Plant cysteine oxidase function in early plants and algae
Keywords: PCOs
oxygen sensing
Plant Cysteine Oxidases

All aerobic organisms require O2 for survival. When O2 is limited (hypoxia), a response is required to reduce demand and/or improve supply. While plants can survive temporary periods of acute hypoxia, prolonged exposure can result in plant damage or death. A concern over food security has driven higher plant hypoxia research, with little contributed to understanding marine or early land plants. The photosynthesis of phytoplankton is the dominant global process that replenishes atmospheric and oceanic O2. In light of global warming, and the reported expansion of oceanic O2 minimums, the impact oceanic deoxygenation will have on algae, or vice versa, must be assessed.
The higher plant hypoxia response is mediated by the group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs). ERF-VII transcription factor stability is regulated by the Arginine/Cysteine (Cys/Arg) branch of the N-degron pathway. It has been shown that the O2-dependent Plant Cysteine Oxidases (PCOs) catalyse oxidation of ERF-VII N-terminal cysteine residues to form cysteine-sulfinic acid. This renders the ERF-VII N-terminus a substrate for arginyl-tRNA protein transferase (ATE); arginylation promotes ubiquitination by E3 Ubiquitin ligase PRT6 (PRT6) and degradation by the proteasome. During hypoxia, the ERF-VII transcription factors are stabilised, resulting in upregulation of genes which promote survival. Genes encoding for putative PCOs are ubiquitous across the plant kingdom. However, the ERF-VII transcription factors are confined to the flowering plants.
Undoubtedly, there are metabolic challenges associated with living as single-celled aquatic organisms, and also with transitioning from single-celled phytoplankton to land-dwelling, multicellular plants. What role the PCOs might have played in this transition, or in what capacity the PCOs help regulate algal health with respect to O2 has yet to be described. This thesis uses a combination of recombinant protein studies, PCO substrate discovery and algal culturing to investigate the evolution of the plant O2 sensing pathway, the effect of hypoxia on algal health, and the role of algal PCOs. To address these questions, a series of model plant species were selected for study. M. polymorpha is a liverwort, with liverworts reputed to be the first land plants; possibly retaining features of its algal ancestors and extant land plants. K. nitens is an undifferentiated freshwater alga and is a model for understanding the plant transition from water to land. E. huxleyi is a globally distributed Haptophyte, with extraordinary adaptive capabilities and therefore a powerful tool for understanding algal adaptation to climate change-driven stresses. All plants contain putative PCOs, with K. nitens and M. polymorpha also containing ATE and PRT6 homologs – essential elements of the Cys/Arg branch of the N-degron pathway.
This thesis describes novel PCO enzymes from Marchantia polymorpha (MpPCO) and Klebsormidium nitens (KnPCO). MpPCO was characterised with an endogenous substrate, MpERF, with homology to the ERF-VII transcription factors. Despite being the only PCO isoform in M. polymorpha, MpPCO catalytic activity was relatively insensitive to changes to O2 concentrations. By contrast, KnPCO had the greatest O2 sensitivity of any PCO characterised thus far. KnPCO did not have as broad a substrate profile as other PCOs, failing to oxidise LITTLE ZIPPER 2, a known substrate of MpPCO and the higher plant PCOs. This thesis also established algal hypoxia culturing facilities and identified potential hypoxia relevant proteins in the C. reinhardtii proteome, which were verified as substrates of the PCOs in vitro.
This study takes the first steps towards understanding the relationship between the basal and higher plant O2 sensing pathways. It characterises a functional PCO with an ERF-VII-related substrate in M. polymorpha, and describes the first algal PCO and substrates. It suggests an O2 sensitive N-degron pathway exists in K. nitens, without any ERF-VII, ZPR2 or MC-initiating VRN2 substrates in the K. nitens genome. Substrate hunting experiments suggest an algal metabolic response to hypoxia, which could have far reaching consequences for global algal health.