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 Km(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.
Highlights
Group VII ethylene response factors (ERF-VIIs) regulate transcriptional adaptation to flooding-induced hypoxia in plants
We found that the most catalytically competent isoform is AtPCO4, both in terms of responding to O2 and oxidizing AtRAP2.2/2,12, which suggests that AtPCO4 plays a central role in ERF-VII regulation
We have previously reported the recombinant production of A. thaliana A. thaliana PCO isoform (AtPCO) 1 and 4 to over 90% purity by immobilized nickel-affinity chromatography (8)
Summary
Hydroxylation of specific prolyl residues in HIF-␣ (coupled with 2-oxoglutarate decarboxylation to succinate and CO2) targets HIF-␣ for proteasomal degradation (14). These enzymes are termed O2 sensors due to the dependence of their rate of activity on O2 availability. For the PCOs to effectively function in the same capacity, their rate of activity must be sensitive to O2 availability in a physiologically relevant manner. We have optimized assay conditions for analyzing PCO activity and determined the kinetic parameters of each A. thaliana PCO isoform (AtPCO) in terms of pH, a peptide representing the N termini of the AtERF-VIIs. values that fall within a physiologically relevant range, implicating their biochemical capacity to act as. Our work provides biochemical evidence supporting the assertion that the AtPCOs act as plant O2 sensors and that suggests that different isoforms could have divergent biological functions
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