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Title: Genetic dissection of the regulation of chloroplast protein import by SP1-type ubiquitin E3 ligases
Author: Ali, Mohammad Sabri Bin Mohd
ISNI:       0000 0004 7654 4354
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Chloroplasts are photosynthetic organelles in plants, and their functions are dependent on the import of thousands of proteins from the cytosol. These proteins engage with the translocon at the outer envelope membrane of chloroplasts (TOC), which acts as a selective conduit for the first stage of their import across the double membrane system. Mutations affecting the TOC components, such as plastid protein import 1 (ppi1) of Arabidopsis thaliana, can hinder the efficiency of import activity, and have consequences for organelle development. A screen for second-site suppressors of ppi1 identified a novel regulator of the TOC machinery called suppressor of ppi1 locus 1 (sp1). Two homologues of SP1, SP1-Like1 (SPL1) and -2 (SPL2), were identified in Arabidopsis based on sequence similarity. In this thesis, the function of these two homologues was characterized in relation to chloroplast biogenesis. While spl1 null mutations had no profound effect on the ppi1 mutant, knockdown of SPL2 exhibited a weak suppression phenotype that could be attributed to a recovery in protein import activity and chloroplast development. Immunoprecipitation results revealed a strong physical interaction between SPL2 and the TOC components. Manipulation of SPL2 gene expression caused phenotypes reminiscent of those caused by SP1 manipulation during the dynamic process of leaf senescence. In vitro ubiquitination analysis showed that SPL1, unlike SP1 and SPL2, does not possess E3 ligase activity, but rather has an inhibitory effect on SP1 activity via an unknown mechanism. Accordingly, spl1 null mutants displayed an opposite phenotype to sp1 mutants during leaf senescence (i.e., senescence was accelerated), suggesting that SPL1 may be a negative regulator of SP1. However, SPL1 is only observed within the Brassicaceae family, which raises questions about the wider relevance of its role across different species. Lastly, a screen for new sp1 enhancer mutants was conducted, and this identified three putative sp1 enhanced chlorophyll (spec) mutants that display elevated chlorophyll levels, intermediate between those of sp1 ppi1 and wild-type plants. Mapping of the spec mutations by whole-genome sequencing identified several gene candidates in each case, which will require further analysis in order to conclude final identification of the causal locus in each case. Overall, the results revealed new regulatory mechanisms governing the chloroplast protein import machinery.
Supervisor: Jarvis, Paul ; Langdale, Jane Sponsor: Ministry of Education ; Government of Brunei
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available