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Mechanisms controlling transcription of the plastid genome

Final Report Summary - POLSPEC (Mechanisms controlling transcription of the plastid genome)

Organellar phage-type RNA polymerases are indispensable for the transcription of the chloroplast genome. They have fundamental roles in the biogenesis of the photosynthetic compartment of plant cells. The project POLSPEC investigates distinct roles of two phage-type RNA polymerases, RPOTp and RPOTmp, which are present in plastids of dicotyledonous plants where they transcribe the plastid genome together with a third, eubacterial-type transcriptase named PEP. The project has two major objectives: 1. Defining the RPOTp- and RPOTmp-specific plastid transcriptomes and generating a plastid genome-wide map of transcription start sites (TSS) utilised by RPOTp or RPOTmp. 2. Identifying cis-regulatory elements on the plastid genome that direct the transcriptional activities of RPOTp and RPOTmp.

Towards these objectives, mutant lines of the model species Arabidopsis thaliana that are defective in RPOTp, RPOTmp or PEP function have been characterised with regard to plant development and morphology. The plastid transcriptomes of these lines have been compared to that of wild-type plants, enabling us to assign distinct transcriptional tasks to the different enzymes. An optimised strategy has been established for next generation sequencing-based, plastid genome-wide TSS mapping. The method was then combined with a reverse-genetic approach to map TSS used by RPOTp, RPOTmp or PEP. Altogether, 157 plastidial TSS were mapped assigned to one or more RNA polymerases. The project discovered a previously unknown extensive overlap between TSS specificities and transcriptional tasks of the two nucleus-encoded phage-type RNA polymerases, RPOTp and RPOTmp. Owing to this extensive overlap, it has not been possible to define cis elements that specifically mediate TSS recognition by RPOTp vs. RPOTmp. Work on TSS activity during seed germination and seedling establishment revealed a developmental switch in PEP activity, from primarily transcribing rRNA and tRNA genes during germination, to additionally transcribing photosynthesis genes in greening tissues. This discovery provided the basis for a more comprehensive study of TSS usage during seed germination, and of transcriptional cofactors that are likely to mediate this switch. The project has generated fundamental knowledge on transcriptional processes in plants and lead to refined models for the transcription of the plastid genome.

Project contact:
kristina.kuehn@pflanzenphys.uni-halle.de
https://www.biologie.uni-halle.de/institutsbereich_pflanzenphys/zellphysiologie/?lang=en