Abstract
Plants not only produce oxygen, but also require molecular oxygen to live. Mitochondrial respiration in plants heavily relies on oxygen, and when there is a lack, the plant undergoes an energy crisis and other stresses, and a balancing act begins to ensure survival. When the plant undergoes hypoxia, plant-specific group VII Ethylene Response Factors (ERF-VIIs) regulate hypoxic response to aid plant survival. These transcription factors promote expression of hypoxia-response genes. These transcription factors are regulated through the N-end pathway. This pathway determines the fate of these proteins based on the amino acid on the N-terminus.Under normoxia, a group of enzymes called Plant Cysteine Dioxygenases (PCO) catalyse the oxidation of the N-terminal cysteine found in the ERF-VII RAP2.12 from Cys to Cys-sulfinic acid with molecular oxygen as the co-substrate. This triggers a degradation pathway for RAP2.12. Arginyl transferase (ATE1) arginylates the acidic N-terminus, and is sent to the proteasome to get degraded. Under hypoxic conditions, there is no molecular oxygen available, and PCO is unable to catalyse the oxidation of RAP2.12.PCO1 was expressed in Escherichia coli cells and purified using StrepTrap™ HP. This purified protein was used to define which PCO1 cysteines are potentially in a disulfide bond. Disulfide bonds are important in redox signalling in the cytoplasm, and some cysteines were found to be “blocked” and could potentially be bound in disulfide bonds. It was concluded that the cysteines found in PCO are interacting with either another cysteine to form disulfides, and it was hypothesised that this was important for redox signalling. Purified PCO was tested using ferrozine and Ellman’s assays, and was unclear if it was kinetically active. PCO1 constructs were fused with green fluorescent protein (GFP) using Multi-site Gateway® Cloning to visualise which part of the protein localises PCO1 into the nucleus. GFP-PCO constructs were transiently expressed in tobacco leaves and were visualised under confocal microscopy. Full length PCO was found to localise exclusively in the nucleus, while the N-terminus (NTD) and C-terminus (CTD) localised in both the nucleus and the cytoplasm. The nuclear localisation signal (NLS) could not be defined exactly, and both the NTD and the CTD are needed to localise the protein in the nucleus. Using a similar approach, RAP2.12 constructs were fused with tandemTOMATO to develop a RAP-reporter system in tobacco leaves, visualised under confocal microscopy and quantified using fluorescence detection. PCOs play a significant role in plant hypoxia response, and characterising these enzymes could contribute to understanding how plants survive submergence tolerance. This is important for agriculture, especially with increasing flooding events, and combining this knowledge with biotechnology, submergent tolerant plants can be developed, to combat the consequences of climate change and population growth.