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Guillaume Allorent

Characterisation of gene expression in photoheterotrophic plastid during seed formation

Published on 4 November 2011

Thesis presented November 04, 2011

Transcription of the plastid genome, one of the three genomes (nuclear, mitochondrial and plastidial) that co-exist in the plant cell, is performed by three ARN polymerases. Two NEPs (Nucleus-Encoded Plastid RNA polymerases) transcribe mainly housekeeping genes and one PEP (plastid-encoded RNA polymerase) transcribes principally photosynthesis related genes. PEP needs transcription factors of the sigma type that are nucleus-encoded. We have previously shown that all three RNA polymerases are present in dry seeds and are necessary for efficient ​germination. These findings raised the question of how theses RNA polymerases come into the dry seeds and what is the importance of plastid gene expression during seed formation. To answer this question my work consisted in the characterization of plastid gene expression profiles and the expression of the components of the plastid transcriptional machinery during the three phases of seed formation, i.e. embryogenesis, maturation (embryonic photosynthesis) and desiccation. The analysis of global plastid transcriptome patterns shows that mRNAs encoding proteins engaged in photosynthesis show the highest quantitative changes during seed formation. Highest mRNA levels are observed during maturation. During desiccation, photosynthesis related mRNA levels as well as the levels of the corresponding proteins strongly decrease. Concerning the expression of NEP and PEP components, we observe also a peak of protein accumulation during maturation that is followed by a strong diminution of the protein levels. On the other hand, the corresponding mRNAs increase continuously during desiccation. This means that these mRNAs accumulate without being translated. We conclude that the storage of mRNAs coding components of the plastid transcriptional machinery in dry seeds is important for efficient germination. Regarding the limited amount of biological material that is available for these types of studies, we have developed a new method for cDNA analyses on microchips that utilizes​ quartz plates and TIRF microscopy. In this way we can visualize single molecules and the amount of necessary material is considerable diminished. Finally, we have also partially characterized the conditions under which embryonic photosynthesis is performed. These studies show that photosynthesis occurs in a special environment that is characterized by hypoxic atmosphere and green enriched light. However, the structure and functioning of the photosynthetic apparatus in seed chloroplasts seems to be very similar to that of chloroplasts in green leaves. This opens the question of how seed photosynthesis can be efficient. On the other hand we have shown that embryonic photosynthesis is indeed very important for efficient germination. Altogether, results provide new information on the functioning of plastid photosynthesis and transcription during seed formation. They underline the importance of the accumulation of NEP and PEP coding mRNAs in dry seeds. We suggest that embryonic photosynthesis influences seed germination not only by providing reserve compounds but also by producing NEP and PEP proteins. Although the majority of these proteins are degraded during desiccation, traces persist and are stored in dry seeds thus assuring immediate transcription of the plastid genome during imbibition/stratification. Our results explain how efficiency of germination is conditioned during seed formation.

Chloroplast, transcription, Arabidopsis thaliana, seed, photosynthesis, gene ​expression

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