Studying chloroplast biogenesis in the basal land plant Marchantia polymorpha: Use of reverse and forward genetics to assess functional conservation and seek novel components

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. In contrast with many other land plants, Marchantia has a life-cycle that is dominated by the haploid gametophyte generation. This, 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, 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 homologous recombination or 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 identified 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.

The student will work alongside experienced postdocs and graduate students in the laboratory, and will learn a range of techniques for molecular and cellular bioscience, including: bioinformatics, molecular cloning, SDS-PAGE, immunoblotting, protein purification, protein-protein interaction analyses, organelle purification and subfractionation, genetic analysis, confocal microscopy, tissue culture, etc.

STUDENT PROFILE

This project would suit candidates with a strong background in one or more of the following areas: biological sciences, molecular biology, cell biology, biochemistry, genetics.