Large, membrane-bound vacuoles of plant cells are suitable organelles for the compartmentation of ions. These vacuoles contain Na+/H+ antiporters for movement of Na+ within the organelle in exchange for H+. They provide an efficient mechanism to prevent the occurance of detrimental outcomes of Na+ accumulation in the cytosol. Identification of AtNHX1 gene that confers resistance to salinity by expressing a Na+/H+ antiport pump facilitates the understanding of the salt stress tolerance mechanisms of plants. The aim of the present study was to isolate and clone the Arabidopsis thaliana AtNHX1 coding sequence for transformation of Nicotiana tabacum plants via Agrobacterium tumefaciens mediated gene transfer. For this purpose, total RNA was isolated from Arabidopsis thaliana plants and cDNA synthesis was performed. AtNHX1 (1614bp) was amplified by using cDNA of Arabidopsis via specific primers. The amplified PCR product was verified by sequencing. AtNHX1 coding sequence was cloned into the plant transformation vector pCVB1 and 10 independent putative transgenic tobacco plants were obtained via Agrobacterium tumefaciens mediated gene transfer sysytem. Transfer of selected 8 putative transgenic plants to soil provided the regeneration of T1 seeds. Germination of the seeds under different salt treatments (0, 50, 100, 150, 200, 250 mM NaCl) was observed for evaluating the salt tolerance of transformed plants. The 82% and 60% of the transgenic T1 seeds were germinated on 150 mM NaCl and 200 mM NaCl containing media, respectively. In contrast the germination percentage of wild type tobacco seeds under 150 mM NaCl and 200 mM NaCl concentrations were 39% and 21%, respectively. The germination rate of the transgenic T1 seeds were significantly higher (p=0,001) when compared to the control seeds especially under high salt stress conditions (150 and 200 mM NaCl). Taken all together, our results demonstrated that the germination efficiencies and growth of the plants transformed with AtNHX1 were higher than the wild type tobacco plants under high salt concentrations.

In this study, 14 days old lentil seedlings (Lens culinaris Medik cv. Sultan), which were subjected to 7 days of drought (20% PEG 6000), and salt (150 mM NaCl ) stress , were examined in a comparative manner for the effects of drought and salt stress treatments. In shoot and root tissues physiological parameters such as wet-dry weight, relative water content, root-shoot lengths, membrane electrolyte leakage, and lipid peroxidation in terms of malondialdehyde (MDA) were determined. H2O2 content, proline accumulation and chlorophyll fluorescence analysis were also performed. Changes in the activity of antioxidant enzymes such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6) ascorbate peroxidase (APX: EC 1.11.1.11) and glutathione reductase (GR: EC 1.6.4.2) were observed upon stress treatments. In salt treated lentil seedlings, significant decreases in wet-dry weight, RWC, shoot-root length and chlorophyll fluorescence measurements indicated a sensitivity, when compared to drought treated plants. Higher MDA concentration and higher electrolyte leakage amounts are supported these results. APX, GR and proline seem to play important roles in antioxidant defense against salt stress for both tissues by removing reactive oxygen species and protecting macromolecules and membranes. GR and proline are also maintains the main protective mechanism against drought stress effects. SOD is active in drought stressed roots and salt stressed shoots, where the H2O2 contents are also observed to be increased.

ABSTRACT TRANSFORMATION OF POTATO WITH MYB4 TRANSCRIPTION FACTOR AND EVALUATION OF ABIOTIC STRESS TOLERANCE AND GENE EXPRESSION PROFILES IN TRANSGENIC PLANTS Kalemtas, G&uuml / ls&uuml / m Ph.D., Department of Biology Supervisor: Prof. Dr. H&uuml / seyin Avni &Ouml / ktem February 2011, 257 pages Potato (Solanum tuberosum L. cv. Kennebec) was transformed via Agrobacterium tumefaciens (EHA105) harbouring two different binary vectors containing Oryza sativa myb4 gene, which encodes MYB4 transcription factor / under the control of CaMV35S promoter or cold inducible COR15a promoter. The transgenic plants were not growth retarded and there was no significant difference (p&lt / 0.05) in their tuber yield compared to wild-type plants. Wild-type and transgenic plants were subjected to abiotic stresses to compare their stress tolerances. There was no significant difference in boron, freezing and drought tolerances of wild-type and transgenic lines. Two of the transgenic lines were more salt tolerant than wild-type with respect to growth parameters. Transcriptomes of wild-type and these two lines, one expressing myb4 under the control of 35S promoter and the other COR15a promoter, were analyzed to elucidate the myb4-regulated processes and downstream target genes in potato. Differentially regulated genes in transgenic lines showed that myb4 controls a large and complex transcriptional network associated with diverse cellular processes, primarily defense and rescue, metabolism and development. Genes involved in sucrose synthesis, some peroxidases and CBF3 transcription factor were up-regulated in transgenic plants upon exposure to freezing stress. This suggested that myb4 may configure freezing response in potato primarily by oxidative stress defence mechanisms, osmotic adjustment or activation of CBF3 regulated genes that may confer cold tolerance. Despite up-regulation of these stress related genes, transgenic potato was not more drought or freezing tolerant compared to WT under the tested conditions. Further experiments should be conducted to better elucidate the involvement of these genes in regulation of stress response in transgenic potato expressing myb4.

In long term, to discover the genes responsible for wood production, genetic control of wood specific gravity (WSG) in Pinus brutia Ten. (Turkish red pine) open pollinated Ceyhan progeny trial, which was established with the seeds collected from 168 clones originated from six clonal Turkish red pine seed orchards was studied. Wood samples were taken by destructive sampling during the rouging of this trial at the age of seven. Specifically / (1) to examine the magnitude of family differences and its components for wood specific gravity (WSG) and growth traits (height, diameter and stem volume) / (2) to determine WSG inheritance and its genetic correlation with growth traits / and (3) to estimate breeding values of 168 families for the WSG and to predict genetic gain if selection is based on phenotypic, rouged and genotypic seed orchard by reselecting the best parents with respect to WSG. Differences among the 168 families for mean WSG was large (ranged from 0.35 to 0.44), as indicated by high individual (0.42+0.07) and family mean (0.55+0.03) heritabilities. Family differences and high heritabilities were also observed for all growth traits. Genetic correlations between WSG and growth traits were statistically insignificant (near zero), while low and insignificant negative phenotypic correlations among the same traits were observed. Realized genetic gain for single trait selection at age seven was insignificant (0.37 %) for WSG and 8.4 % for stem volume in phenotypic seed orchards. Average genetic gain in breeding zone after roguing, by leaving the best 20 clones in each seed orchard, reached 1.7 % for WSG and 16.1 % for stem volume. Genetic gain (relative to controls) at the age of seven obtained from the first generation genotypic seed orchards consisting the best 30 clones was estimated 5.2 % for WSG and 35 % for stem volume. Multi-trait selection was also proposed in this study for the same traits. Selection of best 10 families for the highest WSG and stem volume breeding values produce 5.6 % genetic gain for WSG and 27.7 % genetic gain for stem volume. For the future, the 168 families with known phenotypic and genotypic values regarding to WSG will be screened for the genes responsible for wood production.

ABSTRACT EFFECT OF SALT STRESS ON ANTIOXIDANT DEFENSE SYSTEMS OF SENSITIVE AND RESISTANT CULTIVARS OF LENTIL Cicerali, Iin Nur M.Sc., Department of Biotechnology Supervisor: Prof. Dr. Meral Y&uuml / cel Co-supervisor: Asst. Prof. Dr. F&uuml / sun (nci) Eyidoan June 2004, 90 pages In this study, two lentil cultivars (Lens culinaris, Medik.) (ILL5582-salt tolerant and ILL590) were characterized and compared due to their NaCl susceptibility and antioxidant mechanism was examined under laboratory conditions. Physiological parameters such as wet-dry weight, root-shoot lengths, cell membrane stability, lipid peroxidation in terms of malondialdehyde (MDA), H2O2, proline contents were determined. The activities of antioxidant enzymes such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), ascorbate peroxidase (APX: EC 1.11.1.11) and glutathione reductase (GR: EC 1.6.4.2) were examined and analyzed in 14 days old plant seedlings after 9 days of normal growth and 5 days of 100mM and 200mM NaCl stress conditions. Shoot-root length and wet-dry weight percent decrease were more in ILL590. Especially shoot tissues were affected more from the stress conditions when compared to root tissues. ii According to malondialdehyde (MDA) content and membrane stability results, lipid peoxidation was higher in ILL590 and significant increases were observed in shoot tissues. Proline concentration showed a remarkable increase in salt concentration dependent manner. Higher concentrations of proline in ILL5582 might be the reason of higher salt tolerance when compared to ILL590. Among the antioxidant enzymes SOD was the one which showed highest activity increase. At organ level roots showed highest activity when compared to leaves. In the organelle higher activity percent contribution was achieved by cytosolic Cu/ZnSOD isozyme. Higher percent increase of this isozyme was observed in ILL5582. This might be one of the tolerance mechanisms that get activated against NaCl stress. APX activity showed similar alterations in both cultivars. In leaf tissues significant increase was observed but in root tissues ascorbate peroxidase activity did not change significantly. Glutathione Reductase activity increase was significant in both cultivars leaf tissues but although ILL5582 showed a stress concentration dependent increase, ILL590 did not. The activity of CAT enzyme in leaf and root tissues of both cultivars did not significantly change under increasing salt stress conditions. The results suggested that the leaves were more susceptible to salt stress. Also when two cultivars were compared ILL5582 was found to be more tolerant against salt stress than ILL590 under laboratory conditions and SOD enzyme seemed to be the most active component of the salt tolerant mechanism.

The main objective of this thesis dissertation is functionally characterizing the genes involved in biotic and abiotic stresses of plants at molecular level. Previously, upon pathogen attack Rad6 gene expression was found to be changed in wheat and barley plants. To functionally characterize the Rad6 gene, VIGS (Virus induced gene silencing) system was used. HR (Hypersensitive response) like symptoms was detected in every silenced barley and wheat plants. To figure out, transcriptomes and proteomes of Rad6 silenced plants were analyzed. 2-D PAGE analysis was also performed on silenced and control wheat plants. No pathogen growth was observed in Rad6 silenced barley lines. Additionally, the susceptible wild type Arabidopsis plants showed resistant phenotype when any of the Rad6 gene copies is mutated. This suggests that Rad6 gene has a negative regulatory role in plant disease resistance which was proved for the first time. Yeast two hybrid protein interaction study suggests that RAD6 carrying out its function by interacting with SGT1 protein and regulating resistance related genes. It has been first time reported in this thesis that E2 (Ubiquitin conjugating enzyme) takes role in plant disease resistance. Boron which is the other consideration in the scope of thesis as an abiotic stress factor at a very limited amount is necessary for the normal development of plants. This study is conducted on highly boron tolerant Gypsophila perfoliata L. collected from a location in the boron mining area. The plant samples were tested in the presence of high boron (35 mg/kg) concentrations. The transcriptomes of the plant samples treated with the excess levels of boron to that of the samples grown under normal concentration were compared using differential display PCR method. Thirty bands showing differential expression levels at varying time points were analyzed. 18 of them were confirmed via qRT-PCR.