Editors' ChoicePlant biology

Coupling Oxygen to Protein Stability

Science Signaling  22 Nov 2011:
Vol. 4, Issue 200, pp. ec323
DOI: 10.1126/scisignal.4200ec323

Like animals, plants respond to oxygen deprivation by triggering a genetic program that promotes anaerobic metabolism and adaptation to this stress. Although transcription factors of group VII of the ethylene response factor (ERF) family—HRE1 and HRE2, RAP2.2 and RAP2.12, SUB1A, and SK1 and SK2—enhance plant responses to hypoxia, the mechanism of their regulation is unknown. Two groups (Gibbs et al. and Licausi et al.) show that degradation of these proteins during oxygen-replete conditions occurs through an N-end rule pathway in which modification of the N terminus of the protein targets it for degradation by a ubiquitin-proteasomal mechanism. Gibbs et al. found that Arabidopsis seedlings with mutations in genes important for the N-end rule pathway exhibited constitutively high expression of genes involved in anaerobic metabolism and survival from hypoxia. Seeds from plants with these mutations germinated under low oxygen, and seedlings were more tolerant to prolonged oxygen deprivation than were wild-type seedlings. In vitro transcription and translation of group VII ERFs, in a system containing all of the components necessary for N-end rule pathway degradation, demonstrated that the proteins were short-lived and that their stability was enhanced by the presence of a dipeptide inhibitor of the N-end rule pathway or by mutations of the critical cysteine needed for the posttranslational modification that targets the proteins for degradation. SUB1A, which in rice confers tolerance to submersion in water, was resistant to degradation in vitro and thus was uncoupled from the N-end rule pathway. In vivo, expression of HRE1 or HRE2 or mutant forms that were not subject to N-end rule pathway degradation showed that the mutant forms were constitutively detectable. Wild-type HRE2 protein exhibited an induction in abundance by hypoxia, which was destabilized after a return of the plants to normoxia. In plants compromised in N-end rule pathway degradation, HRE1 and HRE2 were abundant even under normoxic conditions.

Licausi et al. focused on the RAP2.12 member of this ERF family. Overexpression of RAP2.12 increased submergence tolerance of plants, but plant growth and submergence tolerance were reduced if the protein was tagged at the N terminus with hemagglutinin (HA-RAP2.12) or if the first 13 amino acids were deleted. Transcriptome analysis showed that overexpression of RAP2.12 promoted the expression of genes involved in the hypoxia response and that silencing of RAP2.12 (and its homolog RAP2.2) compromised the induction of hypoxia-responsive genes in response to hypoxic conditions. Analysis of the subcellular localization of the protein in leaves showed that a green fluorescent protein (GFP)–tagged RAP2.12 was associated with the plasma membrane under normoxic conditions, accumulated in the nucleus in response to hypoxia, and then was undetectable after a return to oxygen-replete conditions. The deletion mutant was present in both the nucleus and plasma membrane under normoxia, relocalized to the nucleus in response to hypoxia, and then failed to reduce in abundance after a return to normoxia. Both groups showed that the oxygen-dependent changes in the abundance of members of the ERF group VII family were compromised in plants with mutations in the enzymes necessary for N-end rule pathway degradation. Thus, plants produce the transcription factors that induce the hypoxic response all the time, but under normoxic conditions these are degraded by the N-end rule pathway, which couples oxygen to protein stability.

D. J. Gibbs, S. C. Lee, N. M. Isa, S. Gramuglia, T. Fukao, G. W. Bassel, C. S. Correia, F. Corbineau, F. L. Theodoulou, J. Bailey-Serres, M. J. Holdsworth, Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants. Nature 479, 415–418 (2011). [PubMed]

F. Licausi, M. Kosmacz, D. A. Weits, B. Giuntoli, F. M. Giorgi, L. A. C. J. Voesenek, P. Perata, J. T. van Dongen, Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization. Nature 479, 419–422 (2011). [PubMed]