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Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilisation of group VII ETHYLENE RESPONSE FACTORS (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilisation in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N-terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This is the first molecular evidence showing N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and the first ATE1 substrate in plants. The PCOs and ATE1 may be viable intervention targets to stabilise N-end rule substrates, including ERF-VIIs to enhance submergence tolerance in agronomy.
Preprints
Mot, A. C.; Prell, E.; Klecker, M.; Naumann, C.; Faden, F.; Westermann, B.; Dissmeyer, N.;Real-time detection of PRT1-mediated ubiquitination via fluorescently labeled substrate probesbioRxiv(2016)DOI: 10.1101/062067
The N-end rule pathway has emerged as a major system for regulating protein functions by controlling their turn-over in medical, animal and plant sciences as well as agriculture. Although novel functions and enzymes of the pathway were discovered, ubiquitination mechanism and substrate specificity of N-end rule pathway E3 Ubiquitin ligases remained elusive. Taking the first discovered bona fide plant N-end rule E3 ligase PROTEOLYSIS1 (PRT1) as a model, we use a novel tool to molecularly characterize polyubiquitination live, in real-time.We gained mechanistic insights in PRT1 substrate preference and activation by monitoring live ubiquitination by using a fluorescent chemical probe coupled to artificial substrate reporters. Ubiquitination was measured by rapid in-gel fluorescence scanning as well as in real time by fluorescence polarization.Enzymatic activity, substrate specificity, mechanisms and reaction optimization of PRT1-mediated ubiquitination were investigated ad hoc in short time and with significantly reduced reagent consumption.We demonstrated for the first time that PRT1 is indeed an E3 ligase, which was hypothesized for over two decades. These results demonstrate that PRT1 has the potential to be involved in polyubiquitination of various substrates and therefore pave the way to understanding recently discovered phenotypes of prt1 mutants.
Preprints
Dong, H.; Dumenil, J.; Lu, F.-H.; Na, L.; Vanhaeren, H.; Naumann, C.; Klecker, M.; Prior, R.; Smith, C.; McKenzie, N.; Saalbach, G.; Chen, L.; Xia, T.; Gonzalez, N.; Seguela, M.; Inze, D.; Dissmeyer, N.; Li, Y.; Bevan, M. W.;Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in ArabidopsisbioRxiv(2016)DOI: 10.1101/090563
The characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana. The peptidase is activated by two RING E3 ligases, BB and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PRT1 of the N-end rule pathway. DA1 peptidase activity also cleaves the de-ubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TCP15 and TCP22, which promote cell proliferation proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins.