana proizvodnja reaktivnih oblika kisika mogla uzrokovati promjenu morfoloških karakteristika, u ovom je radu istraživan mogući utjecaj kisika na dediferencijaciju i hiperhidriranost u potpuno neorganiziranom tumorskom tkivu i teratomskoj liniji. Uspoređujući povećane vrijednosti malondialdehida i proteinskih karbonila u potpuno neorganiziranom tumorskom tkivu i teratomskoj liniji s onima u listu hrena, uočena je pojava oksidacijskog stresa iako je izmjeren manji udjel H2O2. Aktivnost lipoksigenaze povećana je u teratomskoj liniji, a još više u potpuno neorganiziranom tumorskom tkivu, što pokazuje da bi peroksidacija masnih kiselina ovisna o aktivnosti lipoksigenaze mogla biti jedan od uzroka oksidacijskoga stresa. Štoviše, u transformiranim je tkivima za razliku od lista uočena bitno veća aktivnost peroksidaze i askorbat peroksidaze te veći broj njihovih izoenzima. S druge strane, izmjerena je kudikamo manja aktivnost superoksid dismutaze, s obzirom na smanjeni udjel H2O2. Veća aktivnost katalaze u listu i teratomskoj liniji povezana je s ulogom katalaze u rastu i diferencijaciji. Dobiveni rezultati potvrđuju prisutnost oksidacijskog stresa u dediferenciranim i hiperhidriranim tumorskim tkivima hrena, odnosno da je oksidacijski stres posljedica nesposobnosti teratomske linije da regenerira biljke normalne morfologije