Molecular characterisation of the plant N-end rule, a pathway of agricultural importance.

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Molecular characterisation of the plant N-end rule, a pathway of agricultural importance.

Turnover of regulatory proteins is a hugely important control mechanism in signalling and development in plants. Recently, we discovered that a specialised pathway of targeted protein degradation- the N-end rule pathway- regulates ABA signalling and seed oil mobilisation (Holman et al., 2009 PNAS USA) and acts as a sensor for hypoxia and nitric oxide (Gibbs et al., 2011 Nature; 2014, Mol. Cell). Others have shown important roles in leaf development and senescence (Graciet et al., 2011 TIPS). Thus this pathway has several functions of great importance for agriculture, for example the discovery of the oxygen sensor has important implications for understanding and manipulating flooding tolerance in crops. Despite these exciting advances, much remains to be discovered. The N-end rule pathway has three branches which were originally defined using artificial protein substrates in Arabidopsis: the arginine branch, the leucine branch and the aromatic amino acid branch, of which only the former has been characterised. This project aims to identify the substrates and functions of the aromatic branch. Arabidopsis mutants lacking key N-end rule components are available to the project and we have developed novel proteomics methods to identify proteins which are stabilised in N-end rule mutants. These will be used in parallel with transcriptional profiling to identify both bona fide protein substrates and also downstream targets of these substrates. Selected candidates will be analysed functionally using a range of assays. The following questions will be addressed: Where is the pathway active in the plant? (Promoter-GUS fusions, GFP fusions, Protein stability reporters; X-GUS) What are its physiological roles of the pathway? (Phenotypic and transcriptomic analysis of knockout mutants) What are the protein substrates? (Affinity purification of interacting proteins in vitro and in vivo; Differential proteomics) Validation and role of substrates in planta

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