Project Outline

Like animals, plants need to breathe, and in addition to photosynthesis use oxygen (O2) for respiration and biochemical reactions. Plants are able to sense when they are running low on O2 and change their development and metabolism in response1, allowing them to survive events like being flooded. Scientists have discovered that a key part of this oxygen sensing ability in the laboratory model plant Arabidopsis thaliana is regulated by the N-degron pathway2. However, our knowledge of O2 sensing mechanisms is still incomplete, and understanding them further will be important for adapting our crops to better survive future flooding events that are predicted to increase globally due to climate change.

One important unanswered question is ‘how did oxygen sensing mechanisms evolve?’ Until now investigations have largely been restricted to the youngest group of plants- the flowering plants- but a laboratory model has recently been established in an older lineage, the ferns3. In unpublished work we have shown that the model fern Ceratopteris richardii shows considerable tolerance to complete submergence, but we do not understand how. It also shares some (but not all) of the genes that control the N-degron pathway, suggesting that a more primitive O2 sensing mechanism is at work. This project aims to establish what this fern mechanism is and how closely it resembles that in flowering plants.

This project will address this question through the following methods:

1. Characterise how the fern Ceratopteris survives submergence and what genes change during submergence using RNA-seq and bioinformatic analysis.
2. Genetically engineer a reporter-gene for low oxygen sensing used previously in Arabidopsis into Ceratopteris to compare where, when and how ferns and flowering plants respond to low oxygen.
3. Genetically engineer fern versions of known O2-sensing and signalling genes into Arabidopsis to see if they can perform the same job.
4. Delete candidate O2-sensing genes in Ceratopteris and see if the fern is better or worse at sensing low oxygen

References

1. Weits, van Dongen and Licausi (2021) Molecular Oxygen as a Signalling Component in Plant Development. New Phytologist. 229 (1): 24-35. https://doi.org/10.1111/nph.16424
2. Holdsworth and Gibbs (2020) Comparative Biology of Oxygen Sensing in Animals and Plants. Current Biology. 30 (8): R362-R369. https://doi.org/10.1016/j.cub.2020.03.021
3. Kinosian and Wolf (2022) The Natural History of Model Organisms: The biology of C. richardii as a tool to understand plant evolution. eLife 11: e75019. https://doi.org/10.7554/eLife.75019

Techniques

• Generation of stable transgenic lines in two plant genetic models (Arabidopsis thaliana and Ceratopteris richardii) using two separate plant transformation techniques (Agrobacterium-mediated transformation and microparticle bombardment)
• Manipulation of candidate gene function through functional knockout (RNAi or CRISPR) and heterologous expression of candidate genes.
• Establishment and microscopic imaging of a transgenic reporter line that marks low oxygen either through β-glucoronidase (GUS) or fluorescent protein expression.
• Detailed phenotypic analysis of transgenic lines, with associated statistical analysis where appropriate.
• A wide range of molecular biology techniques to examine candidate gene expression (e.g. quantitative real-time PCR, next-generation sequencing), protein stability analysis (Western blotting) and protein binding (immunoprecipitation).
• Bioinformatic and phylogenetic analysis.