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Studying chloroplast biogenesis in the basal land plant Marchantia polymorpha: Using CRISPR/Cas9 genome editing and forward genetics to assess functional conservation and seek novel components


Project Description

Chloroplasts are responsible for photosynthesis, and are the organelles that define plants [1]. They evolved as a result of an endosymbiotic relationship between a cyanobacterium and an algal progenitor, in a process that began over a billion years ago. Land plants emerged around 500 million years ago, by which time the chloroplast had already become a fully integrated component of the plant cell.

Today, >90% of the ~3000 proteins found inside chloroplasts are encoded by the nuclear genome and synthesized in the cytosol as precursors with N-terminal targeting signals called transit peptides. The import of such precursors into chloroplasts is mediated by multiprotein machines in the chloroplast envelope membranes called TOC and TIC (Translocon at the Outer/Inner envelope membrane of Chloroplasts) [2].

In flowering plants, the TOC complex comprises a channel-forming molecule (Toc75) and multiple receptors in two families (Toc159, Toc34) that recognize precursor proteins as they arrive at the chloroplast surface. Composition of the TOC complex is controlled by a “master regulator” protein called SP1 [3]. The SP1 gene was identified using a forward-genetic approach in the model flowering plant, Arabidopsis thaliana: we screened for extragenic suppressors of a pale-yellow TOC receptor mutant, identifying suppressor mutants by their greener appearance [4]. SP1 is a ubiquitin E3 ligase in the chloroplast outer membrane that targets TOC components for ubiquitination and degradation by the ubiquitin-proteasome system. By controlling protein import in this way, SP1 enables reconfiguration of chloroplast functions in response to developmental and environmental cues [3,4].

Bryophytes, comprising liverworts, mosses and hornworts, are the earliest diverging group of land plants. The liverwort Marchantia polymorpha is an emerging model system for plant biology research [5], which because of its basal position in the land plants enables important evolutionary questions to be addressed. Marchantia has several distinguishing features, such as the dominance of the haploid gametophyte generation over the diploid sporophyte during its life cycle. The latter point, in combination with its low genetic redundancy, means that Marchantia is particularly well suited to forward-genetic screening based on phenotype analysis. Moreover, advanced techniques for generating targeted gene knockouts (including homologous recombination and CRISPR/Cas9 approaches [6,7]) have been successfully applied in Marchantia.

We sequenced the Marchantia genome [8], and bioinformatic analyses indicated the presence of genes encoding SP1 and all major TOC components. The aims of this project will be to elucidate the functions of these genes, assessing the extent of functional conservation with flowering plants, and to seek entirely new components involved in chloroplast protein biogenesis that eluded detection previously due the higher genetic redundancy in flowering plant models:

1. Reverse genetics. Using CRISPR/Cas9 genome editing [6,7], we will generate knockout or knockdown mutants for SP1 and all TOC genes. The phenotypes of the mutants will then be characterized in detail (e.g., in relation to protein import capacity) to elucidate the extent to which the functions of the genes have been conserved during land plant evolution. Functional relationships between the components will be assessed by generating and characterizing all relevant double mutant combinations.

2. Forward genetics. Based on the results from 1, we will establish a forward-genetic screening strategy to identify entirely novel factors involved in chloroplast protein import. We predict that TOC mutants will have visible, pale-yellow phenotypes caused by defective chloroplast biogenesis [2]. This will enable us to conduct a suppressor screen analogous to that which led to the identification of SP1 in Arabidopsis [3]: we will screen for greener plants following UV mutagenesis. The suppressor mutants will be characterized in detail, and the mutated genes will be identified by whole-genome sequencing.

Funding Notes

Prospective students are encouraged to apply to the Oxford Interdisciplinary Bioscience DTP. International applicants are encouraged to explore scholarship options that are available to them through the University of Oxford, their home countries or organisations such as the Commonwealth Scholarship Commission.

References

References:

1. Jarvis, P. and López-Juez, E. (2013) Biogenesis and homeostasis of chloroplasts and other plastids. Nat. Rev. Mol. Cell Biol. 14:787-802.
2. Jarvis, P. (2008) Targeting of nucleus-encoded proteins to chloroplasts in plants (Tansley Review). New Phytol. 179:257-285.
3. Ling, Q., Huang, W., Baldwin, A. and Jarvis, P. (2012) Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system. Science 338:655-659.
4. Ling Q. and Jarvis P. (2015) Regulation of chloroplast protein import by the ubiquitin E3 ligase SP1 Is important for stress tolerance in plants. Curr. Biol. 25:2527-2534..
5. Ishizaki, K. (2016) Molecular genetic tools and techniques for Marchantia polymorpha research. Plant Cell Physiol. 57:262-270.
6. Ishizaki, K. (2013) Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha L. Sci. Rep. 3:1532.
7. Sugano, S. et al. (2014) CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L. Plant Cell Physiol. 55:475-481.
8. Honkanen, S. et al. (2016) The mechanism forming the cell surface of tip-growing rooting cells is conserved among land plants. Curr. Biol. 26:3238-3244.

Recent Publications:

Bédard, J., Trösch, R., Wu, F., Ling, Q., Flores-Pérez, Ú., Töpel, M., Nawaz, F. and Jarvis, P. (2017) Suppressors of the chloroplast protein import mutant tic40 reveal a genetic link between protein import and thylakoid biogenesis. Plant Cell doi: 10.1105/tpc.16.00962. [Epub ahead of print].

Flores-Pérez, Ú., Bédard, J., Tanabe, N., Lymperopoulos, P., Clarke, A.K. and Jarvis, P. (2016) Functional analysis of the Hsp93/ClpC chaperone at the chloroplast envelope. Plant Physiol. 170: 147-162.

Ling, Q. and Jarvis, P. (2015) Regulation of chloroplast protein import by the ubiquitin E3 ligase SP1 is important for stress tolerance in plants. Curr. Biol. 25:2527-2534.

Trösch, R., Töpel, M., Flores-Pérez, Ú. and Jarvis, P. (2015) Genetic and physical interaction studies reveal functional similarities between ALBINO3 and ALBINO4 in Arabidopsis. Plant Physiol. 169: 1292-1306.

Ling, Q. and Jarvis, P. (2013) Dynamic regulation of endosymbiotic organelles by ubiquitination. Trends Cell Biol. 23: 399-408.

Jarvis, P. and López-Juez, E. (2013) Biogenesis and homeostasis of chloroplasts and other plastids. Nat. Rev. Mol. Cell Biol. 14: 787-802.

Ling, Q., Huang, W., Baldwin, A. and Jarvis, P. (2012) Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system. Science 338: 655-659.

How good is research at University of Oxford in Biological Sciences?

FTE Category A staff submitted: 223.80

Research output data provided by the Research Excellence Framework (REF)

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