Hüseyin Çağlar Karakaya

Comparison of ROS formation and antioxidant enzymes in Cleome gynandra (C4) and Cleome spinosa (C3) under drought stress

Differences between antioxidant responses to drought in C(3) and C(4) plants are rather scanty. Even, we are not aware of any research on comparative ROS formation and antioxidant enzymes in C(3) and C(4) species differing in carboxylation pathway of same genus which would be useful to prevent other differences in plant metabolism. With this aim, relative shoot growth rate, relative water content and osmotic potential, hydrogen peroxide (H(2)O(2)) content and NADPH oxidase (NOX) activity, antioxidant defence system (superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR) enzymes and their isoenzymes), CAT1 mRNA level, and lipid peroxidation in seedlings of Cleome spinosa (C(3)) and Cleome gynandra (C(4)) species of Cleome genus exposed to drought stress for 5 and 10 day (d) were comparatively investigated. Constitutive levels of antioxidant enzymes (except SOD) were consistently higher in C. spinosa than in C. gynandra under control conditions. CAT1 gene expression in C. spinosa was correlated with CAT activity but CAT1 gene expression in C. gynandra at 10 d did not show this correlation. Drought stress caused an increase in POX, CAT, APX and GR in both species. However, SOD activity was slightly decreased in C. gynandra while it was remained unchanged or increased on 5 and 10 d of stress in C. spinosa, respectively. Parallel to results of malon dialdehyde (MDA), H(2)O(2) content was also remarkably increased in C. spinosa as compared to C. gynandra under drought stress. These results suggest that in C. spinosa, antioxidant defence system was insufficient to suppress the increasing ROS production under stress condition. On the other hand, in C. gynandra, although its induction was lower as compared to C. spinosa, antioxidant system was able to cope with ROS formation under drought stress.

Identification of a Novel System for Boron Transport: Atr1 Is a Main Boron Exporter in Yeast

ABSTRACT Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Δ mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1Δ cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1Δ cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.

Boron Stress Activates the General Amino Acid Control Mechanism and Inhibits Protein Synthesis

Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance.

Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.

The importance of boron in biological systems

Boron is an essential element for plants and probably essential for human and animal health. Boron has a broad range of physiological effects on biological systems at low concentrations, whereas it is toxic to at high concentrations. Eventhough there are many studies on borons biological effects and toxicity, more information is needed to understand the mechanisms of its action. The aim of the current work is to review borons function, transport and toxicity in different biological systems.

Characterization of two genes encoding metal tolerance proteins from Beta vulgaris subspecies maritima that confers manganese tolerance in yeast

Manganese (Mn2+) is an essential micronutrient in plants. However increased Mn2+ levels are toxic to plant cells. Metal tolerance proteins (MTPs), member of cation diffusion facilitator protein (CDF) family, have important roles in metal homeostatis in different plant species and catalyse efflux of excess metal ions. In this study, we identified and characterized two MTP genes from Beta vulgaris spp. maritima (B. v. ssp. maritima). Overexpression of these two genes provided Mn tolerance in yeast cells. Sequence analyses displayed BmMTP10 and BmMTP11as members of the Mn-CDF family. Functional analyses of these proteins indicated that they are specific to Mn2+ with a role in reducing excess cellular Mn2+ levels when expressed in yeast. GFP-fusion constructs of both proteins localized to the Golgi apparatus as a punctuated pattern. Finally, Q-RT-PCR results showed that BmMTP10 expression was induced threefold in response to the excess Mn2+ treatment. On the other hand BmMTP11 expression was not affected in response to excess Mn2+ levels. Thus, our results suggest that the BmMTP10 and BmMTP11 proteins from B. v. ssp. maritima have non-redundant functions in terms of Mn2+ detoxification with a similar in planta localization and function as the Arabidopsis Mn-CDF homolog AtMTP11 and this conservation shows the evolutionary importance of these vesicular proteins in heavy metal homeostatis among plant species.

Roles of ATR1 paralogs YMR279c and YOR378w in boron stress tolerance

Boron is a necessary nutrient for plants and animals, however excess of it causes toxicity. Previously, Atr1 and Arabidopsis Bor1 homolog were identified as the boron efflux pump in yeast, which lower the cytosolic boron concentration and help cells to survive in the presence of toxic amount of boron. In this study, we analyzed ATR1 paralogs, YMR279c and YOR378w, to understand whether they participate in boron stress tolerance in yeast. Even though these genes share homology with ATR1, neither their deletion rendered cells boron sensitive nor their expression was significantly upregulated by boron treatment. However, expression of YMR279, but not YOR378w, from the constitutive GAPDH promoter on a high copy plasmid provided remarkable boron resistance by decreasing intracellular boron levels. Thus our results suggest the presence of a third boron exporter, YMR279c, which functions similar to ATR1 and provides boron resistance in yeast.

Genome-wide identification of genes that play a role in boron stress response in yeast

Boron is an essential micronutrient for plants and it is either necessary or beneficial for animals. Studies identified only few genes related to boron metabolism thus far and details of how boron is imported into cells and used in cell metabolism are largely unknown. In order to identify genes that play roles in boron metabolism, we screened the entire set of yeast haploid deletion mutants and identified 6 mutants that were resistant to toxic levels of boron, and 21 mutants that were highly sensitive to boron treatment. Furthermore, we performed a proteomic approach to identify additional proteins that are significantly up-regulated by boron treatment. Our results revealed many genes and pathways related to boron stress response and suggest a possible link between boron toxicity and translational control.

Characterization of a cDNA from Beta maritima that confers nickel tolerance in yeast

Nickel is an essential micronutrient due to its involvement in many enzymatic reactions as a cofactor. However, excess of this element is toxic to biological systems. Here, we constructed a cDNA library from Beta maritima and screened it in the yeast system to identify genes that confer resistance to toxic levels of nickel. A cDNA clone (NIC6), which encodes for a putative membrane protein with unknown function, was found to help yeast cells to tolerate toxic levels of nickel. A GFP fused form of Nic6 protein was localized to multivesicular structures in tobacco epidermal cells. Thus, our results suggest a possible role of Nic6 in nickel and intracellular ion homeostasis.