Hakan Terzi

Determination of genotoxic effects of copper sulphate and cobalt chloride in Allium cepa root cells by chromosome aberration and comet assays

We used the anaphase-telophase chromosome aberration and comet (Single Cell Gel Electrophoresis, SCGE) assays to evaluate the genotoxic effects of copper sulphate (CS) and cobalt chloride (CC) chemicals prepared in two concentrations (EC(50), 2xEC(50)), using methyl methanesulfonate (MMS) as a positive control and untreated cells as a negative control. In Allium root growth inhibition test, EC(50) values for CS and CC are 1.5 and 5.5 ppm, respectively. Mitotic index (MI) decreased in all concentrations tested of CS and CC compared to the control at each exposure time. The bridge, stickiness, vagrant chromosomes, fragments, c-anaphase and multipolarity chromosome aberrations were observed in anaphase-telophase cells. The total chromosome aberrations were more frequent with an increasing in the exposure time and the concentrations of both chemicals. The genotoxicity of CS and CC in Allium cepa root cells was analyzed using a mild alkaline comet assay at pH 12.3, which allows the detection of single strand breaks. In all the concentrations, CS and CC induced a significant increase (P<0.05) in DNA damage. No significant difference was found between positive control (300+/-5.81) and 3 ppm CS (280+/-4.61). The methods used are applicable for biological monitoring of environmental pollutants.

Evaluation of 2,4-D and Dicamba genotoxicity in bean seedlings using comet and RAPD assays.

The present study was undertaken to evaluate genotoxic potential of two auxinic herbicides [2,4-dicholorophenoxy acetic acid (2,4-D) and 3,6-dichloro-2-methoxybenzoic acid (Dicamba)] in the roots of common bean (Phaseolus vulgaris L.) seedlings. Two-day-old etiolated seedlings were treated with 10 ppm methyl methanesulfonate (MMS, positive control) or 0.1, 0.2, or 0.3 ppm of either 2,4-D or Dicamba. At the end of a 96 h growth period, root growth, total soluble protein content, DNA damage in individual cells (comet assay scores) and randomly amplified polymorphic DNA (RAPD) profiles were used as endpoints of genotoxicity. 2,4-D and Dicamba were clearly dose-dependent root growth inhibitors. Total soluble protein content was significantly decreased in the positive control and at high concentrations (0.2 and 0.3 ppm) of Dicamba. Soluble protein content increased significantly only at 0.3 ppm 2,4-D (P<0.05). In the comet assay, DNA fragmentation increased in a dose-dependent manner. The diagnostic and phenetic analyzes of appeared and/or disappeared RAPD bands indicated that dose-dependent DNA polymorphism was induced by both herbicides. Genomic template stability was significantly affected at all 2,4-D and Dicamba doses tested. Overall 2,4-D and Dicamba have similar effects on DNA damage detected by comet and RAPD assays.

Proteomic analysis of chromium stress and sulfur deficiency responses in leaves of two canola (Brassica napus L.) cultivars differing in Cr(VI) tolerance

Sulfur (S) is an essential macronutrient for plant growth and development, and it plays an essential role in response to environmental stresses. Plants suffer with combined stress of S deficiency and hexavalent chromium [Cr(VI)] in the rhizosphere. Little is known about the impact of S deficiency on leaf metabolism of canola (Brassica napus L.) under Cr(VI) stress. Therefore, this study is the first to examine the effects of Cr(VI) stress and S deficiency in canola at a molecular level. A comparative proteomic approach was used to investigate the differences in protein abundance between Cr-tolerant NK Petrol and Cr-sensitive Sary cultivars. The germinated seeds were grown hydroponically in S-sufficient (+S) nutrient solution for 7 days and then subjected to S-deficiency (-S) for 7 days. S-deficient and +S seedlings were then exposed to 100μM Cr(VI) for 3 days. Protein patterns analyzed by two-dimensional electrophoresis (2-DE) revealed that 58 protein spots were differentially regulated by Cr(VI) stress (+S/+Cr), S-deficiency (-S/-Cr) and combined stress (-S/+Cr). Of these, 39 protein spots were identified by MALDI-TOF/TOF mass spectrometry. Differentially regulated proteins predominantly had functions not only in photosynthesis, but also in energy metabolism, stress defense, protein folding and stabilization, signal transduction, redox regulation and sulfur metabolism. Six stress defense related proteins including 2-Cys peroxiredoxin BAS1, glutathione S-transferase, ferritin-1, l-ascorbate peroxidase, thiazole biosynthetic enzyme and myrosinase-binding protein-like At3g16470 exhibited a greater increase in NK Petrol. The stress-related proteins play an important role in the detoxification of Cr(VI) and maintaining cellular homeostasis under variable S nutrition.

Small heat shock protein responses in leaf tissues of wheat cultivars with different heat susceptibility

The effect of heat stress on soluble proteins extracted from leaf tissues of bread (Triticum aestivum cv. Gönen-98, tolerant; cv. Cumhuriyet-75, susceptible; genome ABD) and durum (Triticum durum cv. Ege-88, tolerant; cv. Ankara-98, susceptible; genome AB) wheat cultivars differing in sensitivity to high temperature was examined by two-dimensional gel electrophoresis. At acclimation (37°C) and acclimation→high temperature (37°C→50°C) treatments compared to control (25°C), evaluation of gels revealed 31 proteins to be differentially expressed in first leaves as a result of heat stress in heat-susceptible and heat-tolerant cultivars of bread and durum wheats. All of the increased or decreased proteins in amount, newly synthesized and/or disappeared were in low-molecular-weight (LMW, 16.1–24.0 kDa) and generally acidic character (pI 4.8–6.9). The responses of the four cultivars were compared: Twenty-two of 31 proteins were detected as newly synthesized LMW heat shock proteins (LMW HSPs = small HSPs). The number of these sHSPs was different in cultivars which have the same genome. In addition, the number of the sHSPs in heat-tolerant cultivars was higher than in heat-susceptible cultivars. Some of the sHSPs were specific to cultivar. Most of the sHSPs synthesized at 37°C were also detected at 37°C→50°C treatment. It is suggested that sHSPs have special importance in two points: Firstly, sHSPs in cultivars showed abundance and diversity. Secondly, these proteins may play an important role in the acquiring of thermal tolerance.

Proteomic and biochemical responses of canola (Brassica napus L.) exposed to salinity stress and exogenous lipoic acid

To evaluate the mitigating effects of exogenous lipoic acid (LA) on NaCl toxicity, proteomic, biochemical and physiological changes were investigated in the leaves of canola (Brassica napus L.) seedlings. Salinity stress decreased the growth parameters and contents of ascorbate (AsA) and glutathione (GSH), and increased the contents of malondialdehyde (MDA), proline, cysteine and the activities of antioxidant enzymes such as superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX). The foliar application of LA alleviated the toxic effects of salinity stress on canola seedlings and notably decreased MDA content and increased growth parameters, cysteine content, and activities of CAT and POD. In the proteomic analyses, total proteins from the leaves of control, LA, NaCl and NaCl+LA treated-seedlings were separated using two-dimensional gel electrophoresis (2-DE). A total of 28 proteins were differentially expressed. Of these, 21 proteins were successfully identified by MALDI-TOF/TOF MS. These proteins had functions related to photosynthesis, stress defense, energy metabolism, signal transduction, protein folding and stabilization indicating that LA might play important roles in salinity through the regulation of photosynthesis, stress defense and signal transduction related proteins. The proteomic findings have provided new insight to reveal the effect of LA on salinity stress for the first time.