Die epigenetische Regulation des C 4 -Syndroms : belichtungsabhängige Chromatinveränderungen am Promotor der Phosphoenolpyruvat Carboxylase aus Mais (Zea mays L.)
C4 plants have developed a unique mechanism to suppress the undesirable effects of photorespiration by separating primary and secondary CO2 fixation in two different leaf tissues. The primary fixation step takes place in the mesophyll cytoplasm and is controlled by phosphoenolpyruvate carboxylase (PEPC). The enzyme catalyses the formation of oxaloacetate from bicarbonate and phosphoenolpyruvate (PEP). In maize, the reaction product is rapidly converted to malate and transported into the bundle sheath where it is decarboxylated to pyruvate. The liberated CO2 is available for refixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) and PEP is recycled from pyruvate in the mesophyll. By this, CO2 is concentrated inside the bundle sheath and the intrinsic oxygenase activity of RUBISCO is inhibited. We analyzed alterations in the chromatin structure on the promoter of the C4 specific isoform of phosphoenolpyruvate carboxylase (PEPC) after illumination of seedlings. A protocol was established that facilitates the preparation of nuclei from maize leaves with intact chromatin structure and resistance to DNA degradation during prolonged incubation at high temperatures. The presence of non-spliced transcripts from the C4-PEPC gene inside the nuclei was proven by RT-PCR. The chromatin was partially digested with restriction endonucleases. Quantitative PCR analyses revealed a clear increase in the accessibility of the promoter chromatin to restriction dependent on illumination of the seedlings. The data indicate chromatin remodeling on the C4-PEPC promoter during activation. In the second part of the work the technology of the chromatin immunoprecipitation (ChIP) was established with the purpose of investigation of histone modifications. Several experimental approaches were comparatively examined, resulting in establishing a protocol for use the ChIP in the maize system. The method was successfully applied in order to examine exemplary changes in acetylating the histone H3 and H4. The obtained data speak strongly for an anew exposure-dependent acetylation of the histones H4 on the PEPC promoter. Discussion of the data obtained contains their critical analysis in the light of the state of the published biological knowledge. On basis of these findings a model for the activation of the promoter of the C4 specific PEPC in the chromatin organisation was constructed.
info:eu-repo/classification/ddc/580, C4-Pflanzen, Phosphoenolpyruvatcarboxylase, Promotor <Genetik>, Epigenese, Genregulation, Mais, Lichtatmung, Chromatin, Pflanzen (Botanik), Chromatinveränderungen, Pflanzen
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