Hüseyin Avni Öktem

Antioxidant responses of tolerant and sensitive barley cultivars to boron toxicity

Abstract Effect of boron toxicity on antioxidant response of sensitive (Hamidiye) and tolerant (Anadolu) barley ( Holgarium vulgar ) cultivars was investigated. Eight days old hydroponically grown seedlings were subjected to 5 and 10 mM boric acid treatments for 5 days. Growth parameters (dry-wet weight), protein, proline, MDA, H 2 O 2 contents, membrane damage and activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathion reductase (GR) was evaluated in root and shoot tissues. Compared with controls (no boric acid treatment) boron toxicity resulted in a reduction in root weights and did not cause any significant change in protein contents. Boric acid treatment did not cause significant ( P >0.05) changes in proline and H 2 O 2 contents of both tissues and cultivars. On contrary, MDA contents and electrolyte leakage exhibited a dose dependent increase in shoots whereas no significant change was observed in root tissues. Higher extend of membrane damage was observed in leaves of the sensitive cultivar. Total SOD, CAT and GR activities in shoot tissue of both cultivars did not change significantly whereas significantly higher APX activities was observed at 10 mM boric acid treatments. Significant increases in total SOD, CAT and decrease in GR activities was observed in roots of the sensitive cultivar. Roots of resistant cultivar, on contrary, exhibited enhanced CAT and decreased APX activity whereas no significant changes was observed in total SOD and GR activities. Our data suggest that, boron toxicity induced membrane damage in barley leaves, do not involve active oxygen species and antioxidant enzyme activity is not a critical factor in boron toxicity tolerance mechanism. To the best of our knowledge, this is the first report on antioxidant response of barley seedlings under boron toxicity.

Antioxidant responses of shoots and roots of lentil to NaCl-salinity stress

The effect of salt stress (100 mM and 200 mM NaCl) on antioxidant responses in shoots and roots of 14-day-old lentil (Lens culinaris M.) seedlings was investigated. Salt stress caused a significant decrease in length, wet-dry weight and an increase in proline content of both shoot and root tissues. In leaf tissues, high salinity treatment resulted in a 4.4 fold increase in H2O2 content which was accompanied by a significant level of lipid peroxidation and an increase in electrolyte leakage. Root tissues were less affected with respect to these parameters. Leaf tissue extracts exhibited four activity bands, of which two were identified as Cu/Zn-SOD and others as Fe-SOD and Mn-SOD. Fe-SOD activity was missing in root extracts. In both tissues Cu/Zn-SOD activity comprised 70–75% of total SOD activity. Salt stress did not cause a significant increase in total SOD activity of leaf tissues but a significant enhancement (88%) was observed in roots mainly due to an enhancement in Cu/ZnSOD isoforms. Compared to leaf tissues a significantly higher constitutive ascorbate peroxidase (APX) and glutathion reductase (GR) activity was observed in root tissues. Upon salt stress no significant change in the activity of APX, catalase (CAT) and GR was observed in root tissues but a higher APX activity was present when compared to leaf tissues. On the other hand, in leaf tissues, with the exception of CAT, salt stress caused significant enhancement in the activity of other antioxidant enzymes. These results suggested that, root tissues of lentil are protected better from NaCl stress induced oxidative damage due to enhanced total SOD activity together with a higher level of APX activity under salinity stress. To our knowledge this is the first report describing antioxidant enzyme activities in lentil.

Single-Step Purification of Recombinant Thermus aquaticus DNA Polymerase Using DNA-Aptamer Immobilized Novel Affinity Magnetic Beads

A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq‐polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5′‐thiol labeled DNA‐aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq‐polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq‐polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq‐polymerase from crude extracts was also evaluated. For this case, the enzyme “recombinant Taq‐DNA polymerase” was cloned and expressed using an Amersham E. coli GST‐Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag‐polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one‐step purification protocol. Apparently, the system was found highly effective as one step for the low‐cost purification of Taq‐polymerase in bacterial crude extract.

Aptamer-Gated Nanoparticles for Smart Drug Delivery

Aptamers are functional nucleic acid sequences which can bind specific targets. An artificial combinatorial methodology can identify aptamer sequences for any target molecule, from ions to whole cells. Drug delivery systems seek to increase efficacy and reduce side-effects by concentrating the therapeutic agents at specific disease sites in the body. This is generally achieved by specific targeting of inactivated drug molecules. Aptamers which can bind to various cancer cell types selectively and with high affinity have been exploited in a variety of drug delivery systems for therapeutic purposes. Recent progress in selection of cell-specific aptamers has provided new opportunities in targeted drug delivery. Especially functionalization of nanoparticles with such aptamers has drawn major attention in the biosensor and biomedical areas. Moreover, nucleic acids are recognized as an attractive building materials in nanomachines because of their unique molecular recognition properties and structural features. A active controlled delivery of drugs once targeted to a disease site is a major research challenge. Stimuli-responsive gating is one way of achieving controlled release of nanoparticle cargoes. Recent reports incorporate the structural properties of aptamers in controlled release systems of drug delivering nanoparticles. In this review, the strategies for using functional nucleic acids in creating smart drug delivery devices will be explained. The main focus will be on aptamer-incorporated nanoparticle systems for drug delivery purposes in order to assess the future potential of aptamers in the therapeutic area. Special emphasis will be given to the very recent progress in controlled drug release based on molecular gating achieved with aptamers.

Transformation of lentil ( Lens culinaris M.) cotyledonary nodes by vacuum infiltration of Agrobacterium tumefaciens

Lentil cotyledonary nodes are some of the most regenerative tissues in legumes. Attempts to transform them by vacuum filtration have been limitedly successful. This report describes a rapid and convenient transient expression protocol based on vacuum infiltration ofAgrobacterium cells into lentil cotyledonary nodes. Vacuum-infiltrated tissues had significantly (P<.05) higher transient GUS expression than did noninfiltrated tissues. Under optimal conditions (infiltration at 200 mmHg for 20 min), 95% of theAgrobacterium-infiltrated explants exhibited an average of 16 blue foci. We believe this to be the first report of this technique for transient gene expression in lentil cotyledonary nodes.

Pathogen detection by core-shell type aptamer-magnetic preconcentration coupled to real-time PCR.

The presence of pathogenic bacteria is a major health risk factor in food samples and the commercial food supply chain is susceptible to bacterial contamination. Thus, rapid and sensitive identification methods are in demand for the food industry. Quantitative polymerase chain reaction (PCR) is one of the reliable specific methods with reasonably fast assay times. However, many constituents in food samples interfere with PCR, resulting in false results and thus hindering the usability of the method. Therefore, we aimed to develop an aptamer-based magnetic separation system as a sample preparation method for subsequent identification and quantification of the contaminant bacteria by real-time PCR. To achieve this goal, magnetic beads were prepared via suspension polymerization and grafted with glycidylmethacrylate (GMA) brushes that were modified into high quantities of amino groups. The magnetic beads were decorated with two different aptamer sequences binding specifically to Escherichia coli or Salmonella typhimurium. The results showed that even 1.0% milk inhibited PCR, but our magnetic affinity system capture of bacteria from 100% milk samples allowed accurate determination of bacterial contamination at less than 2.0 h with limit of detection around 100 CFU/mL for both bacteria in spiked-milk samples.

Thermo-resistant green microalgae for effective biodiesel production: isolation and characterization of unialgal species from geothermal flora of Central Anatolia.

Oil content and composition, biomass productivity and adaptability to different growth conditions are important parameters in selecting a suitable microalgal strain for biodiesel production. Here, we describe isolation and characterization of three green microalgal species from geothermal flora of Central Anatolia. All three isolates, namely, Scenedesmus sp. METUNERGY1402 (Scenedesmus sp. ME02), Hindakia tetrachotoma METUNERGY1403 (H. tetrachotoma ME03) and Micractinium sp. METUNERGY1405 (Micractinium sp. ME05) are adaptable to growth at a wide temperature range (25-50 °C). Micractinium sp. ME05, particularly has superior properties for biodiesel production. Biomass productivity, lipid content and lipid productivity of this isolate are 0.17 g L(-1) d(-1), 22.7% and 0.04 g L(-1) d(-1), respectively. In addition, Micractinium sp. ME05 and Scenedesmus sp. ME03 mainly contain desirable fatty acid methyl esters (i.e. 16:0, 16:1, 18:0 and 18:1) for biodiesel production. All isolates can further be improved via genetic and metabolic engineering strategies.

Agrobacterium tumefaciens -mediated genetic transformation of a recalcitrant grain legume, lentil ( Lens culinaris Medik)

A simple and reproducible Agrobacterium-mediated transformation protocol for a recalcitrant legume plant, lentil (Lens culinaris M.) is reported. Application of wounding treatments and efficiencies of three Agrobacterium tumefaciens strains, EHA105, C58C1, and KYRT1 were compared for T-DNA delivery into lentil cotyledonary node tissues. KYRT1 was found to be on average 2.8-fold more efficient than both EHA105 and C58C1 for producing transient β-glucuronidase (GUS) gene (gus) expression on cotyledonary petioles. Wounding of the explants, use of an optimized transformation protocol with the application of acetosyringone and vacuum infiltration treatments in addition to the application of a gradually intensifying selection regime played significant roles in enhancing transformation frequency. Lentil explants were transformed by inoculation with Agrobacterium tumefaciens strain, KYRT1 harboring a binary vector pTJK136 that carried neomycin phosphotransferase gene (npt-II) and an intron containing gusA gene on its T-DNA region. GUS-positive shoots were micrografted on lentil rootstocks. Transgenic lentil plants were produced with an overall transformation frequency of 2.3%. The presence of the transgene in the lentil genome was confirmed by GUS assay, PCR, RT-PCR and Southern hybridization. The transgenic shoots grafted on rootstocks were successfully transferred to soil and grown to maturity in the greenhouse. GUS activity was detected in vegetative and reproductive organs of T0, T1, T2 and T3 plants. PCR assays of T1, T2 and T3 progenies confirmed the stable transmission of the transgene to the next generations.

Cu/Zn superoxide dismutase activity and respective gene expression during cold acclimation and freezing stress in barley cultivars

The transcript levels and activities of the superoxide dismutase isoenzyme (Cu/ZnSOD) were assessed in winter (Tarm-92) and spring (Zafer-160) barley cultivars during cold acclimation, freezing stress and after rewarming. Leaf Cu/ZnSOD activity and Cu/ZnSOD expression level were not significantly changed during cold acclimation. The Cu/ZnSOD expression increased evidently at mild freezing stress (−3 °C; F1), while Cu/ZnSOD1 activity did not show any response and Cu/ZnSOD2 activity decreased continuously during F1 and F2 (−7 °C) in Tarm-92. On the other hand, root Cu/ZnSOD2 activity was in accordance with Cu/ZnSOD expression in Zafer-160 after F2 treatment. Rewarming periods did not cause any significant changes in the Cu/ZnSOD activity and expression of Cu/ZnSOD in both cultivars when compared to freezing stresses. These results showed that freezing stress can regulate differently Cu/ZnSOD transcription and enzyme activity.

NITRATE REDUCTASE AND GLUTAMATE DEHYDROGENASE ACTIVITIES OF RESISTANT AND SENSITIVE CULTIVARS OF WHEAT AND BARLEY UNDER BORON TOXICITY

ABSTRACT In this study, changes in activity of key enzymes of nitrogen assimilation, namely nitrate reductase and glutamate dehydrogenase upon boron (B) toxicity in wheat (Triticum aestivum) and barley (Hordeum vulgare) cultivars were investigated. Ten-day-old seedlings were exposed to 10 mM boric acid for a duration of five days. Plants growing on the nutrient solutions and receiving no excess B, were maintained as controls. All experiments were conducted on leaf and root tissues of control and B-treated seedlings of B-tolerant and B-sensitive cultivars. For estimation of activity of nitrate reductase, an in vivo assay was used. Compared to controls (no B treatment) activity of nitrate reductase tended to decrease (15–17%) following B toxicity in root and leaf tissues of all cultivars, however, no significant difference was observed between resistant and sensitive cultivars. Boron stress increased activity of glutamate dehydrogenase in roots and leaves of all cultivars by an average of 81% and 30%, respectively. Compared to sensitive variety, the boron tolerant wheat variety exhibited a significantly higher increase in shoot tissue GDH activity, whereas no significant difference was observed in root tissues. It was concluded that, the increase in activity of glutamate dehydrogenase could be an adaptive mechanism in these species and possibly plays a protective role under boron-stress conditions.