BBSRC MIBTP - Investigating novel branches and substrates of the plant N-end rule pathway of proteolysis

Proteins are major functional components of cells, and regulation of their turnover is essential for controlling cellular responses and developmental outputs across kingdoms. Targeted protein degradation (proteolysis) is especially important in plants, and controls almost all aspects of plant life - for example, the sensing of and response to most plant hormones and a large number of external and internal signals (e.g. light and oxygen) is reliant on protein degradation. Therefore, increasing our understanding of the signals and mechanisms regulating protein stability is a major aim for plant science, in order to identify targets that plant breeders and biotechnologists can focus on to develop improved crop varieties.

The ‘N-end rule pathway’ is a highly evolutionarily conserved proteolytic system that targets proteins for destruction based on their N-terminal amino acid (for a recent review see Gibbs et al. 2014 Trends in Cell Biology). We recently showed that this pathway mediates plant perception and response to low oxygen stress (hypoxia), a situation that frequently occurs during floods (Gibbs et al. 2011 Nature), and have also linked N-end rule control of protein stability to the sensing of nitric oxide (NO), a gaseous molecule that regulates many critical developmental transitions and environmental responses in plants (Gibbs et al. 2014 Molecular Cell). Furthermore, there is increasing evidence that the N-end rule pathway interacts with other hormone signalling networks to control a wide range of plant processes. This pathway therefore represents a promising new system for understanding and manipulating plant growth and survival, a key focus for future food security.

Despite its emergence as a major regulator of plant signal transduction, at present our knowledge of the range of protein targets and cellular processes regulated by the N-end rule pathway in plants is limited. The main aims of this PhD project are:

(i) To screen for, identify and characterise novel protein targets of the N-end rule pathway.
(ii) To investigate whether ‘branches’ of the pathway that were recently identified in yeast are also functional in plants.
(iii) To identify and characterise key developmental-processes and stress-responses regulated by the plant N-end rule pathway.

These studies will provide new mechanistic insight into the structure and targets of this essential proteolytic pathway. Due to the genetic and molecular resources available, the majority of this work will be carried out in the model plant Arabidopsis, with the eventual aim of this research being to translate key findings into crop species, such as barley or rice. The project will be largely molecular based and the PhD candidate will gain expertise in a wide range of cutting edge and transferable techniques.

Key experimental skills involved:

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. 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 as 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. Furthermore, 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.