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Functions for the N-end rule pathway of targeted protein degradation in plant development and stress-response

Principal Supervisor: Dr. Daniel Gibbs - School of Biosciences

Co-supervisor: Prof. George Bassel

PhD project title: Functions for the N-end rule pathway of targeted protein degradation in plant development and stress-response

University of Registration: University of Birmingham

Project outline:

Proteins are major functional components of cells, and regulation of their stability is essential for controlling responses and developmental outputs across kingdoms. Targeted protein degradation (proteolysis) is especially important in plants, and controls almost all aspects of their life - for example, the sensing of most plant hormones and many environmental signals is reliant on protein degradation. Therefore, increasing our understanding of the mechanisms regulating protein stability is a major aim for plant science, because such studies will identify new targets for enhancing yield and improving stress-resilience to ensure future food security.

The ‘N-end rule’ is a specific pathway for protein degradation that targets proteins for destruction based on their N-terminal amino acid (see Gibbs et al. 2014 Trends in Cell Biology). We previously showed that this pathway is important for: (1) sensing low oxygen stress (hypoxia), a situation that frequently occurs during floods (Gibbs et al. 2011 Nature), and; (2) sensing nitric oxide (NO), a gaseous molecule that regulates developmental transitions and environmental responses in plants (Gibbs et al. 2014 Molecular Cell). More recently, we linked this pathway to the regulation of a protein that controls epigenetic responses to the environment, 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), which seems to be important in regulating responses associated with the stress hormone abscisic acid (ABA).

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-end rule 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:

  1. 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.
  2. 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.
  3. Using synthetic biology approaches to artificially engineer new proteins as substrates of the N-end rule pathway and assess the effects on protein stability and plant behaviour. 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 prtoeomics). 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 (2015) Emerging functions for N-terminal protein acetylation in plants. Trends In Plant Science. 20(10): 599-601
  4. 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: Food Security

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: Dr Daniel Gibbs, University of Birmingham