The plant cysteine oxidases from Arabidopsis thaliana are kinetically tailored to act as oxygen sensors

Abstract

Group VII ethylene response factors (ERF-VIIs) regulate transcriptional adaptation to flooding-induced hypoxia in plants. ERF-VII stability is controlled in an O₂-dependent manner by the Cys/Arg branch of the N-end rule pathway whereby oxidation of a conserved N-terminal cysteine residue initiates target degradation. This oxidation is catalyzed by plant cysteine oxidases (PCOs), which use O₂ as cosubstrate to generate Cys-sulfinic acid. The PCOs directly link O₂ availability to ERF-VII stability and anaerobic adaptation, leading to the suggestion that they act as plant O₂ sensors. However, their ability to respond to fluctuations in O₂ concentration has not been established. Here, we investigated the steady-state kinetics of Arabidopsis thaliana PCOs 1-5 to ascertain whether their activities are sensitive to O₂ levels. We found that the most catalytically competent isoform is AtPCO4, both in terms of responding to O₂ and oxidizing AtRAP2.2/2,12 (two of the most prominent ERF-VIIs responsible for promoting the hypoxic response), which suggests that AtPCO4 plays a central role in ERF-VII regulation. Furthermore, we found that AtPCO activity is susceptible to decreases in pH and that the hypoxia-inducible AtPCOs 1/2 and the noninducible AtPCOs 4/5 have discrete AtERF-VII substrate preferences. Pertinently, the AtPCOs had K_m(O2)^app values in a physiologically relevant range, which should enable them to sensitively react to changes in O₂ availability. This work validates an O₂-sensing role for the PCOs and suggests that differences in expression pattern, ERF-VII selectivity, and catalytic capability may enable the different isoforms to have distinct biological functions. Individual PCOs could therefore be targeted to manipulate ERF-VII levels and improve stress tolerance in plants.

Keywords: Arabidopsis; ERF-VII; N-end rule; enzyme kinetics; hypoxia; oxygen-sensing; plant biochemistry; plant cysteine oxidase; post-translational modification (PTM); protein degradation.

Figure 1.

Purification and iron content of AtPCOs 1–5. A, SDS-PAGE analysis showing purified AtPCO isoforms 1–5 following sequential steps of immobilized nickel-affinity and size-exclusion chromatography with M indicating the molecular mass marker. Each AtPCO protein migrated to the expected mass, demonstrating over 95% purity, except for AtPCO3, which was estimated to be ∼50% pure. This was accounted for in all activity measurements. B, the iron content of each AtPCO was quantified using a ferrozine-like assay, allowing metal occupancy to be estimated from known protein concentrations. All samples possessed substoichiometric amounts of cofactor with AtPCO2 containing the least iron. Error bars display S.D. (n = 3).

Figure 2.

Analyzing the influence of exogenous iron and ascorbate on AtPCO activity. The specific activities of AtPCOs 1–5 were determined with and without FeSO₄ (100-fold in excess of enzyme concentration) and sodium ascorbate (1 mm) by measuring the rate of AtRAP2(2–15) cysteine oxidation at regular time intervals (0, 30, 60, and 90 s) by LC-MS. The addition of exogenous additives, particularly ascorbate, significantly increased the specific activities of AtPCO2 and, to a lesser extent, AtPCO5 but had little effect on the other isoforms. AtPCOs 4 and 5 had the greatest specific activity. Reactions were conducted in 50 mm HEPES, 50 mm NaCl, and 1 mm TCEP, pH 7.5, at 25 °C. Statistical analysis was completed using a one-way analysis of variance, post hoc Dunnett test using the “no additive” sample as the control reference with * and *** denoting p ≤ 0.05 and p ≤ 0.001, respectively. Error bars display S.D. (n = 3).

Figure 3.

The activity of AtPCOs 1–5 depends on pH. A, pH profile showing the relative activities of AtPCOs 1–5 with 200 μm RAP2(2–15) at 25 °C and different pH values using 50 mm Bis tris propane, 50 mm NaCl, and 5 mm TCEP as buffer. A significant reduction in AtPCO activity is observed below pH 7.0 for all isoforms. This correlates with the side-group pK_a for the N-terminal cysteine of RAP2(2–15) (B), which was determined by monitoring the chemical shift of cysteine β-protons in D₂O (>95%) using ¹H NMR. This suggests that the thiol needs to be deprotonated for binding and/or turnover. pH* denotes the pH reading given by a pH meter calibrated for solutions in H₂O for D₂O samples. pK_a was corrected to H₂O using the formula pK^H = 0.929pK^H* + 0.42 derived in Ref. . Error bars display S.E. (n = 3).

Figure 4.

Analyzing the dependence of AtPCOs 1–5 activity on AtRAP2(2–15) availability under atmospheric O₂. Michaelis–Menten kinetic plots for AtPCOs 1–5 with respect to AtRAP2(2–15) concentration (calculated from initial rates presented in Fig. S3) are shown. Assays were conducted under aerobic conditions at 25 °C using 50 mm Bis tris propane, 50 mm NaCl, and 5 mm TCEP, pH 8.0, as buffer. Data collected for AtPCO4 and AtPCO5 were fitted to an equation for substrate inhibition to address the decline in rate at high RAP2(2–15) concentrations. Error bars display S.E. (n = 3).

Figure 5.

Dependence of AtPCOs 1–5 activity on O₂ availability. Michaelis–Menten kinetic plots display the activities of AtPCOs 1–5 with respect to O₂ concentration (using single time points within the linear rate range identified in Fig. S3). Assays were conducted at 25 °C using 50 mm Bis tris propane, 50 mm NaCl, and 5 mm TCEP, pH 8.0, as buffer. Peptide concentrations of 5 times the relative K_m for AtRAP2(2–15) were used for analysis (given in the graph titles) to ensure that AtPCO turnover was not limited by peptide availability with the exception of AtPCO4, which was analyzed at 1 mm AtRAP2(2–15) to avoid the substrate inhibition region of this isoform (Fig. 4). Error bars display S.E. (n = 3).

Figure 6.

AtERF-VII substrate preferences of AtPCOs 1–5. A, the relative activities of AtPCOs 1–5 for each AtERF-VII substrate were determined by pooling the four peptides at a concentration equal to the K_m for RAP2(2–15) and normalizing the rate of N-terminal cysteine oxidation to that of the best substrate. Specific activities are given in Table S1. Significant AtERF-VII preferences (relative to the poorest substrate) could be observed for most isoforms despite high conservation in the peptide sequences (B, where black indicates conservation with and red indicates deviation from RAP2.2/2.12). Statistical analysis was completed using a one-way analysis of variance, post hoc Dunnett test using the substrate that generated the lowest activity as the control reference, with * and *** denoting p ≤ 0.05 and p ≤ 0.001, respectively. Error bars display S.D. (n = 3).