Identification of novel plant cysteine oxidase inhibitors from a yeast chemical genetic screen.

Hypoxic responses in plants involve Plant Cysteine Oxidases (PCOs). They catalyze the N-terminal cysteine oxidation of Ethylene Response Factors VII (ERF-VII) in an oxygen-dependent manner, leading to their degradation via the cysteine N-degron pathway (Cys-NDP) in normoxia. In hypoxia, PCO activity drops, leading to the stabilization of ERF-VIIs and subsequent hypoxic gene upregulation. Thus far, no chemicals have been described to specifically inhibit PCO enzymes. In this work, we devised an in vivo pipeline to discover Cys-NDP effector molecules. Budding yeast expressing AtPCO4 and plant-based ERF-VII reporters was deployed to screen a library of natural-like chemical scaffolds and was further combined with an Arabidopsis Cys-NDP reporter line. This strategy allowed us to identify three PCO inhibitors, two of which were shown to affect PCO activity in vitro. Application of these molecules to Arabidopsis seedlings led to an increase in ERF-VII stability, induction of anaerobic gene expression, and improvement of tolerance to anoxia. By combining a high-throughput heterologous platform and the plant model Arabidopsis, our synthetic pipeline provides a versatile system to study how the Cys-NDP is modulated. Its first application here led to the discovery of at least two hypoxia-mimicking molecules with the potential to impact plant tolerance to low oxygen stress.

Structures of Arabidopsis thaliana oxygen-sensing plant cysteine oxidases 4 and 5 enable targeted manipulation of their activity.

In higher plants, molecular responses to exogenous hypoxia are driven by group VII ethylene response factors (ERF-VIIs). These transcriptional regulators accumulate in the nucleus under hypoxia to activate anaerobic genes but are destabilized in normoxic conditions through the action of oxygen-sensing plant cysteine oxidases (PCOs). The PCOs catalyze the reaction of oxygen with the conserved N-terminal cysteine of ERF-VIIs to form cysteine sulfinic acid, triggering degradation via the Cys/Arg branch of the N-degron pathway. The PCOs are therefore a vital component of the plant oxygen signaling system, connecting environmental stimulus with cellular and physiological response. Rational manipulation of PCO activity could regulate ERF-VII levels and improve flood tolerance, but requires detailed structural information. We report crystal structures of the constitutively expressed PCO4 and PCO5 from Arabidopsis thaliana to 1.24 and 1.91 Å resolution, respectively. The structures reveal that the PCOs comprise a cupin-like scaffold, which supports a central metal cofactor coordinated by three histidines. While this overall structure is consistent with other thiol dioxygenases, closer inspection of the active site indicates that other catalytic features are not conserved, suggesting that the PCOs may use divergent mechanisms to oxidize their substrates. Conservative substitution of two active site residues had dramatic effects on PCO4 function both in vitro and in vivo, through yeast and plant complementation assays. Collectively, our data identify key structural elements that are required for PCO activity and provide a platform for engineering crops with improved hypoxia tolerance.

Assessing In Vivo Oxygen Dynamics Using Plant N-Terminal Degrons in Saccharomyces cerevisiae.

The expression of plant cysteine oxidase (PCO) enzyme in Saccharomyces cerevisiae enables the Arg/Cys N-degron pathway (Cys-NDP) for selective protein degradation that, in plants, functions as direct oxygen perception mechanism. A synthetic construct based on the plant Cys-NDP substrate related to apetala 2.12 (RAP2.12), the dual luciferase oxygen reporter (DLOR), exploits the N-terminal Cys of RAP2.12, and its oxygen-dependent degradation through the Cys-NDP. The luminescent output of DLOR can be used as a proxy for intracellular oxygen dynamics in budding yeast. Replacement of the luciferase reporter of the DLOR with fluorescent proteins would furthermore facilitate the imaging of reporter dynamics in living cells. In this chapter, we describe the methods for delivering the DLOR synthetic construct to yeast and calibrating its output by means of oxygen quantification in the culture with a physical oxygen sensor. We explain the setup needed to carry out hypoxic treatments with several colonies as replicates. We also describe the method to measure oxygen concentration in the culture, the closest indication of intracellular oxygen levels, as a way that would serve to calibrate the DLOR output. Finally, we propose a strategy to replace the luminescent reporters in the DLOR with fluorescent proteins to visualize oxygen dynamics in vivo.