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Build and destroy: investigating targeted protein degradation in plant development and stress-response

Primary Supervisor: Professor Daniel Gibbs, School of Biosciences

Secondary supervisor: Dr Juliet Coates

PhD project title: Build and destroy: investigating targeted protein degradation in plant development and stress-response

University of Registration: University of Birmingham

Project outline:

In order to effectively co-ordinate their growth and development, plants need to be able to respond to changes in their environment, whether it is adapting to the seasons or coping with stresses. Such responses are often orchestrated at the cellular level through altering the amounts and activities of proteins, which can be achieved through changes in gene transcription and translation, but also through targeted protein degradation (proteolysis). Proteolysis is especially important in plants, and controls almost all aspects of their life; the perception and transduction of most plant hormones and many environmental signals is reliant on protein degradation. Therefore, increasing our understanding of the mechanisms regulating where and when proteins are selectively destroyed is a major aim for plant science, because such studies could identify new targets for enhancing yield and improving stress-resilience to ensure future food security.

The ‘N-degron pathway’ is a specific pathway for protein degradation that targets proteins for destruction based on their N-terminal amino acid residue (see Gibbs et al. 2014 Trends in Cell Biology). It was previously shown that regulation of transcription factor stability via this pathway is important for sensing low oxygen stress (hypoxia), a situation that frequently occurs during floods, and nitric oxide (NO), a gaseous molecule that regulates developmental transitions and environmental responses (Gibbs et al. 2011 Nature; Gibbs et al. 2014 Molecular Cell). More recently, we have linked this pathway to the regulation of a conserved protein complex that controls longer-term epigenetic responses to the environment by modifying histones in target genes (Gibbs et al. 2018 Nature Communications), and we are also investigating a new ‘branch’ of the system based on N-terminal acetylation of proteins (Gibbs et al. 2015 Trends in Plant Science). Currently our projects are supported by a 5 year ERC-STG grant.

Despite its emergence as a major regulator of plant signal transduction, our knowledge of the range of protein targets and cellular processes regulated by the N-degron pathway in plants is currently limited. A PhD project in my lab would be focussed on one of several key areas of study relating to the N-end rule pathway in plants:

(i) Investigating the role of protein degradation via the N-end rule pathway in controlling the epigenome, and its subsequent effects on development and environmental responsiveness.

(ii) Investigating links between N-terminal acetylation and protein stability in plants, by functionally characterising the enzymes involved, identifying protein targets, and linking the pathway to plant growth and stress-responses.

(iii) Investigating how components of the N-degron pathway regulate co-translational quality control to ensure the efficient production of proteins to maintain cellular function.

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 (such as ChIP-Seq and proteomics). The PhD candidate will therefore gain expertise in a wide range of cutting edge and transferable techniques.

References:

  1. Gibbs DJ, Lee SC, Isa NM, Gramuglia S, Fukao T, Bassel GW, Correia CS, Corbineau F, Theodoulou FL, Bailey-Serres J, Holdsworth MJ (2011) Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants. Nature. 479(73):415-8.
  2. Gibbs DJ, Isa NM, Movahedi M, Lozano-Juste J, Mendiondo GM, Berckhan S, Marín-de la Rosa N, Vicente Conde J, Sousa Correia C, Pearce, SP, Bassel GW, Hamali B, Talloji P, Tomé DFA, Coego A, Beynon J, Alabadí D, Bachmair A, León J, Gray JE, Theodoulou FL, Holdsworth MJ (2014) Nitric oxide sensing in plants is mediated by proteolytic control of Group VII ERF transcription factors. Molecular Cell. 53(3): 369-379
  3. 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
  4. Gibbs, DJ (2015) Emerging functions for N-terminal protein acetylation in plants. Trends In Plant Science. 20(10): 599-601
  5. Gibbs DJ*, Bacardit J, Bachmair A, Holdsworth MJ* (2014) The eukaryotic N-end rule pathway: conserved mechanisms and diverse functions. Trends in Cell Biology. http://dx.doi.org/10.1016/j.tcb.2014.05.001

BBSRC Strategic Research Priority: Understanding the Rules of Life: Plant Science

        Techniques that will be undertaken during the project:

        This project will use a wide range of state-of-the-art molecular biology, genetic and protein biochemistry approaches to identify and characterise new substrates and branches of the N-end rule, and to link the pathway 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. Crucially, since this pathway 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.

        Contact: Professor Dan Gibbs, University of Birmingham