Plant Cysteine Oxidase Oxygen-Sensing Function Is Conserved in Early Land Plants and Algae

All aerobic organisms require O2 for survival. When their O2 is limited (hypoxia), a response is required to reduce demand and/or improve supply. A hypoxic response mechanism has been identified in flowering plants: the stability of certain proteins with N-terminal cysteine residues is regulated in an O2-dependent manner by the Cys/Arg branch of the N-degron pathway. These include the Group VII ethylene response factors (ERF-VIIs), which can initiate adaptive responses to hypoxia. Oxidation of their N-terminal cysteine residues is catalyzed by plant cysteine oxidases (PCOs), destabilizing these proteins in normoxia; PCO inactivity in hypoxia results in their stabilization. Biochemically, the PCOs are sensitive to O2 availability and can therefore act as plant O2 sensors. It is not known whether oxygen-sensing mechanisms exist in other phyla from the plant kingdom. Known PCO targets are only conserved in flowering plants, however PCO-like sequences appear to be conserved in all plant species. We sought to determine whether PCO-like enzymes from the liverwort, Marchantia polymorpha (MpPCO), and the freshwater algae, Klebsormidium nitens (KnPCO), have a similar function as PCO enzymes from Arabidopsis thaliana. We report that MpPCO and KnPCO show O2-sensitive N-terminal cysteine dioxygenase activity toward known AtPCO ERF-VII substrates as well as a putative endogenous substrate, MpERF-like, which was identified by homology to the Arabidopsis ERF-VIIs transcription factors. This work confirms functional and O2-dependent PCOs from Bryophyta and Charophyta, indicating the potential for PCO-mediated O2-sensing pathways in these organisms and suggesting PCO O2-sensing function could be important throughout the plant kingdom.


Expression and Purification of MpPCO and KnPCO
The gene encoding MpPCO was amplified from initial constructs donated by Professor Francesco Licausi and cloned into pET28a (Novagen). The gene encoding KnPCO was synthesised and inserted into pET28a (Genscript). Recombinant proteins were expressed and purified as described previously 1 . Briefly, enzymes were expressed in E. coli BL21 (DE3) competent cells induced with 0.5 mM isopropyl 1-thio-β-Dgalactopyranoside for 16 hours 18 °C. Soluble protein was purified via Ni-affinity chromatography and size exclusion chromatography. Protein purity was assessed by SDS-PAGE.

MpPCO/KnPCO metal content determination
Trace element analyses of metal content in enzyme samples were conducted using inductively coupled plasma mass-spectrometry (ICP-MS) on a NexION 350D ICP-MS (PerkinElmer) coupled with a prepFAST Flow Injection Automation System autosampler (Elemental Scientific). The instrument was calibrated from a series of several standards, which were robotically prepared by the autosampler. Each solution was injected into the instrument nine times at 100ms intervals, and the concentration calculated from the average peak height of the nine injections by comparison to the calibration response curve. Rhodium, indium iridium and rhenium were also added into each measured solution as internal standards, to correct for any instrumental drift that may be caused by matrix suppression. Ratios of Fe per protein molecule were calculated.

KnPCO/MpPCO activity assays
Unless indicated otherwise, the activities of KnPCO and MpPCO were examined by incubating synthesized peptide with 0.1 μM enzyme at 25 °C under aerobic conditions in the presence of 1 mM TCEP, 50 mM Bis tris propane and 50 mM NaCl at pH as specified in the text. Reactions were quenched by the addition of 1% formic acid. For qualitative analysis, oxidation was monitored by ultrahigh-performance LC (UPLC)-MS using an Acquity UPLC system coupled to a Xevo G2-S Q-ToF mass spectrometer (Waters) operated in positive electrospray mode. Instrument parameters, data acquisition and data processing were controlled by Masslynx 4.1 with source conditions adjusted to maximise sensitivity and minimise fragmentation. Samples were injected on to a Chromolith Performance RP-18e 100 -2 mm column (Merck) heated to 40 o C and eluted at 0.3 mL/min using a gradient of 95 % deionized water supplement with 0.1 % (v/v) formic acid to 95 % acetonitrile.
High-throughput steady-state kinetic assays were conducted as described above but were analysed using a RapidFire RF 365 high-throughput sampling robot (Agilent) attached to an iFunnel Agilent 6550 accurate mass quadrupole time-of-flight (Q-TOF) mass spectrometer operating in the positive ionization mode with the parameters: capillary voltage (4000 V), nozzle voltage (1000 V), fragmentor voltage (365 V), gas temperature (225 °C), gas flow (13 L/min), sheath gas temperature (350 °C), sheath gas flow (12 L/min). Samples were aspirated under vacuum for 0.4 s and loaded onto a C4 solid phase extraction (SPE) cartridge at a flow rate of 1.25 mL/min. The C4 SPE was then washed with aqueous 0.1% (v/v) formic acid in LCMS grade water for 5.5 s at a flow rate of 1.25 mL/min followed by elution from the C4 SPE with 85% (v/v) acetonitrile, 15% (v/v) LCMS water containing 0.1% (v/v) formic acid at a flow rate of 1.5 mL/min for 5.5 s. Peptide oxidation was quantified using RapidFire Integrator software (Agilent), where the charged ion with the highest intensity was chosen for deconvolution and peptide quantification. Turnover was quantified by comparing the areas underneath the product and substrate ions. All spectra were assessed manually in Masshunter Qualitative Analysis B.07.00 (Agilent) to ensure the correct ion was chosen for quantification. All figures and kinetic parameters were generated using Prism (GraphPad).

Determining the O 2 Dependence of KnPCO and MpPCO
The activities of KnPCO and MpPCO were examined at different O 2 concentrations using a previously described method [22]. Briefly, silicone-sealed vials containing the desired concentration of peptide and TCEP were equilibrated with different ratios of N 2 and O 2 gas (to give final defined % O 2 concentrations) for 10 min using a mass flow controller (Brooks Instruments). Reactions were initiated by injecting 1.5 μL of enzyme using a gas-tight syringe (Hamilton), incubated at 25 °C for 45-60s and quenched with 80 μL of 1% (v/v) formic acid. The period of incubation was selected based on parameters where it is known that the enzyme is catalysing peptide oxidation under initial (linear) rate conditions (typically from previous kinetic measurements to determine dependence of rate on peptide substrate concentration). Peptide oxidation was analysed by RapidFire Q-TOF MS as described above.