Screening of drought oxidative stress tolerance in Serbian melliferous plant species

This study was designed to examine and compare antioxidant and free-radical scavenging activities of leaves of six different melliferous plant species (Populus alba, Robinia pseudoacacia, Sophora japonica, Euodia hupehensis, Tilia sp., Fraxinus sp.) from Serbia in order to evaluate their drought oxidative stress tolerance. Experiment was conducted during June, July and August. In this study, we reported the results concerning proline accumulation, soluble protein content, quantities of malonyldialdehyde,total antioxidant capacity determined by FRAP method and scavenger activity determined by DPPH method. According to our results, all melliferous plant species were subjected to drought oxidative stress during July when soil humidity decreased. During July, proline content and MDA quantity increased and soluble proteins decreased in all investigated species. High and permanent antioxidant activity during the whole investigated period was observed in P. alba, but insufficient to protect itsleaves from oxidative injury during the period of drought in July. The highest ability to accumulate proline and highest protein content under severe drought stress in July was observed in Fraxinus sp. Other investigated antioxidant parameters (total antioxidant and DPPH radical scavenger capacities) were high and accumulation of MDA was low which indicate high drought oxidative stress tolerance. Therefore, highest ability to adapt under severe drought stress and highest drought oxidative stresstolerance were observed in Fraxinus sp.Key words: Melliferous trees, lipid peroxidation, DPPH, FRAP, proline accumulation

UTJECAJ ONEČIŠĆENOSTI TLA TEŠKIM METALIMA NA KLONOVE TOPOLA IZ SRBIJE

Oxidative stress is known as disturbed balance between antioxidative protection mechanism and production of reactive oxygen species, which can negatively influence on normal biological and metabolical processes in living organisms, such as poplar species. Phytoremediation is promising biotechnical method of cleaning of polluted soils by various pollutants: heavy metals, organic contaminants, pesticides, oil etc. Until today, poplars showed potential for regenerating polluted soils during phytoremediation process. This study represents results of oxidative stress profiles of three poplar clones (M1, B229 and PE 19/66) shoots from Institute of Lowland Forestry and Environment, University of Novi Sad, Serbia, while being treated by different concentration of three heavy metals in soil: Ni3+, Cu2+ and Cd2+. Biochemical parameters of oxidative stress which have been analyzed were: content of soluble proteins, intensity of lipid peroxidation, antioxidative capacity by ferric reducing antioxidative power assay and activity of superoxid dismutase. Results showed that the most acceptable phytoremediation response to heavy metal pollution in soil showed clone M1. Great differences between B229 and PE 19/66 clones were in response on soil heavy metal contamination, directly suggesting of not being suitable for possible phytoremediation application.Oksidacijski stres je poznat kao narušena ravnoteža između antioksidacijskog mehanizma zaštite i proizvodnje reaktivnih kisikovih vrsta, što može negativno utjecati na normalne biološke i metaboličke procese u živim organizmima, kao na primjer kod topola. U cilju opstanka, aerobni organizmi su stekli mehanizme antioksidacijske zaštite, gdje su od enzimskih najistraženije aktivnosti antioksidativnih enzima kao što su katalaze, peroksidaze, glutation peroksidaze i superoksid dismutaza. Fitoremedijacija je obećavajuća biotehnička metoda čišćenja zagađenih tala raznim onečišćujućim tvarima poput: teških kovina, organskih kontaminanata, pesticida i dr. Do danas, topole su pokazale potencijal za regeneraciju onečišćenih tala tijekom fitoremedijacijskog procesa.Biokemijsko profiliranje statusa oksidacijskog stresa u drvenastim biljkama nije često istraživana u Srbiji i stoga je cilj ovoga pokusa bio ispitati utjecaj različitih koncentracija tri jona teških kovina, Ni3+, Cu2+ i Cd2+ na razinu oksidacijskog stresa tri klona, dvije različite vrste topole (Populus euramericana-M1; PE 19/66 i B-229 oba Populus deltoides vrsta). Biokemijski parametri za određivanje razine oksidacijskog stresa su: lipidna peroksi­dacija (LPx), test redukcijske snage željeza (FRAP), superoksid dismutaze (SOD) aktivnost i sadržina topivih bjelančevina. Ovi rezultati mogli bi dati smjernice u odabiru klonova iz kolekcije Instituta za nizijsko šumarstvo i životnu sredinu Univerziteta u Novom Sadu za buduće fitoremedijacijske uporabe ovih genotipova topola. Pješčano fluvisol tlo u pokusnim posudama je zagađeno različitim tretmanima teških kovina, koji su prikazani u tablici 1. Biljni ekstrakti izrađeni su od 2 g biljnog materijala (izbojci) i homogenizirani s kvarcnim pijeskom i suspenzirani u 10 cm3 0,1 mol/dm3 K2HPO4 pH 7,0. Homogenati su centrifugirani za 10 min na 4000 g (Quy Hai sur., 1975). Sva očitavanja apsorbancija za sadržaj topivih proteina, SOD i FRAP odrađeno pomoću Janway UV / VIS spektrofotometra 6505 i za čitanje količine nastalog MDA korišten je višefrekvencijski Spectrum Termo Corporation.Sve analize izvedene su u tri ponavljanja. Statistička usporedba između uzoraka izvedena je u programu Statistica 9, koristeći Duncan test, s statističkom značajnosti p <0,05. Dobiveni rezultati prikazani su grafički preko histograma (slike 1. do 4.) i iznad njih su slova koja označuju statistički značajne razlike između rezultata i kontrola (od a do e).Usporedni postotak je izračunat prema formuli: Δ (%) = (100 * uzorak / kontrola) –100, gdje vrijednosti mogu rezultirati pozitivno (+) ako je došlo do povećanja u odnosu na kontrolu i rezultiralo negativno (–), ako je došlo do smanjenja u odnosu na kontrolu.Trudić i sur. (2012) analizirali su kroz iste metode okdisacijskog stresa ekstrakte lišća i korijena spomenutih klonova topola. Uspoređujući ove rezultate, B229 klon je kroz lišće i korijenje pokazao više prihvatljiv antioksidacijski odgovor, dok je, u ovom istraživanju, M1 klon pokazao najprihvatljivije antioksidacijske odgovore kroz ekstrakte izbojaka. Parametri iz Pilipović i sur. (2012), Trudić i sur. (2012) i naše studije, mogu predstavljati udružen fiziološki i antioksidacijski marker sustav za buduće različite fitoremedijacijske primjene ovih klonova na tlima onečišćenim raznim tvarima.Rezultati ovog istraživanja mogu ukazati na genotipske specifičnosti svih ispitivanih biokemijskih parametara i obilježava klonove topola, kao što je M1 kao primarni, a B229 klon kao potencijalno sljedeći u primjeni fitoremedijacije tala onečišćenih teškim kovinama. Ipak, za stjecanje novih, dubljih rezultata u vezi s oksidacijskim stresom izazvanim onečišćavanjem tala teškim kovinama, potrebne su dalje in vitro antioksidacijske analize

Chemical parameters of oxidative stress adaptability in beech

e antioxidant activity, lipid peroxidation, and contents of free proline and soluble proteins were investigated on six-year-old beech plants. Provenance Avala, in Serbia, had the best adaptability to environmental factors on locality Fruska Gora due to low lipid peroxidation, high FRAP value, and free proline and soluble proteins contents. Provenances Scharnstein and Mitterndorf, in Austria, had the best adaptability to environmental factors on locality Debeli Lug due to high FRAP value and free proline and soluble proteins contents. FRAP values in majority of provenances from locality Debeli Lug were higher. Correlations parameters were much higher between provenances in locality Debeli Lug, situated at higher altitude, which is the consequence of better adaption to environmental factors in�uence

Biochemical and gene expression analyses in different poplar clones: the selection tools for afforestation of halomorphic environments

Halomorphic soils cover a significant area in the Vojvodina region and represent ecological and economic challenges for agricultural and forestry sectors. In this study, four economically important Serbian poplar clones were compared according to their biochemical and transcriptomic responses towards mild and severe salt stress to select the most tolerant clones for afforestation of halomorphic soils. Three prospective clones of Populus deltoides (Bora-B229, Antonije-182/81 and PE19/66) and one of hybrid genetic background P. nigraxP. deltoides, e.g., P. x euramericana (Pannonia-M1) were hydroponically subjected to NaCl as a salt stress agent in a concentration range from 150 mM to 450 mM. Plant responses were measured at different time periods in the leaves. Biochemical response of poplar clones to salt stress was estimated by tracking several parameters such as different radical scavenging capacities (estimated by DPPH, FRAP and ABTS assays), accumulation of total phenolic content and flavonoids. Furthermore, accumulation of two osmolytes, glycine betaine and proline, were quantified. The genetic difference of those clones has been already shown by single nucleotide polymorphisms (SNPs) but this paper emphasized their differences regarding biochemical and transcriptomic salt stress responses. Five candidate genes, two putative poplar homologues of GRAS family TFs (PtGRAS17 and PtGRAS16), PtDREB2 of DREB family TFs and two abiotic stress-inducible genes (PtP5SC1, PtSOS1), were examined for their expression profiles. Results show that most salt stress-responsive genes were induced in clones M1 and PE19/66, thus showing they can tolerate salt environments with high concentrations and could be efficient in phytoremediation of salt environments. Clone M1 and PE19/66 has ABA-dependent mechanisms expressing the PtP5CS1 gene while clone 182/81 could regulate the expression of the same gene by ABA-independent pathway. To improve salt tolerance in poplar, two putative GRAS/SCL TFs and PtDREB2 gene seem to be promising candidates for genetic engineering of salt-tolerant poplar clones

Biological activities of the lignicolous fungus Meripilus giganteus (Pers.: Pers.) Karst.

Crude extracts (methanolic-ME andwater-WE) of the fungus Meripilus giganteus (Pers.: Pers.) Karst. were analyzed for their antioxidative, antibacterial, cytotoxic, neurotoxic, and hemolytic activities using tests in vitro. The highest scavenging activity was exhibited by WE on OH• radicals, showing a 50% effective concentration (EC50) at 292.83±2.5 μg/ml and ferric-reducing ability at 14.24±2.9 mg eq of ascorbic acid per g of dried extract (mg/g).An anti­bacterial effect was detected mostly against Gram-positive strains of bacteria. Toxicity assays exhibited higher effects for ME, reaching EC50=403.43±5.8 μg/ml against estrogen-dependent breast cancer cell lines (MCF-7); 50% hemolytic activity at a concentration of 30±0.03 μg/ml against erythrocytes; andEC50=4.5±0.05 mg/ml for acetyl cholinesterase-inhibitory activity. These results suggest possible use of this fungus as a new source of pharmaceuticals

Biochemical and Gene Expression Analyses in Different Poplar Clones: The Selection Tools for Afforestation of Halomorphic Environments

Halomorphic soils cover a significant area in the Vojvodina region and represent ecological and economic challenges for agricultural and forestry sectors. In this study, four economically important Serbian poplar clones were compared according to their biochemical and transcriptomic responses towards mild and severe salt stress to select the most tolerant clones for afforestation of halomorphic soils. Three prospective clones of Populus deltoides (Bora-B229, Antonije-182/81 and PE19/66) and one of hybrid genetic background P. nigra x P. deltoides, e.g., P. x euramericana (Pannonia-M1) were hydroponically subjected to NaCl as a salt stress agent in a concentration range from 150 mM to 450 mM. Plant responses were measured at different time periods in the leaves. Biochemical response of poplar clones to salt stress was estimated by tracking several parameters such as different radical scavenging capacities (estimated by DPPH, FRAP and ABTS assays), accumulation of total phenolic content and flavonoids. Furthermore, accumulation of two osmolytes, glycine betaine and proline, were quantified. The genetic difference of those clones has been already shown by single nucleotide polymorphisms (SNPs) but this paper emphasized their differences regarding biochemical and transcriptomic salt stress responses. Five candidate genes, two putative poplar homologues of GRAS family TFs (PtGRAS17 and PtGRAS16), PtDREB2 of DREB family TFs and two abiotic stress-inducible genes (PtP5SC1, PtSOS1), were examined for their expression profiles. Results show that most salt stress-responsive genes were induced in clones M1 and PE19/66, thus showing they can tolerate salt environments with high concentrations and could be efficient in phytoremediation of salt environments. Clone M1 and PE19/66 has ABA-dependent mechanisms expressing the PtP5CS1 gene while clone 182/81 could regulate the expression of the same gene by ABA-independent pathway. To improve salt tolerance in poplar, two putative GRAS/SCL TFs and PtDREB2 gene seem to be promising candidates for genetic engineering of salt-tolerant poplar clones