Real-time detection of N-end rule-mediated ubiquitination via fluorescently labeled substrate probes

The majority of eukaryotic organisms rely on molecular oxygen for respiratory energy production. When the supply of oxygen is compromised, a variety of acclimation responses are activated to reduce the detrimental effects of energy depletion. Various oxygen-sensing mechanisms have been described that are thought to trigger these responses, but they each seem to be kingdom specific and no sensing mechanism has been identified in plants until now. Here we show that one branch of the ubiquitin-dependent N-end rule pathway for protein degradation, which is active in both mammals and plants, functions as an oxygen-sensing mechanism in Arabidopsis thaliana. We identified a conserved amino-terminal amino acid sequence of the ethylene response factor (ERF)-transcription factor RAP2.12 to be dedicated to an oxygen-dependent sequence of post-translational modifications, which ultimately lead to degradation of RAP2.12 under aerobic conditions. When the oxygen concentration is low-as during flooding-RAP2.12 is released from the plasma membrane and accumulates in the nucleus to activate gene expression for hypoxia acclimation. Our discovery of an oxygen-sensing mechanism opens up new possibilities for improving flooding tolerance in crops. © 2011 Macmillan Publishers Limited. All rights reserved

E3 ligases carry out the final step in the ubiquitination cascade, catalyzing transfer of ubiquitin from an E2 enzyme to form a covalent bond with a substrate lysine. Three distinct classes of E3 ligases have been identified that stimulate transfer of ubiquitin and ubiquitin-like proteins through either a direct or an indirect mechanism. Only recently have the catalytic mechanisms of E3 ligases begun to be elucidated.

Plants and animals are obligate aerobes, requiring oxygen for mitochondrial respiration and energy production. In plants, an unanticipated decline in oxygen availability (hypoxia), as caused by root waterlogging or foliage submergence, triggers changes in gene transcription and mRNA translation that promote anaerobic metabolism and thus sustain substrate-level ATP production 1 . In contrast to animals 2 , oxygen sensing has not been ascribed to a mechanism of gene regulation in response to oxygen deprivation in plants. Here we show that the N-end rule pathway of targeted proteolysis acts as a homeostatic sensor of severe low oxygen in Arabidopsis, through its regulation of key hypoxia response transcription factors. We found that plants lacking components of the N-end rule pathway constitutively express core hypoxia response genes and are more tolerant of hypoxic stress. We identify the hypoxia-associated Ethylene Response Factor (ERF) Group VII transcription factors of Arabidopsis as substrates of this pathway. Regulation of these proteins by the N-end rule pathway occurs through a characteristic conserved motif at the N-terminus initiating with MetCys- (MC-). Enhanced stability of one of these proteins, HRE2, under low oxygen conditions improves hypoxia survival and reveals a molecular mechanism for oxygen sensing in plants via the evolutionarily conserved N-end rule pathway. SUB1A-1, a major determinant of submergence tolerance in rice 3 , was shown not to be a substrate for the N-end rule pathway despite containing the N-terminal motif, suggesting that it is uncoupled from N-end rule pathway regulation, and that enhanced stability may relate to the superior tolerance of Sub1 rice varieties to multiple abiotic stresses 4 .