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Öğe Formation of wheat (Triticum aestivum L.) embryogenic callus involves peroxide-generating germin-like oxalate oxidase(2004) Caliskan, Mahmut; Turet, Müge; Cuming, Andrew C.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. © Springer-Verlag 2004.Öğe Germin-like oxalate oxidase activity increase in auxin-treated wheat coleoptiles(2000) Çalışkan, Mahmut; Cuming, Andrew C.Oxalic acid or more commonly its salts, oxalates, are widely distributed throughout the plant kingdom. Oxalic acid is generally considered an inert product of metabolism and its accumulation is toxic to tissues. Therefore, the enzymes degrading oxalic acid have received considerable attention. Oxalate oxidase is an oxidoreductase which degrades oxalate into $CO_2$ and $H_2O_2$ . Recently, oxalate oxidase was shown to have high amino acid homology with germin proteins and at the same time cereal germin proteins were shown to have oxalate oxidase activity. Germin gene expression was shown to be auxin responsive. It was well demonstrated that coleoptile cells underwent extensive elongation upon auxin treatment. In the current study, it was shown that during auxin-induced wheat coleoptile elongation there was an increase in the activity of germin-like oxalate oxidase. This activity which produce $H_2O_2$ may restrict cell elongation by mediating cross-linking of cell wall polymers.Öğe Monitoring of Germin synthesis by pre-absorbed anti-Germin serum(2000) Çalışkan, Mahmut; Cuming, Andrew C.Bitki embriyoları dormant halden aktif bir büyüme dönemine girdikleri zaman, bazı yeni gen ürünleri sentezlerler. Bu ürünlerden germin proteini karakterize edilmistir. Germin, suda çözünen, homopentamerik bir glikoproteindir. GermiN sentezi ve birikimi bugday embriyosunun ilk büyüme evreleri ile yakından ilgilidir. Germin sentezi spesifik olmayan baglanmaları önlemek için bugday–ekstraktı ile on muamele edilmis anti–germin antikoru kullanılarak analiz edilmistir. Germin proteini 16 saat ile 6 gün arası tüm filizlenen bugday embriyolarında bulunmustur. Germin az bulunan bir protein olmasına ragmen, 20 ?L protein ekstraktında bile X–Ray filmi üzerinde koyu bir band olarak antikorlar tarafından belirlenmistir. Germin olgun bugday bitkisi organlarında ve 16 saatten önceki filizlenen embriyolarda bulunamamıstır.Öğe A plasma membrane H+ATPase gene is germination-induced in wheat embryos(Academic Journals, 2010) Caliskan, Mahmut; Bashiardes, Stavros; Cuming, Andrew C.The expression pattern of a germination specific plasma membrane H+-ATPase was analyzed by RTPCR and in situ RNA hybridization methods. RT-PCR results revealed that germination specific plasma membrane H+-ATPase accumulation was detectable in all organs and tissues of germinating wheat embryos. H+-ATPase expression was not observed in dry wheat embryos and in immature wheat embryos. In situ RNA hybridization indicated that germination specific plasma membrane H+-ATPase gene expression was associated with all organs of germinating wheat tissues. The accumulation of H+-ATPase mRNA was more heavily on the cells of vascular bundles and epidermal cells of coleoptiles. Since germination specific plasma membrane H+-ATPase gene was identified as a growth related gene, interest was focused on the activity of growth regulators (GA, IAA, ABA) and stress factors, NaCl and Mannitol, on H+-ATPase gene expression. The results indicated that there were not any dramatic changes in the accumulation of germination specific plasma membrane H+-ATPase gene in any case. More rigorous analysis is necessary to evaluate the effect of growth regulators on germination specific plasma membrane H+-ATPase.Öğe Temporal and spatial determination of germin bosynthesis in wheat tissues(2000) Çalışkan, Mahmut; Cuming, Andrew C.Bu çalışmada germin proteininin çimlenen buğday embriyo organlarındaki biosentezi belirlenmiştir. Germin protein sentezinin yersel ve zamansal analizi germin antikor serumu kullanılarak gel-blot tekniği ile belirlenmiştir. Germin proteini 2, 3, ve 6 gün süreyle çimlendirilmiş olan buğday fidelerinin koleoriza, koleoptile ve kök bölgelerinde belirlenmiştir. Detaylı çalışmalar germin protein sentezinin yaprak dokusunda ancak çimlenmenin ileri devresinde olduğunu göstermiştir. Değişik yaşlardaki embriyolardan alınan protein homowenatları içinde pellet fraksiyonlarının supernatant fraksiyonlarına göre daha çok germin içerdiği belirlenmiştir.
