To minimize damage caused by low levels of oxygen (hypoxia), cells rely on oxygen-sensing mechanisms, which drive downstream adaptive responses. Two studies now show that the N-end rule degradation pathway is part of the oxygen-sensing mechanism in Arabidopsis thaliana.
Proteins carrying particular amino-terminal sequences (known as N degrons) can be targeted for proteasomal degradation via the N-end rule pathway, following Arg conjugation by the Arg transferase ATE and subsequent ubiquitylation by the ligase PROTEOLYSIS 6 (PRT6). Gibbs et al. found that ate and prt6 mutant seedlings constitutively expressed genes important for anaerobic metabolism and hypoxia at high levels, and were more resistant to long-term oxygen deprivation. Licausi et al. also showed that ate and prt6 mutants had altered hypoxia responses, which demonstrates a link between the N-end rule pathway and the response to hypoxia.
group VII ERFs are substrates of the N-end rule pathway and sense oxygen levels
In A. thaliana, tolerance to hypoxia is in part mediated by members of subgroup VII of the ETHYLENE RESPONSE FACTOR (ERF) transcription factor family, which are potential substrates of the N-end rule pathway. To confirm whether subgroup VII ERFs are N-end rule substrates, both groups examined the importance of the N-terminal sequence in protein stability and function. Gibbs et al. showed that the stability of HRE1 and HRE2, which are subgroup VII ERFs, was dependent on their N terminus. Furthermore, Licausi et al. observed that constitutive overexpression of RAP2-12 resulted in higher expression of hypoxia-responsive genes in hypoxic conditions; by contrast, overexpression of RAP2-12 with a modified or truncated N terminus led to constant induction of hypoxia genes even after re-oxygenation, ultimately impairing plant growth. Licausi et al. also found that RAP2-12 lacking the N-end rule N degron remained active in the nucleus after re-oxygenation. Thus, the N-terminal residues of subgroup VII ERFs are important for their stability and function during hypoxia.
Further findings from both groups show that group VII ERFs are regulated by the N-end rule pathway. Licausi et al. found that the hypoxia responses of ate and prt6 mutants were impaired in a manner similar to that of plants expressing RAP2-12 proteins modified at their N terminus. Furthermore, they revealed that RAP2-12 is sequestered to the plasma membrane and is released to the nucleus only upon hypoxia, activating the subsequent expression of hypoxia-related genes. Consistently, Gibbs et al. observed that the stability of all group VII ERFs was enhanced following mutation of Cys2 — which is part of the N degron — or inhibition of the N-end rule pathway in vitro. Furthermore, HRE2 stability was increased in ptr6 mutants.
These studies show that group VII ERFs are substrates of the N-end rule pathway and sense oxygen levels, probably through oxidation of their Cys2 residue. Both groups demonstrated that degradation of these proteins by the N-end rule pathway was oxygen-dependent, and that they were stabilized under hypoxia in plants. Whether oxidation is directly related to molecular oxygen or other cellular changes remains to be determined.
ORIGINAL RESEARCH PAPERS
Gibbs, D. J. et al. Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants. Nature 23 Oct 2011 (doi:10.1038/nature10534)
Licausi, F. et al. Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization. Nature 23 Oct 2011 (doi:10.1038/nature10536)
Sriram, S. M., Kim, B. Y. & Kwon, Y. T. The N-end rule pathway: emerging functions and molecular principles of substrate recognition. Nature Rev. Mol. Cell Biol. 12, 735–747 (2011)
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Baumann, K. Sensing oxygen. Nat Rev Mol Cell Biol 12, 770 (2011). https://doi.org/10.1038/nrm3235
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