Thesis presented December 06, 2019

Abstract:
Chloroplast biogenesis from undifferentiated proplastids is a process that requires a complex, largely not understood, molecular regulation network. One decisive is the plastid-encoded plastid RNA polymerase (PEP). PEP consists of four subunits encoded by the plastidial RPO genes. Upon illumination, the PEP core enzyme becomes decorated with 12 PEP-associated proteins (PAPs) that are all encoded in the nucleus.
The genetic inactivation of the majority of these PAPs blocks plastid differentiation and disrupts chloroplast biogenesis leading to albinism. The Arabidopsis albino mutant pap7-1, here in this work, is used as a genetic tool to understand the early steps of the chloroplast biogenesis.
The PAP7/pTAC14 protein (that has been identified as a subunit of the PEP complex) exhibits a dual localization in plastids and nucleus. Functional complementation of the pap7-1 mutant with a full length coding sequence construction could rescue the albino mutant. Transgenic plants could develop a wild-type phenotype in the mutant background. This confirms the importance of PAP7 in the early stages of chloroplast biogenesis. Nevertheless, hemi-complementation tests showed that a separation between nuclear and plastid functions is accompanied with loss of functionality. On the other hand, the absence of PAP7 destroyed the formation of late nuclear photobodies, which suggests that the nuclear fraction of PAP7 can interact with other molecular regulators in order to mediate light signaling by activated phytochrome B. This pathway likely controls the expression of the nuclear transcription factors (such as GLK1) that is known to control nuclear encoded photosynthesis associated genes. The proteome of the pap7-1 mutant suggested that the majority of proteins are strongly down-regulated strictly following the corresponding transcript profile. Albino plants showed a complete absence of photosynthetic apparatus, pigment accumulation (chlorophyll and carotenoid), thylakoid membrane lipids and selected hormones. The tetrapyrrole biosynthesis pathway remained partially functional in the mutant producing heme, however, the chlorophyll biosynthesis branch was found to be selectively blocked.
In sum, these data suggest the action of a retrograde signal that prevents expression of some PhANGs and genes for other related processes. The carotenoid biosynthesis should be functional in the pap7-1 mutant due to normal expression of carotenoid biosynthesis genes and corresponding enzymes. Nevertheless, carotenoids were absent likely caused by the absence of photosynthesis that usually produces the carotenoid precursor glyceraldehyde-3-phosphate. As consequence abscisic acid likely does not accumulate. In addition, in the absence of thylakoid membranes establishment, membrane lipids such as MGDG are absent. This and the down-regulation of the allene oxide synthesis pathway most likely cause a block in synthesis of OPDA, the plastid precursor of jasmonic acid. Jasmonic acid, in turn, is known to be a repressor of hypocotyl elongation during early seedling development, a phenotype observed when PAP7 is missing. The present study provides evidence that PAP7 is essential for chloroplast biogenesis being a central component responsible for proper plastid gene expression. Furthermore, it acts as a retrograde signaling component that couples phytochrome-mediated light signaling towards the nucleus with chloroplast biogenesis and integrates metabolic feedback loops from the plastid into this complex process.

Keywords:
Arabidopsis, chloroplast biogenesis, albinism, pap7-1 mutant, dual nucleus-plastid localization, proteomics, systems biology, retrograde signaling

On-line thesis.