Vuk Maksimović

Silicon alleviates iron deficiency in cucumber by promoting mobilization of iron in the root apoplast

· Root responses to lack of iron (Fe) have mainly been studied in nutrient solution experiments devoid of silicon (Si). Here we investigated how Si ameliorates Fe deficiency in cucumber (Cucumis sativus) with focus on the storage and utilization of Fe in the root apoplast. · A combined approach was performed including analyses of apoplastic Fe, reduction-based Fe acquisition and Fe-mobilizing compounds in roots along with the expression of related genes. · Si-treated plants accumulated higher concentrations of root apoplastic Fe, which rapidly decreased when Fe was withheld from the nutrient solution. Under Fe-deficient conditions, Si also increased the accumulation of Fe-mobilizing compounds in roots. Si supply stimulated root activity of Fe acquisition at the early stage of Fe deficiency stress through regulation of gene expression levels of proteins involved in Fe acquisition. However, when the period of Fe deprivation was extended, these reactions further decreased as a consequence of Si-induced enhancement of the Fe status of the plants. · This work provides new evidence for the beneficial role of Si in plant nutrition and clearly indicates that Si-mediated alleviation of Fe deficiency includes an increase of the apoplastic Fe pool in roots and an enhancement of Fe acquisition.

Generation of hydroxyl radical in isolated pea root cell wall, and the role of cell wall-bound peroxidase, Mn-SOD and phenolics in their production.

The hydroxyl radical produced in the apoplast has been demonstrated to facilitate cell wall loosening during cell elongation. Cell wall-bound peroxidases (PODs) have been implicated in hydroxyl radical formation. For this mechanism, the apoplast or cell walls should contain the electron donors for (i) H(2)O(2) formation from dioxygen; and (ii) the POD-catalyzed reduction of H(2)O(2) to the hydroxyl radical. The aim of the work was to identify the electron donors in these reactions. In this report, hydroxyl radical (.OH) generation in the cell wall isolated from pea roots was detected in the absence of any exogenous reductants, suggesting that the plant cell wall possesses the capacity to generate .OH in situ. Distinct POD and Mn-superoxide dismutase (Mn-SOD) isoforms different from other cellular isoforms were shown by native gel electropho-resis to be preferably bound to the cell walls. Electron paramagnetic resonance (EPR) spectroscopy of cell wall isolates containing the spin-trapping reagent, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), was used for detection of and differentiation between .OH and the superoxide radical (O(2)(-).). The data obtained using POD inhibitors confirmed that tightly bound cell wall PODs are involved in DEPMPO/OH adduct formation. A decrease in DEPMPO/OH adduct formation in the presence of H(2)O(2) scavengers demonstrated that this hydroxyl radical was derived from H(2)O(2). During the generation of .OH, the concentration of quinhydrone structures (as detected by EPR spectroscopy) increased, suggesting that the H(2)O(2) required for the formation of .OH in isolated cell walls is produced during the reduction of O(2) by hydroxycinnamic acids. Cell wall isolates in which the proteins have been denaturated (including the endogenous POD and SOD) did not produce .OH. Addition of exogenous H(2)O(2) again induced the production of .OH, and these were shown to originate from the Fenton reaction with tightly bound metal ions. However, the appearance of the DEPMPO/OOH adduct could also be observed, due to the production of O(2)(-). when endogenous SOD has been inactivated. Also, O(2)(-). was converted to .OH in an in vitro horseradish peroxidase (HRP)/H(2)O(2) system to which exogenous SOD has been added. Taken together with the discovery of the cell wall-bound Mn-SOD isoform, these results support the role of such a cell wall-bound SOD in the formation of .OH jointly with the cell wall-bound POD. According to the above findings, it seems that the hydroxycinnamic acids from the cell wall, acting as reductants, contribute to the formation of H(2)O(2) in the presence of O(2) in an autocatalytic manner, and that POD and Mn-SOD coupled together generate .OH from such H(2)O(2).

Silicon ameliorates manganese toxicity in cucumber by decreasing hydroxyl radical accumulation in the leaf apoplast

This work was focused on the role of silicon (Si) in amelioration of manganese (Mn) toxicity caused by elevated production of hydroxyl radicals (·OH) in the leaf apoplast of cucumber (Cucumis sativus L.). The plants were grown in nutrient solutions with adequate (0.5 μM) or excessive (100 μM) Mn concentrations with or without Si being supplied. The symptoms of Mn toxicity were absent in the leaves of Si-treated plants subjected to excess Mn, although the leaf Mn concentration remained extremely high. The apoplastic concentration of free Mn(2+) and H(2)O(2) of high Mn-treated plants was significantly decreased by Si treatment. Si supply suppressed the Mn-induced increased abundance of peroxidase (POD) isoforms in the leaf apoplastic fluid, and led to a rapid suppression of guaiacol-POD activity under excess Mn. The spin-trapping reagent 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide was used to detect ·OH by electron paramagnetic resonance spectroscopy. Although supplying Si markedly decreased the accumulation of ·OH in the leaf apoplast with excess Mn, adding monosilicic acid to the Mn(2+)/H(2)O(2) reaction mixture did not directly affect the Fenton reaction in vitro. The results indicate that Si contributes indirectly to a decrease in ·OH in the leaf apoplast by decreasing the free apoplastic Mn(2+), thus regulating the Fenton reaction. A direct inhibitory effect of Si on guaiacol-POD activity (demonstrated in vitro) may also contribute to decreasing the POD-mediated generation of ·OH.

Anti-tumor effect of Coriolus versicolor methanol extract against mouse B16 melanoma cells: in vitro and in vivo study.

Numerous studies have shown immunostimulatory and anti-tumor effects of water and standardized aqueous ethanol extracts derived from the medicinal mushroom, Coriolus versicolor, but the biological activity of methanol extracts has not been examined so far. In the present study we investigated the anti-tumor effect of C. versicolor methanol extract (which contains terpenoids and polyphenols) on B16 mouse melanoma cells both in vitro and in vivo. In vitro treatment of the cells with the methanol extract (25-1600 microg/ml) reduced melanoma cell viability in a dose-dependent manner. Furthermore, in the presence of the methanol extract (200 microg/ml, concentration IC(50)) the proliferation of B16 cells was arrested in the G(0)/G(1) phase of the cell cycle, followed by both apoptotic and secondary necrotic cell death. In vivo methanol extract treatment (i.p. 50 mg/kg, for 14 days) inhibited tumor growth in C57BL/6 mice inoculated with syngeneic B16 tumor cells. Moreover, peritoneal macrophages collected 21 days after tumor implantation from methanol extract-treated animals exerted stronger tumoristatic activity ex vivo than macrophages from control melanoma-bearing mice. Taken together, our results demonstrate that C. versicolor methanol extract exerts pronounced anti-melanoma activity, both directly through antiproliferative and cytotoxic effects on tumor cells and indirectly through promotion of macrophage anti-tumor activity.

Contrasting effect of silicon on iron, zinc and manganese status and accumulation of metal-mobilizing compounds in micronutrient-deficient cucumber.

Although the beneficial role of silicon (Si) in alleviation of abiotic stress is well established, little is known of the relevance of Si nutrition under microelement deficiency. The aim of our work was to investigate the physiological role of Si in relation to micronutrient (Fe, Zn and Mn) deficiencies in cucumber (Cucumis sativus L.). Cucumber (cv. Semkross) plants were grown hydroponically in a complete nutrient solution (control) and in nutrient solutions free from Fe, Zn or Mn, with or without Si supply. Plant tissue concentrations of microelements, organic acids and phenolics were measured. Si supply effectively mitigated the symptoms of Fe deficiency, but only in part, the symptoms of Zn- or Mn deficiency. Leaf Fe concentration significantly increased in plants deprived of Fe but treated with Si, whereas the concentrations of other microelements were not affected by Si supply. The effects of Si supply in increasing accumulation of both organic acids and phenolic compounds in cucumber tissues were exclusively related to Fe nutrition. Enhancement of Fe distribution towards apical shoot parts, along with the tissue accumulation of Fe-mobilizing compounds such as citrate (in leaves and roots) or cathechin (in roots) appears to be the major alleviating effect of Si. Si nutrition, however, was without effect on the mobility and tissue distribution of either Zn or Mn.

Effects of 5-fluorouracil on erythrocytes in relation to its cardiotoxicity : Membrane structure and functioning

In the present study, we showed that the antineoplastic drug 5-fluorouracil (5-FU) induces in vitro exposure-time/dose-dependent changes on the level of an erythrocytes morphology, ionic balance, and membrane fluidity. These changes are partially or fully irreversible, and we suggest that they are provoked by an irreversible depletion of ATP. Because of these changes that could also occur in vivo during 5-FU infusion, a certain amount of erythrocytes with echinocytic shape and diminished ability to deliver oxygen is present in blood for longer periods of time. This renders oxygen transport and delivery more difficult, leaving the heart with an insufficient supply of oxygen, thus leading to cardiotoxicity.

Rutin, a flavonoid with antioxidant activity, improves plant salinity tolerance by regulating K+ retention and Na+ exclusion from leaf mesophyll in quinoa and broad beans

The causal relationship between salinity and oxidative stress tolerance is well established, but specific downstream targets and the role of specific antioxidant compounds in controlling cellular ionic homeostasis remains elusive. In this work, we have compared antioxidant profiles of leaves of two quinoa genotypes contrasting in their salt tolerance, with the aim of understanding the role of enzymatic and non-enzymatic antioxidants in salinity stress tolerance. Only changes in superoxide dismutase activity were correlated with plant adaptive responses to salinity. Proline accumulation played no major role in either osmotic adjustment or in the tissue tolerance mechanism. Among other non-enzymatic antioxidants, rutin levels were increased by over 25 fold in quinoa leaves. Exogenous application of rutin to glycophyte bean leaves improved tissue tolerance and reduced detrimental effects of salinity on leaf photochemistry. Electrophysiological experiments revealed that these beneficial effects were attributed to improved potassium retention and increased rate of Na+ pumping from the cell. The lack of correlation between rutin-induced changes in K+ and H+ fluxes suggest that rutin accumulation in the cytosol scavenges hydroxyl radical formed in response to salinity treatment thus preventing K+ leak via one of ROS-activated K+ efflux pathways, rather than controlling K+ flux via voltage-gated K+-permeable channels.

Nepetalactone content in shoot cultures of three endemic Nepeta species and the evaluation of their antimicrobial activity.

Rapid micropropagation of Nepeta rtanjensis, N. sibirica and N. nervosa was performed. Qualitative and quantitative nepetalactone content in methanol extracts of in vitro grown plants was analysed by reverse-phase HPLC coupled with UV and MS detection. Only trans,cis-nepetalactone was detected in shoots of N. rtanjensis, while cis,trans-nepetalactone stereoisomer was present in N. sibirica. No nepetalactone was observed in shoots of N. nervosa. The antimicrobial activity of methanol extracts, against eight bacterial and eight fungal species, was evaluated. All the tested extracts showed significant antibacterial and strong antifungal activity. However, N. rtanjensis extract exhibited the best antimicrobial potential.

Differential response of antioxidative systems of maize (Zea mays L.) roots cell walls to osmotic and heavy metal stress.

An analysis of peroxidase and ascorbate oxidase activity, phenolic content and antioxidant capacity of isolated maize root cell walls was performed in controls and plants stressed with polyethylene glycol (PEG) or heavy metals, zinc or copper. Peroxidase activity (oxidative and peroxidative) was more pronounced in the ionic than in the covalent cell wall fraction. PEG induced an increase and Zn(2+) a decrease of both ionically bound peroxidase activities. In the covalent fraction, Cu(2+) decreased oxidative and increased peroxidative activity of peroxidase. Isoelectric focusing of ionically bound proteins and activity staining for peroxidase demonstrated increased intensities and appearance of new acidic isoforms, especially in Zn(2+) and PEG treatments. Most pronounced basic isoforms (pI ~ 7.5) in controls, decreased in intensity or completely disappeared in stressed plants. Ascorbate oxidase activity was significantly increased by PEG and decreased by Zn(2+) treatments, and highly correlated with peroxidase activity. Antioxidant capacity and total phenolics content increased in heavy metal-treated and decreased in PEG-treated plants. Analysis of individual phenolic components revealed p-coumaric and ferulic acids, as the most abundant, as well as ferulic acid dimers, trimers and tetramers in the cell walls; their quantity increased under stress conditions. Results presented demonstrate the existence of diverse mechanisms of plant response to different stresses.

Classification and fingerprinting of different berries based on biochemical profiling and antioxidant capacity

O objetivo deste trabalho foi avaliar a composicao bioquimica de seis especies de pequenas frutas dos generos Fragaria, Rubus, Vaccinium e Ribes. As amostras das frutas foram coletadas em triplicata (com 50 frutas cada) de 18 diferentes especies ou cultivares dos generos mencionados, durante tres anos (2008 a 2010). Os teores individuais de acucares, acidos orgânicos, flavonois e acidos fenolicos foram determinados por cromatografia liquida de alta performance (HPLC), e os fenois totais (FT) e a capacidade antioxidante total (CAT), por meio de espectrofotometria. As analises de componente principal (ACP) e de agrupamento hierarquico (AH) foram utilizadas para avaliar as diferencas no perfil bioquimico das frutas. Os maiores teores de compostos bioativos foram encontrados em Ribes nigrum e em Fragaria vesca, Rubus plicatus e Vaccinium myrtillus. ACP e AH foram capazes de discriminar parcialmente as frutas com base em sua composicao bioquimica. Os acucares totais e individuais, a miricetina, o acido elagico, FT e CAT foram os parâmetros que mais impactaram na avaliacao da composicao bioquimica das pequenas frutas. AH fez a separacao de amora-preta, framboesa e mirtilo como grupos isolados, porem a classificacao do morango, da groselha-preta e da groselha-vermelha, em um grupo especifico, nao ocorreu. Ha grande variabilidade entre e dentro dos diferentes tipos de frutas vermelhas. A identificacao de metabolitos das pequenas frutas mostrou perfis bioquimicos unicos e combinacoes especificas de teores de compostos bioativos.