Mahmut Caliskan

Formation of wheat (Triticum aestivum L.) embryogenic callus involves peroxide-generating germin-like oxalate oxidase

In wheat (Triticum aestivum L.), embryogenic callus formation comprises suppression of precocious germination by the zygotic embryo and the initiation of dedifferentiated cellular proliferation within it. Embryogenic calli are induced by treating immature embryos with 2,4-dichlorophenoxyacetic acid (2,4-D). Upon withdrawal from 2,4-D, somatic embryos develop from the periphery of the callus. Prior to visible callus formation, there is a striking induction of “germin-like” oxalate oxidase (“gl-OXO”: EC 1.2.3.4) gene expression. Accumulation of gl-OXO mRNA is rapidly stimulated upon auxin treatment, with a consequent development of apoplastic enzyme activity producing H2O2 within the cell wall. Within the dedifferentiated calli, gl-OXO enzyme activity becomes widespread over the surface of embryogenic calli. Differentiation of somatic embryos is initiated in regions of densely cytoplasmic, meristematic cells that are marked by highly localised expression of gl-OXO activity within these embryogenic cell masses. We suggest that this localised generation of H2O2 by gl-OXO promotes peroxidative cross-linking of cell wall components, thereby preventing cellular expansion and maintaining these cell masses in an embryogenically competent condition.

Phylogenetic relationships of nine mullet species (Mugilidae) in the Mediterranean Sea

Phylogenetic relationships among four genera and nine species (Mugil cephalus, Mugil soiuy, Liza ramada, Liza aurata, Liza abu, Liza saliens, Liza carinata, Chelon labrosus, Oedalechilus labeo) of the Mediterranean mullets (Mugilidae) were investigated by means of allozyme electrophoresis using a seven-enzyme system (CK*, GAPDH*, G3PDH*, IDHP*, ME*, MDH*, PGM*) comprising eleven putative loci. The highest genetic divergence was 1.299, detected between M. cephalus and L. aurata and the lowest (0.280) was found between L. carinata and L. saliens. The amount of genetic divergence between the genera Chelon and Oedalechilus did not appear to be high (0.285). In a UPGMA tree, all nine species were grouped in two main branchings. In the first branch, C. labrosus and O. labeo clustered as closest taxa and were sister group to L. ramada. The other four Liza species produced two sub-branching in this group; L. carinata branched together with L. saliens, and L. aurata branched together with L. abu. In the second branch the two species of the genus Mugil, M. cephalus and M. soiuy, clustered together and were clearly isolated from the other three genera.

Characterization of extremely halophilic Archaea isolated from saline environment in different parts of Turkey

Ninety-five extremely halophilic strains were isolated from six distinct saline regions of Turkey by using complex medium containing 25% NaCl. The selected regions are Tuz Golu (salt lake), Ankara; Aci Lake, Denizli; Salda Lake, Denizli; Seyfe Lake, Kyrsherhir; Tuzla Lake, Kayseri; and Bolluk Lake, Konya. The isolated strains were tested for motility, gram reaction, cell and colony morphologies, pigmentation, biochemical characteristics, and antibiotic sensitivities. According to membrane glycerol diether moieties and antibiotic susceptibilities, all isolated strains were found to belong to the domain Archaea. All isolates were examined for the presence of plasmids by agarose gel electrophoresis and it was established that most isolates contained plasmids that varied in number and whose molecular sizes ranged from 1 to 36.9 kbp. Whole-cell protein profiles from isolates were analyzed by SDS-PAGE and a similarity dendogram was constructed using the UPGMA method. Significant similarities and differences were observed among the isolates. The strains were clustered in eight groups and ten of our isolates were placed in the same group with the standard strains. The current study represents the first isolation and characterization of such a large collection of archeal strains from Turkey.

Antimicrobial and Antioxidant Activities of Various Extracts of Verbascum antiochium Boiss. (Scrophulariaceae)

Verbascum antiochium Boiss., a member of the Scrophulariaceae family, is endemic to Turkey. The extracts obtained from V. antiochium by increased polarity and direct methanol extraction were tested by the agar well diffusion method against various Gram-positive and Gram-negative bacteria and one fungus. The methanol/water extract exhibited a larger inhibition zone against both the Gram-negative and Gram-positive bacteria than the other extracts. Haemophilus influenzae was found to be the most sensitive bacterium among the bacteria tested. The antioxidant activities of the methanolic extract of V. antiochium were examined by two complementary test systems. The 50% inhibition activity of the methanolic extract of V. antiochium against the free radical 2,2-diphenyl-1-picrylhydrazyl was determined as 4.80 mg/mL. In the case of the linoleic acid system, oxidation of linoleic acid was inhibited by the methanolic extract of V. antiochium with 79.92% inhibition, which is close to the value of the synthetic antioxidant reagent, tert-butylated hydroxytoluene. The total phenolic components of V. antiochium were determined to be 92.71 mg of gallic acid equivalents/g. Iridoid glycosides, flavonoids, and saponins were detected as the major chemical constituents in the extract.

Phylogenetic analysis and characterization of lipolytic activity of halophilic archaeal isolates

Five isolates designated as B45, D83A, A206A, A85 and E49 found to possess lipolytic activities were taxonomically classified on the basis of their phylogenetic, phenotypic and chemotaxonomic characteristics. The isolates were determined to be gram-negative, catalase and oxidase positive, hydrolyzing Tween 80 and 60 but not starch, need 3.5–4 M NaCl for optimal growth and lack of anaerobic growth with arginine or DMSO. All isolates had the highest lipolytic activity at pH 8.5. Lipase and esterase activities increased with salt concentration up to 3–4.5 M NaCl, and decreased at 5 M NaCl. Esterase and lipase showed their maximal activities at 50–55°C and 60–65°C, respectively. The phylogenetic tree constructed by the neighbor-joining method indicated that the strain B45 and A85 were closely related to the members of genera Halovivax and Natrinema, respectively. The closest relative of the strain A206A and D83A were found to be Haloterrigena saccharevitans. The strain E49 displayed a more distant relationship to known strains.

The Roles of Germin Gene Products in Plants Under Salt Stress

The members of plants response various internal and external signals differently. The responses of plants to biotic and abiotic stress factors involve biochemical, physiological, morphological and developmental changes. Among the various circumstances salt stress is particularly under extensive studies due to low salt tolerance of crop plants. Germin and germin-like gene products were previously announced to be involved in various aspects of plant development such as plant defence, embryonic development and they are responsive to biotic and abiotic stress including salt. The responses of germin and germin like genes to salt stress are found to be various in different plants. The salinity of soil is an important problem in agriculture, particularly since the majority of crop plants have low salt tolerance. The response of plants to salt stress is a complex phenomenon that involves biochemical and physiological processes as well as morphological and developmental changes (Flowers et al., 1977; Greenway & Munns, 1980). The identification of genes whose expression enables plants to adapt to or tolerate to salt stress is essential for breeding programs, but little is known about the genetic mechanisms for salt tolerance. One approach in clarifying the molecular mechanisms involved in salt stress is to identify the genes whose levels change as a result of salt stress. In this aspect, Hurkman et al. (1989) reported that in barley, gene regulation is altered by salt stress and the levels of translatable mRNAs change with salt treatment. Among the salt stress responsive gene products, germin and germin-like proteins (GLP) were identified (Caliskan, 1997; Hurkman et al., 1989). Cereal germin protein is a homopentameric apoplastic glycoprotein whose synthesis is associated with the onset of growth in germinating wheat embryos (Lane, 1991). Germin genes and their proteins were first detected in germinating cereals (Grzelczak et al., 1985), but subsequently, germin-like proteins were also identified in dicotyledonous angiosperms (Michalowski and Bohnerd, 1992), gymnosperms (Domon et al., 1995) and mosses (Yamahara et al., 1999). Germins are suggested to be a member of “superfamily” which comprises various growth-related genes (Dunwell et al., 2000). Cereal germin proteins display strong oxalate oxidase activity (Lane et al., 1993), an activity that generates one mole of H2O2 and two moles of CO2 from the degradation of oxalic acid. It is reported that H2O2 might act as a signaling molecule at low concentration (Luthell, 1993) or a component of cell wall modifications at high concentrations (Showalter, 1993). Another germin-like protein isolated from the cells of a moss, Barbula unguiculata, was

A Novel Application for Determination of Germin Gene Products In Situ

ABSTRACT In situ nucleic acid hybridization is one of the most powerful techniques developed for localizing the expression site of a particular gene at the cell, tissue and organ levels. This method is especially useful in understanding the function of specific gene products in particular tissues and the relation between tissue function and its localization in the whole structure of an organ. In this system, labeled anti-sense RNA probes were used to hybridize with desired mRNA while labeled sense probes were used as negative control. In general, each of these labeled probes applied to successive sections rather then the same section. That might cause some uncertainty if there are any spatial differences in the expression of gene products. In the current study, in our knowledge for the first time, anti-sense and sense probes were applied to the same sample to overcome any spatial differentiation of gene expression. The technique was successfully used to localize a plant gene product by applying anti-sense probes to the one site of the section while the other site of the same section was reacted with sense probes for control hybridization.

Glu-370 in the large subunit influences the substrate binding, allosteric, and heat stability properties of potato ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. In this study, we showed that the conversion of Glu to Gly at position 370 in the LS of AGPase alters the heterotetrameric stability along with the binding properties of substrate and effectors of the enzyme. Kinetic analyses revealed that the affinity of the LSE370GSSWT AGPase for glucose-1-phosphate is 3-fold less than for wild type (WT) AGPase. Additionally, the LSE370GSSWT AGPase requires 3-fold more 3-phosphogyceric acid to be activated. Finally, the LSE370GSSWTAGPase is less heat stable compared with the WT AGPase. Computational analysis of the mutant Gly-370 in the 3D modeled LS AGPase showed that this residue changes charge distribution of the surface and thus affect stability of the LS AGPase and overall heat stability of the heterotetrameric AGPase. In summary, our results show that LSE370 intricately modulate the heat stability and enzymatic activity of potato the AGPase.

Spatial Localization of Germin-Like Oxalate Oxidase Genes in Developing Wheat Shoots

ABSTRACT When quiescent embryos in dry grains of ripened wheat are isolated and provided with ample water and oxygen at an appropriate temperature, they begin to grow very rapidly. The onset of the growth is signaled by nascent synthesis of germin genes which encodes a relatively rare, water-soluble homop entameric glycoprotein. Germin is resistant to pepsin digestion under conditions that lead to hydrolysis of virtually all other proteins in wheat embryos. Germin proteins have oxalate oxidase activity, an activity that degrades oxalic acid to generate hydrogen peroxide which involves in many aspects of plant development. Following 48-hour imbibition on water, wheat embryos give rise to distinguishable shoot and roots. Shoots comprise coleoptile, leaf primordium and shoot apex. In the current study, non-radioactively labeled germin riboprobes were prepared by in vitro transcription. The riboprobes were used to search and localize germin mRNAs in sections taken from throughout of shoots. The results revealed that although leaf primordium and shoot apex did not show any signals of the presence of germin mRNAs, coleoptiles as a whole tissue displayed germin gene expression on epidermal cells and vascular bundle sheath cells. Among the sections taken from different parts of shoots, the sections from middle part gave the strongest signals on coleoptile cells.