Build or destroy? Investigating how plants connect protein synthesis and degradation to regulate protein quality control

Project Outline

How do plant cells make sure that the proteins they produce are error free and fully functional?

Proteins are essential components of cells, and the proteome - i.e., the complete cohort of proteins and their modification status - must be effectively regulated to maintain cellular integrity. A critical contributory step to proteome function is protein synthesis, where mRNAs are decoded and translated into polypeptides by the ribosome. However, problems can arise during this process, leading to the production of aberrant proteins that negatively impact cellular function. Consequently, defective mRNAs and proteins must be recognised and destroyed, and dedicated machineries exist to facilitate this. Despite their central importance for ensuring proteome homeostasis (proteostasis), mechanisms controlling co-translational mRNA and protein quality in the plant kingdom are still largely unknown. This represents a major ‘black-box’ in the field of plant cell biology, since precise protein translation is essential for coordinating growth, development and resistance to environmental stresses that negatively impact plant productivity and survival. Increasing our understanding of the breadth of mechanisms employed by plants to ensure effective mRNA translation under stress has strong potential to identify novel targets for manipulating traits of agronomic importance and could facilitate the development of improved crop varieties with enhanced performance. As such, this project aligns with the BBSRC priority area of sustainable agriculture and food.

We recently identified a family of E3 ubiquitin ligases in Arabidopsis that function at the interface of mRNA translation and protein destruction. Here, we will investigate the hypothesis that these ligases are components of an expanded and plant-specific “toolkit” that provides dynamic and stress-responsive functions in translational quality control.

Using a range of cutting edge molecular and biochemical techniques, we will decipher roles for these regulators in controlling global translation. There are several key areas where a PhD candidate would focus their investigations:

• Defining how these E3 ligases dynamically associate with ribosomes, proteasomes and other cellular machineries to form ‘translasomes’ that act as quality surveillance hubs.
• Characterising their direct proteolytic and mRNA targets.
• Determining how they collectively contribute to the control of global and stress-responsive mRNA translation and degradation.
• Defining their roles in the proteolytic turnover of stalled or misfolded proteins that form when translational errors arise.

The majority of our work is carried out in the genetic model plant Arabidopsis, with the eventual aim of this research being to translate key findings into crop species, such as barley or rice. The research will be largely molecular based and will also include ‘omics’ approaches. The PhD candidate will therefore gain expertise in a wide range of cutting edge and transferable techniques.

Techniques

• Standard molecular genetics (PCR, qPCR, transgenic generation etc).
• Western Blotting.
• Immunoprecipitation.
• Yeast two hybrid.
• RNAseq and RIP-seq.
• Polysome profiling.
• mRNA degradome analyses.

Investigating the epigenetic control of development and stress responses in plants

Project Outline

To survive and thrive in dynamic environments, plants must be able to continually sense their surroundings and modify gene expression to orchestrate appropriate growth and developmental responses. One way in which gene expression is controlled is through methylation of histones in the chromatin of target genes by a conserved eukaryotic protein complex called the polycomb repressive complex 2 (PRC2). This methylation leads to epigenetic repression of gene expression, which is important for regulating cell identity, developmental fate, and environmental memory. Although many agronomically relevant plant processes are regulated by the PRC2, fundamental knowledge about how the complex directly senses and transduces environmental signals into chromatin changes is still lacking.

We recently showed that the stability/abundance of a flowering plant specific subunit of the PRC2 - a protein called Vernalization 2 – is regulated by oxygen availability (Gibbs et al. 2018 Nature Communications; Labandera et al. 2021 New Phytologist). This post-translational regulation means that VRN2 is degraded when oxygen is plentiful, but stabilised under low oxygen conditions (e.g., in specific hypoxic tissues, such as root and shoot meristems, and in response to stresses such as floods, which are increasing in frequency as climate change takes hold). We now want to explore the consequence of this regulation further, to understand how VRN2 contributes to diverse aspects of plant development, stress tolerance and environmental memory. A PhD project in my lab would be focussed on one of several key areas of inquiry, including:

1. Investigating a role for VRN2 and other histone modifying proteins in establishing stress tolerance and memory (especially during flooding stress), and exploring how manipulating their function can improve stress-resilience.
2. Investigating how post-translational accumulation of VRN2 in hypoxic niches of the root and shoot meristems helps to co-ordinate plant development.
3. Using synthetic biology approaches to rewire protein stability and function to create programmable plants that have desired developmental and stress-associated traits.

The exact focus of the project is flexible and can be tailored to specific interests. Most of our work is carried out in the genetic model plant Arabidopsis, with the eventual aim of this research being to translate key findings into crop species, such as barley or rice. As such, this project aligns with the BBSRC priority area of Sustainable Agriculture and Food. The research will be largely molecular based and will also include ‘omics’ approaches (such as ChIP-Seq and proteomics). The PhD candidate will therefore gain expertise in a wide range of cutting edge and transferable techniques.

References

Gibbs DJ, Tedds HM, Labandera A-M, Bailey M, White MD, Hartman S, Sprigg C, Mogg SL, Osborne R, Dambire C, Boeckx T, Paling Z, Voesenek LACJ, Flashman E, Holdsworth MJ (2018) Oxygen-dependent proteolysis regulates the stability of angiosperm Polycomb Repressive Complex 2 subunit VERNALIZATION2.Nature Communications. 9:5438 DOI:10.1038/s41467-018-07875-7

Labandera AM, Tedds H, Bailey M, Sprigg C, Etherington R, Akintewe O, Kalleechurn G, Holdsworth MJ andGibbs DJ(2021) The PRT6 N-degron pathway restricts VERNALIZATION 2 to endogenous hypoxic niches to modulate plant development.NEW PHYTOLOGIST. https://doi.org/10.1111/nph.16477

Techniques

This project will use a wide range of state-of-the-art molecular biology, genetic and protein biochemistry approaches to characterise a recently identified environment sensing histone modifier, and to link its function to important physiological and growth processes in plants. Depending on the specific project, candidates will gain experience in gene cloning and the generation of mutant and transgenic plants to assist in the dissection of gene function, gene expression analysis (e.g. qRT-PCR) and phenotypic assessment at the physiological and molecular level. This will also inclue transcriptomics and ChIP-seq approaches. Crucially, since this project is related to protein degradation, protein biochemical approaches (including western blotting, protein stability assays and immunoprecipitation/pull down techniques) will also be heavily utilised, as well as mass-spectrometry based proteomics methods for protein screening and assessing protein stability/modifications. Candidates will gain skills in confocal microscopy and image analysis. This project will therefore provide the candidate with training in a wide range of varied, important and highly transferable molecular laboratory-based skills.